G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
valosin containing protein
G00001108 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000019855 (Vega human gene)
ENSG00000165280 (Ensembl human gene)
7415 (Entrez Gene)
1145 (G2Cdb plasticity & disease)
VCP (GeneCards)
601023 (OMIM)
Marker Symbol
HGNC:12666 (HGNC)
Protein Sequence
P55072 (UniProt)

Synonyms (2)

  • p97

Literature (114)

Pubmed - other

  • New genes, new dilemmas: FTLD genetics and its implications for families.

    Goldman JS, Adamson J, Karydas A, Miller BL and Hutton M

    Sergievsky Center and Taub Institute, Columbia University, New York, NY 10036, USA. jg2673@columbia.edu

    After Alzheimer's disease, frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in persons less than 65 years of age. Up to 40% of FTLD cases have a positive family history. Research on these families has led to the discovery of four disease-causing genes: microtubule-associated protein tau (MAPT), progranulin (PGRN), valosin-containing protein (VCP), and charged multivesicular body protein 2B (CHMP2B). MAPT and PGRN are responsible for the largest number of familial cases. Each of these genes differs by disease mechanism. Moreover mutations in both genes are associated with significant interfamilial and intrafamilial phenotypic variation. Genetic counseling needs to address the differences between the PGRN and MAPT mutations as well as the variation in clinical symptoms. The aims of this article are to describe the genetics of the FTLD spectrum and aid in the genetic counseling of individuals who may carry genetic mutations.

    Funded by: NIA NIH HHS: P01 AG019724

    American journal of Alzheimer's disease and other dementias 

  • Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease.

    Ju JS, Fuentealba RA, Miller SE, Jackson E, Piwnica-Worms D, Baloh RH and Weihl CC

    Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM), Paget's disease of the bone, and frontotemporal dementia (IBMPFD). Patient muscle has degenerating fibers, rimmed vacuoles (RVs), and sarcoplasmic inclusions containing ubiquitin and TDP-43 (TARDNA-binding protein 43). In this study, we find that IBMPFD muscle also accumulates autophagosome-associated proteins, Map1-LC3 (LC3), and p62/sequestosome, which localize to RVs. To test whether VCP participates in autophagy, we silenced VCP or expressed adenosine triphosphatase-inactive VCP. Under basal conditions, loss of VCP activity results in autophagosome accumulation. After autophagic induction, these autophagosomes fail to mature into autolysosomes and degrade LC3. Similarly, IBMPFD mutant VCP expression in cells and animals leads to the accumulation of nondegradative autophagosomes that coalesce at RVs and fail to degrade aggregated proteins. Interestingly, TDP-43 accumulates in the cytosol upon autophagic inhibition, similar to that seen after IBMPFD mutant expression. These data implicate VCP in autophagy and suggest that impaired autophagy explains the pathology seen in IBMPFD muscle, including TDP-43 accumulation.

    Funded by: NCI NIH HHS: P50 CA094056, P50 CA94056; NIA NIH HHS: 5K08AG026271, K08 AG026271, P50 AG005681, P50AG05681, R01 AG031867, R01AG031867; NINDS NIH HHS: P30 NS057105

    The Journal of cell biology 2009;187;6;875-88

  • Valosin containing protein associated inclusion body myopathy: abnormal vacuolization, autophagy and cell fusion in myoblasts.

    Vesa J, Su H, Watts GD, Krause S, Walter MC, Martin B, Smith C, Wallace DC and Kimonis VE

    Department of Pediatrics, Division of Genetics and Metabolism, University of California, Irvine, CA 92868, USA.

    Inclusion body myopathy associated with Paget's disease and frontotemporal dementia (IBMPFD) is caused by mutations in the valosin containing protein (VCP) gene. The disease is associated with progressive proximal muscle weakness, inclusions and vacuoles in muscle fibers, malfunction in the bone remodeling process resulting in Paget's disease, and premature frontotemporal dementia. VCP is involved in several cellular processes related to the endoplasmic reticulum associated degradation of proteins. To understand the pathological mechanisms underlying the myopathy in IBMPFD, we have studied the cellular consequences of VCP mutations in human primary myoblasts. Our results revealed that patients' myoblasts accumulate large vacuoles. Lysosomal membrane proteins Lamp1 and Lamp2 show increased molecular weights in patients' myoblasts due to differential N-glycosylation. Additionally, mutant myoblasts show increased autophagy when cultured in the absence of nutrients, as well as defective cell fusion and increased apoptosis. Our results elucidate that VCP mutations result in disturbances in several cellular processes, which will help us in the understanding of the pathological mechanisms resulting in muscle weakness and other features of VCP associated disease.

    Funded by: NIA NIH HHS: AG025159, AG13154, AG16573, AG24373; NIAMS NIH HHS: R01 AR050236, R01 AR050236-01A1, R01 AR050236-02, R01 AR050236-03, R01 AR050236-04, R01 AR050236-05, R01AR050236; NINDS NIH HHS: NS21328, NS41850; Telethon: GTB07001

    Neuromuscular disorders : NMD 2009;19;11;766-72

  • Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD.

    Lim PJ, Danner R, Liang J, Doong H, Harman C, Srinivasan D, Rothenberg C, Wang H, Ye Y, Fang S and Monteiro MJ

    Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.

    Unwanted proteins in the endoplasmic reticulum (ER) are exported into the cytoplasm and degraded by the proteasome through the ER-associated protein degradation pathway (ERAD). Disturbances in ERAD are linked to ER stress, which has been implicated in the pathogenesis of several human diseases. However, the composition and organization of ERAD complexes in human cells is still poorly understood. In this paper, we describe a trimeric complex that we propose functions in ERAD. Knockdown of erasin, a platform for p97/VCP and ubiquilin binding, or knockdown of ubiquilin in human cells slowed degradation of two classical ERAD substrates. In Caenorhabditis elegans, ubiquilin and erasin are ER stress-response genes that are regulated by the ire-1 branch of the unfolded protein response pathway. Loss of ubiquilin or erasin resulted in activation of ER stress, increased accumulation of polyubiquitinated proteins, and shortened lifespan in worms. Our results strongly support a role for this complex in ERAD and in the regulation of ER stress.

    Funded by: NIA NIH HHS: AG016839; NIGMS NIH HHS: GM066287, GM06696, R01 GM066287-09

    The Journal of cell biology 2009;187;2;201-17

  • The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER.

    Ernst R, Mueller B, Ploegh HL and Schlieker C

    Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA.

    YOD1 is a highly conserved deubiquitinating enzyme of the ovarian tumor (otubain) family, whose function has yet to be assigned in mammalian cells. YOD1 is a constituent of a multiprotein complex with p97 as its nucleus, suggesting a functional link to a pathway responsible for the dislocation of misfolded proteins from the endoplasmic reticulum. Expression of a YOD1 variant deprived of its deubiquitinating activity imposes a halt on the dislocation reaction, as judged by the stabilization of various dislocation substrates. Accordingly, we observe an increase in polyubiquitinated dislocation intermediates in association with p97 in the cytosol. This dominant-negative effect is dependent on the UBX and Zinc finger domains, appended to the N and C terminus of the catalytic otubain core domain, respectively. The assignment of a p97-associated ubiquitin processing function to YOD1 adds to our understanding of p97's role in the dislocation process.

    Funded by: NIAID NIH HHS: R37 AI033456-16, R37 AI033456-17; NIGMS NIH HHS: R01 GM062502-05A1, R01 GM062502-06

    Molecular cell 2009;36;1;28-38

  • Clinical heterogeneity in 3 unrelated families linked to VCP p.Arg159His.

    van der Zee J, Pirici D, Van Langenhove T, Engelborghs S, Vandenberghe R, Hoffmann M, Pusswald G, Van den Broeck M, Peeters K, Mattheijssens M, Martin JJ, De Deyn PP, Cruts M, Haubenberger D, Kumar-Singh S, Zimprich A and Van Broeckhoven C

    Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, University of Antwerp-CDE, Universiteitsplein 1, B-2610 Antwerpen, Belgium.

    Background: Families associated with missense mutations in the valosin-containing protein (VCP) present with a rare autosomal dominant multisystem disorder of frontotemporal lobar degeneration (FTLD), inclusion body myopathy (IBM), and Paget disease of bone (PDB), referred to as IBMPFD.

    Methods: We used exon-based genomic DNA sequencing to test for VCP mutations in 123 unrelated Belgian patients with FTLD and their relatives, and the absence of such mutations in 157 control individuals. We analyzed haplotype sharing among mutation carriers by genotyping 8 microsatellite markers in the VCP locus. We obtained family history and clinical and pathologic data using established diagnostic instruments.

    Results: Mutation analysis of VCP identified 2 Belgian patients with FTLD carrying the p.Arg159His mutation, which segregated in their families. In one family, patients presented with FTLD only, whereas in the other family, patients developed FTLD, PDB, or both without signs of IBM for any of the mutation carriers. We had previously identified p.Arg159His in an Austrian family with patients exhibiting both IBM and PDB. Haplotype sharing analysis indicated that the 3 p.Arg159His families are unrelated. Clinical follow-up of the Austrian family identified dementia symptoms in 1 patient. Autopsy data of 3 patients of the 2 Belgian families revealed FTLD pathology with numerous ubiquitin-immunoreactive, intranuclear inclusions and dystrophic neurites staining positive for TDP-43 protein.

    Conclusions: In 3 unrelated families with IBMPFD segregating VCP p.Arg159His, we observed a high degree of clinical heterogeneity and variable penetrance of the 3 cardinal clinical phenotypes: inclusion body myopathy, Paget disease of bone, and frontotemporal lobar degeneration. In contrast, the neuropathologic phenotype was consistent with FTLD-TDP type 4.

    Neurology 2009;73;8;626-32

  • Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation.

    Halawani D, LeBlanc AC, Rouiller I, Michnick SW, Servant MJ and Latterich M

    The Nicholas Conor Institute for Pediatric Cancer Research, 9710 Scranton Road, Suite 170, San Diego, CA 92121, USA.

    Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH(2) domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97(R155P) and p97(A232E) formed hexameric ring-shaped structures of approximately 600 kDa. p97(R155P) and p97(A232E) exhibited an approximately 3-fold increase in ATPase activity compared to wild-type p97 (p97(WT)) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97(R155P) and p97(A232E), but not p97(WT) or the ATP-binding mutant p97(K524A). Therefore, we propose that hIBMPFTD p97/VCP mutants p97(R155P) and p97(A232E) possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes.

    Molecular and cellular biology 2009;29;16;4484-94

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non-small cell lung carcinoma.

    Qiu Y, Morii E, Tomita Y, Zhang B, Matsumura A, Kitaichi M, Okumura M and Aozasa K

    Department of Pathology, Tongji University School of Medicine, Shanghai 200092, China.

    Previous studies on the mammary carcinoma cell line have shown that the pre B cell leukemia transcription factor 1 (PBX1) was a transcription factor for valosin-containing protein (VCP), which is involved in invasion and metastasis of cancers. The roles of PBX1 and PBX2, a highly homologous transcription factor to PBX1, for expression of VCP were examined in the cell lines from non-small cell lung cancer (NSCLC). The effects of PBX1 and PBX2 on VCP expression were examined with siRNA in A549 and PC14 NSCLC cell lines. Expression levels of PBX2 and VCP were immunohistochemically examined and compared with each other in 206 NSCLC cases. Subsequently, significance of PBX expression in clinical behavior of NSCLC patients was evaluated. Expression levels of VCP mRNA significantly decreased when PBX2 but not PBX1 expression was knocked down in NSCLC cell lines. Immunohistochemically, staining intensity of PBX2 was correlated with that of VCP in clinical samples. Then correlation of PBX2 expression and clinical behavior of NSCLC patients was evaluated. Univariate analysis revealed high expression levels of PBX2 and VCP to be poor prognosticators for overall and disease-free survival. Multivariate analysis revealed that high expression of VCP but not PBX2 to be an independent prognostic factor. PBX2 is a transcription factor for VCP in NSCLC. Because high levels of PBX2 expression correlated with prognosis of NSCLC, PBX2 could be a target molecule for treatment of NSCLC.

    Cancer science 2009;100;7;1198-209

  • UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation.

    Nagahama M, Ohnishi M, Kawate Y, Matsui T, Miyake H, Yuasa K, Tani K, Tagaya M and Tsuji A

    Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan. nagahama@bio.tokushima-u.ac.jp

    AAA ATPase VCP and its yeast ortholog Cdc48, in a complex with the Ufd1-Npl4 heterodimer as an adaptor, play an essential role in endoplasmic reticulum-associated degradation (ERAD). Several UBX domain-containing proteins function to recruit ubiquitylated substrates to VCP/Cdc48 by binding both VCP/Cdc48 and other ERAD components such as ubiquitin ligases. Here we show that mammalian UBXD1 is an additional UBX domain-containing protein involved in the ERAD process. UBXD1 is a cytosolic protein that interacts with VCP and Derlin-1. Overexpression of UBXD1 in cells causes selective dissociation of Ufd1 from VCP, resulting in inhibition of mutant cystic fibrosis transmembrane conductance regulator (CFTR) degradation by ERAD. Additionally, depletion of endogenous UBXD1 protein by RNA interference also results in a defect in CFTR degradation. Collectively, these findings suggest that UBXD1 is a regulatory component of ERAD that may modulate the adaptor binding to VCP.

    Biochemical and biophysical research communications 2009;382;2;303-8

  • VCP mutations causing frontotemporal lobar degeneration disrupt localization of TDP-43 and induce cell death.

    Gitcho MA, Strider J, Carter D, Taylor-Reinwald L, Forman MS, Goate AM and Cairns NJ

    Alzheimer's Disease Research Center and the Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.

    Funded by: NIA NIH HHS: P01-AG03991, P50-AG05681; NINDS NIH HHS: P30-NS057105

    The Journal of biological chemistry 2009;284;18;12384-98

  • Clinical outcome in 19 French and Spanish patients with valosin-containing protein myopathy associated with Paget's disease of bone and frontotemporal dementia.

    Stojkovic T, Hammouda el H, Richard P, López de Munain A, Ruiz-Martinez J, Camaño P, Gonzalez PC, Laforêt P, Pénisson-Besnier I, Ferrer X, Lacour A, Lacomblez L, Claeys KG, Maurage CA, Fardeau M and Eymard B

    AP-HP, Institute of Myology, G-H Pitié-Salpêtrière, Paris, France. Tanya.Stojkovic@psl.aphp.fr

    We report the clinical, histological and genetic findings in 10 families (19 patients) presenting mutations in the valosin-containing protein (VCP). The mean age at onset was 42 years. The clinical pattern was characterized by an early involvement of the proximal upper limbs with scapular winging. Axial and lower limb muscles were often affected, whereas facial, oculobulbar muscles were spared. Ten patients were wheelchair bound after a mean disease course of 9 years and six patients required canes for walking. Two patients required mechanically assisted ventilation and seven patients had reduced vital capacity. There was no cardiac involvement. Paget's disease of bone was observed in eight patients and cognitive impairment in nine patients. Seven patients died as a consequence of weakness and respiratory distress. Muscle biopsy showed rimmed vacuolar myopathy. Genetic analysis revealed missense heterozygous mutations mostly located in exon 5 of the VCP gene, four of which were not previously reported. We observed intrafamilial and interfamilial variability in terms of severity, distribution of weakness and presence or not of Paget's disease or cognitive impairment.

    Neuromuscular disorders : NMD 2009;19;5;316-23

  • Inclusion body myopathy and frontotemporal dementia caused by a novel VCP mutation.

    Bersano A, Del Bo R, Lamperti C, Ghezzi S, Fagiolari G, Fortunato F, Ballabio E, Moggio M, Candelise L, Galimberti D, Virgilio R, Lanfranconi S, Torrente Y, Carpo M, Bresolin N, Comi GP and Corti S

    Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation Ospedale Maggiore Policlinico Mangiagalli and Regina Elena, Via F. Sforza 35, 20122 Milan, Italy.

    Hereditary inclusion body myopathy (IBM) with Paget's disease of the bone (PDB) and frontotemporal dementia (FTD) is a rare autosomal dominant disease caused by mutations in the valosin-containing protein (VCP) gene. We report a novel heterozygous VCP gene mutation (R159C) in a 69-year-old Italian patient presenting with slowly progressive muscle weakness of the distal upper and proximal lower limbs since the age of 50 years, 18 years later FTD supervened. No dementia or myopathies were revealed in the family history covering two generations. Degenerative changes and rimmed vacuoles together with VCP- and ubiquitin-positive cytoplasmic and nuclear aggregates were observed at the muscle biopsy. Several elements support the pathogenic role of the R159C VCP gene mutation: the occurrence at the same codon of a different, previously identified pathogenic mutation within a VCP gene mutational hot-spot, the histopathological and biochemical evidence of muscle VCP accumulation and the combined clinical presentation of IBM and FTD. These findings suggest VCP gene investigation even in apparently sporadic cases.

    Funded by: Telethon: GTF02008

    Neurobiology of aging 2009;30;5;752-8

  • Mechanisms targeting apolipoprotein B100 to proteasomal degradation: evidence that degradation is initiated by BiP binding at the N terminus and the formation of a p97 complex at the C terminus.

    Rutledge AC, Qiu W, Zhang R, Kohen-Avramoglu R, Nemat-Gorgani N and Adeli K

    Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.

    Objective: In lipid-poor states, the ubiquitin-proteasomal pathway rapidly degrades misfolded apolipoprotein B100 (apoB) cotranslationally, although the mechanism of delivery from the ER to cytosolic proteasomes is poorly understood. Here we demonstrate key roles of BiP, an ER luminal chaperone, and p97, a cytosolic ATPase anchored to the ER membrane, in the targeting of apoB for proteasomal degradation.

    Using coimmunoprecipitations, we observed associations of apoB with BiP, p97, Derlin-1, VIMP, and the E3 ubiquitin ligase Hrd1 in HepG2 cells. BiP and p97 were found to bind apoB cotranslationally. Expression of C-terminal truncated apoB molecules in COS-7 cells showed an N-terminal region outside apoB15 and a C-terminal region found in apoB72 were required for BiP and p97 binding, respectively. Interestingly, overexpression of dominant negative p97 demonstrated that the ATPase activity of p97 was essential for proteasomal degradation of apoB but not for apoB binding. However, p97 activity did not appear to affect the N terminus of apoB, which may be cleaved before degradation.

    Conclusions: These data suggest that p97 and BiP play critical roles in the cotranslational delivery of apoB to proteasomes and formation of a degradative complex. Proteasomal degradation appears to selectively target apoB molecules with large C-terminal domains.

    Arteriosclerosis, thrombosis, and vascular biology 2009;29;4;579-85

  • The Salmonella effector SptP dephosphorylates host AAA+ ATPase VCP to promote development of its intracellular replicative niche.

    Humphreys D, Hume PJ and Koronakis V

    Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK.

    Virulence effectors delivered into intestinal epithelial cells by Salmonella trigger actin remodeling to direct pathogen internalization and intracellular replication in Salmonella-containing vacuoles (SCVs). One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake. SptP also harbors a C-terminal protein tyrosine phosphatase (PTPase) domain with no clear host substrates. Investigating SptP's longevity in infected cells, we uncover a late function of SptP, showing that it associates with SCVs, and its PTPase activity increases pathogen replication. Direct SptP binding and specific dephosphorylation of the AAA+ ATPase valosin-containing protein (VCP/p97), a facilitator of cellular membrane fusion and protein degradation, enhanced pathogen replication in SCVs. VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase. Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

    Funded by: Medical Research Council: G0500583; Wellcome Trust

    Cell host & microbe 2009;5;3;225-33

  • UBXD1 binds p97 through two independent binding sites.

    Kern M, Fernandez-Sáiz V, Schäfer Z and Buchberger A

    Max Planck Institute of Biochemistry, Department of Molecular Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.

    The chaperone-related p97 protein plays a central role in various cellular processes involving the ubiquitin-proteasome system. The diverse functions of p97 are controlled by a large number of cofactors that recruit specific substrates or influence their ubiquitylation state. Many cofactors bind through a UBX or PUB domain, two major p97 binding modules. However, the recently identified UBXD1 cofactor possesses both domains. To elucidate the molecular basis underlying the interaction between UBXD1 and p97, we analyzed the contribution of both domains to p97 binding biochemically and in living cells. The PUB domain mediated robust binding to the carboxy-terminus of p97, while the UBX domain did not contribute to p97 binding. Importantly, we identified an additional p97 binding site in UBXD1 that competed with the p47 cofactor for binding to the N domain of p97. This novel, bipartite binding mode suggests that UBXD1 could be an efficient regulator of p97 cofactor interactions.

    Biochemical and biophysical research communications 2009;380;2;303-7

  • A novel mutation in the VCP gene (G157R) in a German family with inclusion-body myopathy with Paget disease of bone and frontotemporal dementia.

    Djamshidian A, Schaefer J, Haubenberger D, Stogmann E, Zimprich F, Auff E and Zimprich A

    Department of Neurology, Medical University of Vienna; Währinger Gürtel 18-20, Vienna A-1097, Austria.

    Mutations in the valosin-containing protein (VCP) are known to cause autosomal-dominant inclusion-body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD). We report a novel missense mutation (G157R) in the N-terminal region of the VCP gene in a German family. Family members presented with mild to moderate proximal muscle weakness, Paget disease of bone, and signs of early cognitive decline, with onset in the fourth decade. Two family members also showed signs of early hearing impairment, which was confirmed to be sensorineural in one person, a symptom not yet described in the context of IBMPFD.

    Muscle & nerve 2009;39;3;389-91

  • No association of common VCP variants with sporadic frontotemporal dementia.

    Schumacher A, Friedrich P, Diehl J, Ibach B, Schoepfer-Wendels A, Mueller JC, Konta L, Laws SM, Kurz A, Foerstl H and Riemenschneider M

    Neurochemistry and Neurogenetics Laboratory, Department of Psychiatry, TU-Munich, Germany.

    Mutations in the gene for valosin containing protein (VCP) cause autosomal dominant inclusion body myopathy associated with Paget disease and frontotemporal dementia (IBMPFD). To investigate the role of this novel gene in sporadic forms of frontotemporal dementia (FTD), we genotyped 27 single nucleotide polymorphisms covering the entire VCP genomic region in 198 patients with sporadic FTD and 184 matched controls from Germany. No significant association could be demonstrated. There is no evidence, that common variants in VCP confer a strong risk to the development of sporadic FTD.

    Neurobiology of aging 2009;30;2;333-5

  • Unsaturated fatty acids inhibit proteasomal degradation of Insig-1 at a postubiquitination step.

    Lee JN, Zhang X, Feramisco JD, Gong Y and Ye J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Proteasomes mediate the regulated degradation of Insig-1, a membrane protein of the endoplasmic reticulum (ER) that plays a crucial role in lipid metabolism. We showed previously that sterols inhibit this degradation by blocking ubiquitination of Insig-1. Here we show that unsaturated fatty acids stabilize Insig-1 without affecting its ubiquitination. Instead unsaturated fatty acids inhibit extraction of ubiquitinated Insig-1 from membranes, a process known to be mediated by valosin-containing protein and necessary for ER-associated degradation. Valosin-containing protein is recruited to Insig-1 through the action of another protein, Ubxd8. Unsaturated fatty acids block the binding between Ubxd8 and Insig-1, thereby abrogating the membrane extraction of Insig-1. Unsaturated fatty acid-mediated stabilization of Insig-1 enhances the ability of sterols to inhibit proteolytic activation of SREBP-1, which activates transcription of genes involved in fatty acid synthesis. The current study provides a molecular mechanism for regulation of proteasome-mediated ER protein degradation at a postubiquitination step.

    Funded by: NHLBI NIH HHS: HL-20948

    The Journal of biological chemistry 2008;283;48;33772-83

  • Tyrosine phosphorylation of ATPase p97 regulates its activity during ERAD.

    Li G, Zhao G, Schindelin H and Lennarz WJ

    Department of Biochemistry and Cell Biology, 450 Life Science Building, Stony Brook University, Stony Brook, NY 11794-5215, USA.

    In eukaryotic cells, the endoplasmic reticulum-associated degradation (ERAD) pathway is essential for the disposal of misfolded proteins. Recently, we demonstrated the existence of a higher order complex consisting of the ER bound E3 ligase gp78, p97, PNGase, and HR23B in mammals. This complex may serve to facilitate the routing of misfolded glycoproteins out of the ER to the cytosol where they are degraded by the proteasome. In this complex, p97 functions as an organizer to mediate the interactions with gp78 and the deglycosylating enzyme PNGase. A novel protein-binding motif of mouse p97 was identified that consists of its last 10 amino acid residues; this motif is sufficient to mediate the interaction of p97 with PNGase and Ufd3. Phosphorylation of p97's highly conserved penultimate tyrosine residue, completely blocks binding of both PNGase and Ufd3 to mp97. We have found that c-Src kinase directly and selectively phosphorylated the penultimate tyrosine of p97 in vitro, and that overexpression of c-Src significantly increased the phosphorylation level of p97 in cells and caused accumulation of the ERAD substrate TCRalpha-GFP, as well as ubiquitin-conjugated substrates. These results suggest a role for p97 phosphorylation in the degradation of misfolded glycoproteins.

    Funded by: NIDDK NIH HHS: DK54835; NIGMS NIH HHS: GM33814

    Biochemical and biophysical research communications 2008;375;2;247-51

  • Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway.

    Maruyama Y, Yamada M, Takahashi K and Yamada M

    Department of Psychogeriatrics, National Institute of Mental Health, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8553, Japan.

    Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer's disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1DeltaR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1DeltaR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.

    Biochemical and biophysical research communications 2008;374;4;737-41

  • The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway.

    Fisher EA, Lapierre LR, Junkins RD and McLeod RS

    Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.

    The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.

    Journal of lipid research 2008;49;10;2149-60

  • UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

    Alexandru G, Graumann J, Smith GT, Kolawa NJ, Fang R and Deshaies RJ

    Division of Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.

    p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.

    Funded by: Howard Hughes Medical Institute

    Cell 2008;134;5;804-16

  • Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD.

    Lass A, McConnell E, Fleck K, Palamarchuk A and Wójcik C

    Department of Anatomy and Cell Biology, Indiana University School of Medicine, 8600 University Boulevard, Evansville, IN 47712, USA.

    Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP(Ufd1-Npl4) promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate alphaTCR. This favors the model where the Ufd1-Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.

    Experimental cell research 2008;314;14;2715-23

  • Inclusion body myopathy, Paget's disease of the bone and frontotemporal dementia: recurrence of the VCP R155H mutation in an Italian family and implications for genetic counselling.

    Viassolo V, Previtali SC, Schiatti E, Magnani G, Minetti C, Zara F, Grasso M, Dagna-Bricarelli F and Di Maria E

    Department of Neuroscience, Ophthalmology and Genetics, University of Genova, Genova, Italy, and Laboratory of Genetics, Galliera Hospital, Genova, Italy.

    The acronym IBMPFD denotes a syndrome including inclusion body myopathy, Paget's disease of the bone (PDB) and frontotemporal dementia (FTD) as cardinal features, which is caused by missense mutations in the VCP gene. We studied the clinical characteristics and the histopathological features in two siblings and their mother who presented with adult-onset myopathy and presenile, rapidly progressive FTD. One sibling also showed PDB. Light and electron microscopy performed on muscle biopsies demonstrated degenerative changes with inclusion bodies and abnormal aggregates. Mutation analysis of the VCP gene on affected siblings revealed a heterozygous missense mutation (R155H) in a hot spot. This is the first Italian family with multiple individuals diagnosed as having IBMPFD and carrying the recurrent R155H mutation. The implications for genetic counselling were also discussed, with regard to the procedures that may be offered to families suffering from a multisystem disorder with high risk of cognitive decline.

    Funded by: Telethon: GTF04003

    Clinical genetics 2008;74;1;54-60

  • E74-like factor 2 transactivates valosin-containing protein gene, a gene involved in cancer growth.

    Qiu Y, Morii E, Zhang B, Tomita Y and Aozasa K

    Department of Pathology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

    Enhanced expression of valosin-containing protein (VCP) correlates with invasiveness and metastatic potential of cancer cells; therefore its transcriptional regulation could be a new target for cancer therapy. Here, the mechanism for transactivation of VCP promoter by E74-like factor 2 (ELF2), a transcription factor which enhances VCP transcription, was investigated. There was a highly conserved region among species in VCP promoter with one proven motif for ELF2 binding. However, the mutation at this motif did not obviously change the promoter activity. Further survey revealed another ELF2 binding motif in the upstream of the conserved region. The promoter activity was modestly reduced when this upstream motif was mutated, and completely abolished when both upstream and downstream motifs were mutated. Chromatin immunoprecipitation assay revealed that ELF2 bound both upstream and downstream motifs. These findings indicate that ELF2 transactivates VCP promoter through binding to two motifs, with a predominant contribution of the upstream one.

    Experimental and molecular pathology 2008;84;3;226-9

  • Inhibition of p97-dependent protein degradation by Eeyarestatin I.

    Wang Q, Li L and Ye Y

    Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2008;283;12;7445-54

  • Derlin-1 and p97/valosin-containing protein mediate the endoplasmic reticulum-associated degradation of human V2 vasopressin receptors.

    Schwieger I, Lautz K, Krause E, Rosenthal W, Wiesner B and Hermosilla R

    Leibniz-Institut für Molekulare Pharmakologie (FMP), Campus Buch, Robert-Rössle Str. 10, 13125 Berlin, Germany.

    The endoplasmic reticulum-associated degradation (ERAD), the main quality control pathway of the cell, is crucial for the elimination of unfolded or misfolded proteins. Several diseases are associated with the retention of misfolded proteins in the early secretory pathway. Among them is X-linked nephrogenic diabetes insipidus, caused by mutations in the gene encoding the V2 vasopressin receptor (V2R). We studied the degradation pathways of three intracellularly retained V2R mutants with different misfolded domains in human embryonic kidney 293 cells. At steady state, the wild-type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, whereas core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the nonglycosylated and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild-type receptor were found to be polyubiquitinylated. Nonglycosylated and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Distinct Derlin-1-dependent and -independent ERAD pathways have been proposed for proteins with different misfolded domains (cytosolic, extracellular, and membrane) in yeast. Here, we show for the first time that V2R mutants with different misfolded domains are able to coprecipitate the ERAD components p97/valosin-containing protein, Derlin-1 and the 26S proteasome regulatory subunit 7. Our results demonstrate the presence of a Derlin-1-mediated ERAD pathway degrading wild-type and disease-causing V2R mutants with different misfolded domains in a mammalian system.

    Molecular pharmacology 2008;73;3;697-708

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • An Italian family with inclusion-body myopathy and frontotemporal dementia due to mutation in the VCP gene.

    Gidaro T, Modoni A, Sabatelli M, Tasca G, Broccolini A and Mirabella M

    Department of Neuroscience, Catholic University, L.go A. Gemelli 8, 00168 Rome, Italy.

    Mutations of the valosin-containing protein gene (VCP) are responsible for autosomal-dominant hereditary inclusion-body myopathy associated with frontotemporal dementia and Paget's disease of bone. We identified the p.R155C missense mutation in the VCP gene segregating in an Italian family with three affected siblings, two of whom had a progressive myopathy associated with dementia, whereas one exhibited a progressive myopathy and preclinical signs of Paget's disease of bone. Our study demonstrates that VCP mutations are found in patients of Italian background and may lead to a variable clinical phenotype even within the same kinship.

    Muscle & nerve 2008;37;1;111-4

  • The differentiation/retrodifferentiation program of human U937 leukemia cells is accompanied by changes of VCP/p97.

    Bertram C, von Neuhoff N, Skawran B, Steinemann D, Schlegelberger B and Hass R

    Dept. of Gynecology, Biochemistry and Tumor Biology Lab, Medical School Hannover, Carl-Neuberg-Str, 1, D - 30625 Hannover, Germany. bertram.catharina@mh-hannover.de

    Background: Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.

    Results: Separation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G0/G1 cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G0/G1 growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.

    Conclusion: The findings demonstrated that monocytic differentiation and G0/G1 growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.

    BMC cell biology 2008;9;12

  • Ubxd1 is a novel co-factor of the human p97 ATPase.

    Madsen L, Andersen KM, Prag S, Moos T, Semple CA, Seeger M and Hartmann-Petersen R

    Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark.

    The AAA ATPase complex known as p97 or VCP in mammals and Cdc48 in yeast is connected to a multitude of cellular pathways, including membrane fusion, protein folding, protein degradation and activation of membrane-bound transcription factors. The mechanism by which p97 participates in such a broad spectrum of cellular functions appears to be via recruiting certain specific co-factors. Here we isolate and characterize the human protein Ubxd1, a novel co-factor of p97. We show that Ubxd1 is a stable protein that localizes to the cytoplasm and nucleus and is highly enriched in centrosomes. In mice Ubxd1 is widely expressed, but especially abundant in brain. Curiously, Ubxd1 does not associate with p97 via its UBX domain, but via its PUB domain which binds the extreme C-terminus of p97. Phosphorylation of the penultimate tyrosine residue in p97 completely abolishes Ubxd1 interaction. Ternary complexes of Ubxd1, p47, and p97 were detected in vitro. Inhibition of Ubxd1 expression by siRNA did not affect the degradation of bulk protein or a model substrate of the ERAD pathway, indicating that Ubxd1 directs p97 activity to specialized functions in vivo.

    The international journal of biochemistry & cell biology 2008;40;12;2927-42

  • Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation.

    Ballar P, Zhong Y, Nagahama M, Tagaya M, Shen Y and Fang S

    University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA.

    Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.

    Funded by: NIGMS NIH HHS: R01 GM 69967, R01 GM069967

    The Journal of biological chemistry 2007;282;47;33908-14

  • Novel VCP mutations in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia.

    Watts GD, Thomasova D, Ramdeen SK, Fulchiero EC, Mehta SG, Drachman DA, Weihl CC, Jamrozik Z, Kwiecinski H, Kaminska A and Kimonis VE

    Division of Genetics, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA.

    Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD, OMIM 167320) has recently been attributed to eight missense mutations in valosin-containing protein (VCP). We report novel VCP mutations N387H and L198W in six individuals from two families who presented with proximal muscle weakness at a mean age of diagnosis of 40 years, most losing the ability to walk within a few years of onset. Electromyographic studies in four individuals were suggestive of 'myopathic' changes, and neuropathic pattern was identified in one individual in family 1. Muscle biopsy in four individuals showed myopathic changes characterized by variable fiber size, two individuals showing rimmed vacuoles and IBM-type cytoplasmic inclusions in muscle fibers, and electron microscopy in one individual revealing abundant intranuclear inclusions. Frontotemporal dementia associated with characteristic behavioral changes including short-term memory loss, language difficulty, and antisocial behavior was observed in three individuals at a mean age of 47 years. Detailed brain pathology in one individual showed cortical degenerative changes, most severe in the temporal lobe and hippocampus. Abundant ubiquitin-positive tau-, alpha-synuclein-, polyglutamine repeat-negative neuronal intranuclear inclusions and only rare intracytoplasmic VCP positive inclusions were seen. These new mutations may cause structural changes in VCP and provide some insight into the functional effects of pathogenic mutations.

    Funded by: NIA NIH HHS: K08 AG026271; NIAMS NIH HHS: R01AR050236, R03 AR 46869

    Clinical genetics 2007;72;5;420-6

  • Nuclear localization of valosin-containing protein in normal muscle and muscle affected by inclusion-body myositis.

    Greenberg SA, Watts GD, Kimonis VE, Amato AA and Pinkus JL

    Department of Neurology, Division of Neuromuscular Disease, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115, USA. sagreenberg@partners.org

    Inclusion-body myopathy with Paget's disease and frontotemporal dementia (IBMPFD) is a disease of muscle, bone, and brain that results from mutations in the gene encoding valosin-containing protein (VCP). The mechanism of disease resulting from VCP mutations is unknown. Previous studies of VCP localization in normal human muscle samples have found a capillary and perinuclear distribution, but not a nuclear localization. Here we demonstrate that VCP is present in both myonuclei and endothelial cell nuclei in normal human muscle tissue. The immunodetection of VCP varies with acetone or paraformaldehyde fixation. Within the nucleus, VCP associates with the nucleolar protein fibrillarin and Werner syndrome protein (Wrnp) in normal and IBMPFD muscle. In patients with inclusion-body myositis (IBM), normal nuclear localization is present and some rimmed vacuoles are lined with VCP. These findings suggest that impairment in the nuclear function of VCP might contribute to the muscle pathology occurring in IBMPFD.

    Funded by: NIAMS NIH HHS: R01 AR050236

    Muscle & nerve 2007;36;4;447-54

  • Brain imaging and neuropsychology in late-onset dementia due to a novel mutation (R93C) of valosin-containing protein.

    Krause S, Göhringer T, Walter MC, Schoser BG, Reilich P, Linn J, Pöpperl GE, Frölich L, Hentschel F, Lochmüller H and Danek A

    Neurologische Klinik, Ludwig-Maximilians-Universität München, Germany.

    Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD, MIM 167320) is a recently identified autosomal dominant disorder due to mutations in the valosin-containing protein (VCP) that affects muscle, bone and brain. Brain involvement and neuropsychological findings of IBMPFD have not been described in detail. A patient carried a novel heterozygous base pair change, 47832C>T, in the VCP gene that resulted in substitution of an arginine residue by cysteine at position 93 (R93C). He presented first with myopathy while bone involvement remained subclinical. The patient developed behavioral abnormalities in his 60s and showed frank personality change with fluent empty speech at the age of 74 years. This syndrome was best classified as semantic dementia. Magnetic resonance imaging disclosed slight but progressive cerebral atrophy with prominent callosal and frontal white matter loss. Positron emission tomography demonstrated glucose hypometabolism of the frontal and temporal lobes disproportionate to their structural involvement. This first comprehensive clinical and neuroimaging study in IBMPFD may raise the awareness among clinicians as well as basic scientists for this exemplary genetic model of dementia.

    Clinical neuropathology 2007;26;5;232-40

  • Ufd1 is a cofactor of gp78 and plays a key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase.

    Cao J, Wang J, Qi W, Miao HH, Wang J, Ge L, DeBose-Boyd RA, Tang JJ, Li BL and Song BL

    State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.

    The membrane-anchored ubiquitin ligase gp78 promotes degradation of misfolded endoplasmic reticulum (ER) proteins and sterol-regulated degradation of HMG-CoA reductase. It was known previously that Ufd1 plays a critical role in ER-associated degradation (ERAD) together with Npl4 and VCP. The VCP-Ufd1-Npl4 complex recognizes polyubiquitin chains and transfers the ubiquitinated proteins to the proteasome. Here we show that Ufd1 directly interacts with gp78 and functions as a cofactor. Ufd1 enhances the E3 activity of gp78, accelerates the ubiquitination and degradation of reductase, and eventually promotes receptor-mediated uptake of low-density lipoprotein. Furthermore, we demonstrate that the monoubiquitin-binding site in Ufd1 is required for the enhancement of gp78 activity and that the polyubiquitin-binding site in Ufd1 is critical for a postubiquitination step in ERAD. In summary, our study identifies Ufd1 as a cofactor of gp78, reveals an unappreciated function of Ufd1 in the ubiquitination reaction during ERAD, and illustrates that Ufd1 plays a critical role in cholesterol metabolism.

    Cell metabolism 2007;6;2;115-28

  • Valosin-containing protein and the pathogenesis of frontotemporal dementia associated with inclusion body myopathy.

    Guinto JB, Ritson GP, Taylor JP and Forman MS

    Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 422 Curie Blvd., 605B Stellar-Chance Building, Philadelphia, PA, 19104-6140, USA.

    Frontotemporal dementia with inclusion body myopathy and Paget's disease of bone (IBMPFD) is a rare, autosomal dominant disorder caused by mutations in the gene valosin-containing protein (VCP). The CNS pathology is characterized by a novel pattern of ubiquitin pathology distinct from sporadic and familial frontotemporal lobar degeneration with ubiquitin-positive inclusions without VCP mutations. Yet, the ubiquitin-positive inclusions in IBMPFD also stain for TAR DNA binding protein, a feature that links this rare disease with the pathology associated with the majority of sporadic FTD as well as disease resulting from different genetic alterations. VCP, a member of the AAA-ATPase gene family, associates with a plethora of protein adaptors to perform a variety of cellular processes including Golgi assembly/disassembly and regulation of the ubiquitin-proteasome system. However, the mechanism whereby mutations in VCP lead to CNS, muscle, and bone disease is largely unknown. In this report, we review current literature on IBMPFD, focusing on the pathology of the disease and the biology of VCP with respect to IBMPFD.

    Funded by: NIA NIH HHS: AG000255, AG10124; NINDS NIH HHS: NS053825

    Acta neuropathologica 2007;114;1;55-61

  • A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum.

    Lass A, McConnell E, Nowis D, Mechref Y, Kang P, Novotny MV and Wójcik C

    Department of Anatomy and Cell Biology, Indiana University School of Medicine-Evansville, Evansville, IN 47712, USA.

    alpha-Chain of T-cell receptor (TCR) is a typical ERAD (ER-associated degradation) substrate degraded in the absence of other TCR subunits. Depletion of derlin 1 fails to induce accumulation of alphaTCR despite inducing accumulation of alpha1-antitrypsin, another ERAD substrate. Furthermore, while depletion of VCP does not affect levels of alpha1-antitrypsin, it induces an increase in levels of alphaTCR. RNAi of VCP induces preferential accumulation of alphaTCR with less mannose residues, suggesting its retention within the ER. Mass spectrometric analysis of cellular N-linked glycans revealed that depletion of VCP decreases the level of high-mannose glycoproteins, increases the levels of truncated low-mannose glycoproteins and induces changes in the abundance of complex glycans assembled in post-ER compartments. Since proteasome inhibition was unable to mimic those changes, they cannot be regarded as a simple consequence of inhibited ERAD but represent a complex effect of VCP on the function of the ER.

    Funded by: NCRR NIH HHS: P41 RR018942, P41 RR018942-01A29001, RR018942

    Archives of biochemistry and biophysics 2007;462;1;62-73

  • VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator.

    Goldstein RF, Niraj A, Sanderson TP, Wilson LS, Rab A, Kim H, Bebok Z and Collawn JF

    Department of Cell Biology, Comprehensive Cancer Center, and Gregory Fleming James Cystic Fibrosis Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.

    Funded by: NHLBI NIH HHS: HL076587; NIAID NIH HHS: T32 AI007051, T32AI07051, T32AI07493; NIDDK NIH HHS: DK60065

    American journal of respiratory cell and molecular biology 2007;36;6;706-14

  • ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.

    Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, Shiloh Y, Gygi SP and Elledge SJ

    Department of Genetics and Center for Genetics and Genomics, Brigham and Women's Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.

    Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

    Funded by: PHS HHS: 1U19A1067751

    Science (New York, N.Y.) 2007;316;5828;1160-6

  • E74-like factor 2 regulates valosin-containing protein expression.

    Zhang B, Tomita Y, Qiu Y, He J, Morii E, Noguchi S and Aozasa K

    Department of Pathology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.

    Enhanced expression of valosin-containing protein (VCP) correlates with invasion and metastasis of cancers. To clarify the transcription mechanism of VCP, human and mouse genomic sequence was compared, revealing a 260 bp DNA sequence in the 5'-flanking region of VCP gene to be highly conserved between the two, in which binding motif of E74-like factor 2/new Ets-related factor (ELF2/NERF) was identified. Chromatin immunoprecipitation assay showed binding of ELF2/NERF to the 5'-flanking region of VCP gene. Knock-down of ELF2/NERF by siRNA decreased expression level of VCP. Viability of cells under tumor necrosis factor-alpha treatment significantly reduced in ELF2/NERF-knock-down breast cancer cell line. Immunohistochemical analysis on clinical breast cancer specimens showed a correlation of nuclear ELF2/NERF expression with VCP expression and proliferative activity of cells shown by Ki-67 immunohistochemistry. These findings indicate that ELF2/NERF promotes VCP transcription and that ELF2/NERF-VCP pathway might be important for cell survival and proliferation under cytokine stress.

    Biochemical and biophysical research communications 2007;356;3;536-41

  • Proteomics analysis of the interactome of N-myc downstream regulated gene 1 and its interactions with the androgen response program in prostate cancer cells.

    Tu LC, Yan X, Hood L and Lin B

    Institute for Systems Biology, Seattle, Washington 98103, USA.

    NDRG1 is known to play important roles in both androgen-induced cell differentiation and inhibition of prostate cancer metastasis. However, the proteins associated with NDRG1 function are not fully enumerated. Using coimmunoprecipitation and mass spectrometry analysis, we identified 58 proteins that interact with NDRG1 in prostate cancer cells. These proteins include nuclear proteins, adhesion molecules, endoplasmic reticulum (ER) chaperons, proteasome subunits, and signaling proteins. Integration of our data with protein-protein interaction data from the Human Proteome Reference Database allowed us to build a comprehensive interactome map of NDRG1. This interactome map consists of several modules such as a nuclear module and a cell membrane module; these modules explain the reported versatile functions of NDRG1. We also determined that serine 330 and threonine 366 of NDRG1 were phosphorylated and demonstrated that the phosphorylation of NDRG1 was prominently mediated by protein kinase A (PKA). Further, we showed that NDRG1 directly binds to beta-catenin and E-cadherin. However, the phosphorylation of NDRG1 did not interrupt the binding of NDRG1 to E-cadherin and beta-catenin. Finally, we showed that the inhibition of NDRG1 expression by RNA interference decreased the ER inducible chaperon GRP94 expression, directly proving that NDRG1 is involved in the ER stress response. Intriguingly, we observed that many members of the NDRG1 interactome are androgen-regulated and that the NDRG1 interactome links to the androgen response network through common interactions with beta-catenin and heat shock protein 90. Therefore we overlaid the transcriptomic expression changes in the NDRG1 interactome in response to androgen treatment and built a dual dynamic picture of the NDRG1 interactome in response to androgen. This interactome map provides the first road map for understanding the functions of NDRG1 in cells and its roles in human diseases, such as prostate cancer, which can progress from androgen-dependent curable stages to androgen-independent incurable stages.

    Funded by: NCI NIH HHS: 1U54CA119347, 5P01CA085859, 5P50CA097186; NIDA NIH HHS: 1U54DA021519; NIGMS NIH HHS: 1P50GM076547, P50 GM076547

    Molecular & cellular proteomics : MCP 2007;6;4;575-88

  • Mutations in p97/VCP induce unfolding activity.

    Rothballer A, Tzvetkov N and Zwickl P

    Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.

    A comparison of the protein sequences of various two-domain AAA+ ATPases revealed a striking difference in the residues lining the central pore of the D1 domain. The protein unfoldases of the bacterial Clp family and the archaeal VAT protein have at least one aromatic residue in the central D1 pore. In contrast, none of the members of the eukaryotic p97/VCP protein family has an aromatic residue in the D1 pore. The protein unfolding activity of VAT and other AAA+ ATPases is critically dependent on the presence of aromatic residues in this central pore. Unfoldase activity has not been demonstrated for the p97/VCP family in vitro. Thus, we exchanged the two aliphatic residues leucine and alanine of the D1 pore for aromatic tyrosine residues in full length p97 and in p97DeltaN, a truncated form of p97 lacking the N domain. We found that the mutant p97DeltaN variants with a single tyrosine or with two tyrosine residues in the central pore of D1 unfold the Clp family and VAT model substrate YFP-ssrA, whereas full length p97 with aromatic pore residues and wild-type p97 or p97DeltaN do not. Thus, p97 can exert unfoldase activity in vitro, provided that a single tyrosine residue is introduced into the D1 pore and that the N domain is deleted.

    FEBS letters 2007;581;6;1197-201

  • Pre-B-cell leukemia transcription factor 1 regulates expression of valosin-containing protein, a gene involved in cancer growth.

    Qiu Y, Tomita Y, Zhang B, Nakamichi I, Morii E and Aozasa K

    Department of Pathology, Medical School of Tongji University, Shanghai, China.

    Valosin-containing protein (VCP) is involved in a wide variety of cellular functions. Our previous studies showed that the enhanced expression of VCP in cancer cells correlated with invasion and metastasis of cancers. Here, the regulatory mechanism for VCP transcription was investigated. Luciferase reporter constructs containing serially deleted 5'-flanking region of the VCP gene were transfected into MCF7 mammary carcinoma cell line, in which VCP was abundantly expressed. The deletion and mutation at the two binding motifs for pre-B-cell leukemia transcription factor 1 (PBX1) reduced the luciferase activity, indicating that these two PBX1 motifs mediated the transactivation of the VCP gene. Chromatin immunoprecipitation assay showed the binding of PBX-1 to the 5'-flanking region of the VCP gene. The knockdown of PBX1 by siRNA decreased the expression level of VCP. VCP is reported to maintain cell viability after the treatment of tumor necrosis factor-alpha. The viability of tumor necrosis factor-alpha-treated cells was significantly reduced in PBX1 knockdown MCF7. These findings indicate that PBX1 plays a crucial role in VCP expression and function and that the PBX-VCP pathway might be important for cell survival under cytokine stress.

    The American journal of pathology 2007;170;1;152-9

  • Identification of a novel valosin-containing protein polymorphism in late-onset Alzheimer's disease.

    Kaleem M, Zhao A, Hamshere M and Myers AJ

    Laboratory of Neurogenetics, National Institutes of Health, Bethesda, MD 20892, USA.

    Recently, mutations in the valosin-containing protein gene (VCP) were found to be causative for a rare form of dementia [Watts GDJ, et al.: Nat Genet 2004;36:377-381]. This gene lies within a region on the genome that has been linked to late onset Alzheimer's disease (LOAD) [Myers A, et al.: Am J Med Genet 2002;114:233-242]. In this study, we investigated whether variation within VCP could account for the LOAD linkage peak on chromosome 9.

    Methods: We sequenced 188 individuals from the set of sibling pairs we had used to obtain the linkage results for chromosome 9 to look for novel polymorphisms that could explain the linkage signal. Any variant that was found was then typed in 2 additional sets of neuropathologically confirmed samples to look for associations with Alzheimer's disease.

    Results: We found 2 variants when we sequenced VCP. One was a novel rare variant (R92H) and the other is already reported within the publicly available databases (rs10972300). Neither explained the chromosome 9 linkage signal for LOAD.

    Conclusions: We have found a novel rare variant within the VCP gene, but we did not find a variant that could explain the linkage signal for LOAD on chromosome 9.

    Funded by: Intramural NIH HHS; NIA NIH HHS: AG 05128, AG 05146, P50 AG 08671, P50 AG 16570; NIMH NIH HHS: MH 60451, U01 MH 46281, U01 MH 46290, U01 MH 46373; NINDS NIH HHS: NS 39764

    Neuro-degenerative diseases 2007;4;5;376-81

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis.

    Uchiyama K, Totsukawa G, Puhka M, Kaneko Y, Jokitalo E, Dreveny I, Beuron F, Zhang X, Freemont P and Kondo H

    Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511, Japan.

    We previously reported that p97/p47-assisted membrane fusion is important for the reassembly of organelles at the end of mitosis, but not for their maintenance during interphase. We have now identified a p97 adaptor protein, p37, which forms a complex with p97 in the cytosol and localizes to the Golgi and ER. siRNA experiments revealed that p37 is required for Golgi and ER biogenesis. Injection of anti-p37 antibodies into cells at different cell cycle stages showed that p37 plays an important role in both Golgi and ER maintenance during interphase as well as in their reassembly at the end of mitosis. In an in vitro Golgi reassembly assay, the p97/p37 complex has membrane fusion activity. In contrast to the p97/p47 pathway, this pathway requires p115-GM130 tethering and SNARE GS15, but not syntaxin5. Interestingly, although VCIP135 is also required, its deubiquitinating activity is unnecessary for p97/p37-mediated activities.

    Funded by: Wellcome Trust

    Developmental cell 2006;11;6;803-16

  • Valosin-containing protein (p97) is a regulator of endoplasmic reticulum stress and of the degradation of N-end rule and ubiquitin-fusion degradation pathway substrates in mammalian cells.

    Wójcik C, Rowicka M, Kudlicki A, Nowis D, McConnell E, Kujawa M and DeMartino GN

    Department of Anatomy and Cell Biology, Indiana University School of Medicine, Evansville, IN 47712, USA.

    Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)-proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas approximately 30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-(G76V)-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of alpha chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, alpha1-antitrypsin and deltaCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.

    Funded by: NIDDK NIH HHS: DK-46181, R01 DK046181, R56 DK046181

    Molecular biology of the cell 2006;17;11;4606-18

  • The PUB domain functions as a p97 binding module in human peptide N-glycanase.

    Allen MD, Buchberger A and Bycroft M

    Centre for Protein Engineering, Medical Research Council, Hills Road, Cambridge CB2 2QH, United Kingdom.

    The AAA ATPase p97 is a ubiquitin-selective molecular machine involved in multiple cellular processes, including protein degradation through the ubiquitin-proteasome system and homotypic membrane fusion. Specific p97 functions are mediated by a variety of cofactors, among them peptide N-glycanase, an enzyme that removes glycans from misfolded glycoproteins. Here we report the three-dimensional structure of the aminoterminal PUB domain of human peptide N-glycanase. We demonstrate that the PUB domain is a novel p97 binding module interacting with the D1 and/or D2 ATPase domains of p97 and identify an evolutionary conserved surface patch required for p97 binding. Furthermore, we show that the PUB and UBX domains do not bind to p97 in a mutually exclusive manner. Our results suggest that PUB domain-containing proteins constitute a widespread family of diverse p97 cofactors.

    The Journal of biological chemistry 2006;281;35;25502-8

  • Endoplasmic reticulum vacuolization and valosin-containing protein relocalization result from simultaneous hsp90 inhibition by geldanamycin and proteasome inhibition by velcade.

    Mimnaugh EG, Xu W, Vos M, Yuan X and Neckers L

    Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, 9000 Rockville Pike, Building 10, Room 1-5940, Bethesda, MD 20892-1107, USA.

    Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER--associated protein degradation, a sequential process requiring valosin-containing protein (VCP)-dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from geldanamycin and Velcade exposure overwhelms the ability of the VCP--centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the "small VCP--interacting protein" exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP--enhanced yellow fluorescent protein fluorescent construct, geldanamycin plus Velcade treatment redistributed VCP--enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination.

    Funded by: Intramural NIH HHS

    Molecular cancer research : MCR 2006;4;9;667-81

  • Selective inhibition of endoplasmic reticulum-associated degradation rescues DeltaF508-cystic fibrosis transmembrane regulator and suppresses interleukin-8 levels: therapeutic implications.

    Vij N, Fang S and Zeitlin PL

    Division of Pediatric Respiratory Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is the major quality control pathway of the cell. The most common disease-causing protein folding mutation, DeltaF508-cystic fibrosis transmembrane regulator (CFTR), is destroyed by ERAD to cause cystic fibrosis (CF). p97/valosin-containing protein (VCP) physically interacts with gp78/autocrine motility factor receptor to couple ubiquitination, retrotranslocation, and proteasome degradation of misfolded proteins. We show here that p97/VCP and gp78 form complexes with CFTR during translocation from the ER for degradation by the cytosolic proteasome. Interference in the VCP-CFTR complex promoted accumulation of immature CFTR in the ER and partial rescue of functional chloride channels to the cell surface. Moreover, under these conditions, interleukin-8 (IL8), the expression of which is regulated by the proteasome, was reduced. Inhibition of the proteasome with bortezomib (PS-341/Velcade) also rescued CFTR, but with less efficiency, and suppressed NFkappaB-mediated IL8 activation. The inhibition of the major stress-inducible transcription factor CHOP (CCAAT/enhancer-binding protein homologous protein)/GADD153 together with bortezomib was most effective in repressing NFkappaB-mediated IL8 activation compared with interference of VCP, MLN-273 (proteasome inhibitor), or 4-phenylbutyrate (histone deacetylase inhibitor). Immunoprecipitation of DeltaF508-CFTR from primary CF bronchial epithelial cells confirmed the interaction with VCP and associated chaperones in CF. We conclude that VCP is an integral component of ERAD and cellular stress pathways induced by the unfolded protein response and may be central to the efficacy of CF drugs that target the ubiquitin-proteasome pathway.

    Funded by: NHLBI NIH HHS: R01 HL59410; NIGMS NIH HHS: R01 GM069967

    The Journal of biological chemistry 2006;281;25;17369-78

  • Novel ubiquitin neuropathology in frontotemporal dementia with valosin-containing protein gene mutations.

    Forman MS, Mackenzie IR, Cairns NJ, Swanson E, Boyer PJ, Drachman DA, Jhaveri BS, Karlawish JH, Pestronk A, Smith TW, Tu PH, Watts GD, Markesbery WR, Smith CD and Kimonis VE

    Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA. formanm@mail.med.upenn.edu

    Frontotemporal dementia (FTD) with inclusion body myopathy and Paget disease of bone (IBMPFD) is a rare, autosomal-dominant disorder caused by mutations in the valosin-containing protein (VCP) gene, a member of the AAA-ATPase gene superfamily. The neuropathology associated with sporadic FTD is heterogeneous and includes tauopathies and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). However, there is limited information on the neuropathology in IBMPFD. We performed a detailed, systematic analysis of the neuropathologic changes in 8 patients with VCP mutations. A novel pattern of ubiquitin pathology was identified in IBMPFD that was distinct from sporadic and familial FTLD-U without VCP gene mutations. This was characterized by ubiquitin-positive neuronal intranuclear inclusions and dystrophic neurites. In contrast to FTLD-U, only rare intracytoplasmic inclusions were identified. The ubiquitin pathology was abundant in the neocortex, less robust in limbic and subcortical nuclei, and absent in the dentate gyrus. Only rare inclusions were detected with antibodies to VCP and there was no biochemical alteration in the VCP protein. VCP is associated with a variety of cellular activities, including regulation of the ubiquitin-proteasome system. Our findings are consistent with the hypothesis that the pathology associated with VCP gene mutations is the result of impairment of ubiquitin-based degradation pathways.

    Funded by: NIA NIH HHS: K08 AG20073, P30 AG10124, P30 AG21300, P50 AG05144, P50 AG05681; NIAMS NIH HHS: R01 AR050236, R03 AR46869

    Journal of neuropathology and experimental neurology 2006;65;6;571-81

  • The valosin-containing protein (VCP) is a target of Akt signaling required for cell survival.

    Vandermoere F, El Yazidi-Belkoura I, Slomianny C, Demont Y, Bidaux G, Adriaenssens E, Lemoine J and Hondermarck H

    ERI-8 INSERM, Growth factor signaling in breast cancer. Functional proteomics, University of Sciences and Technologies Lille, 59655 Villeneuve d'Ascq, France.

    The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappaB and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

    The Journal of biological chemistry 2006;281;20;14307-13

  • An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.

    Boeddrich A, Gaumer S, Haacke A, Tzvetkov N, Albrecht M, Evert BO, Müller EC, Lurz R, Breuer P, Schugardt N, Plassmann S, Xu K, Warrick JM, Suopanki J, Wüllner U, Frank R, Hartl UF, Bonini NM and Wanker EE

    Department of Neuroproteomics, Max Delbrueck Center for Molecular Medicine (MDC), Berlin, Germany.

    Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

    The EMBO journal 2006;25;7;1547-58

  • Identification of VCP/p97, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners using membrane-based human proteome arrays.

    Grelle G, Kostka S, Otto A, Kersten B, Genser KF, Müller EC, Wälter S, Böddrich A, Stelzl U, Hänig C, Volkmer-Engert R, Landgraf C, Alberti S, Höhfeld J, Strödicke M and Wanker EE

    Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13125 Berlin-Buch, Germany.

    Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

    Molecular & cellular proteomics : MCP 2006;5;2;234-44

  • Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation.

    Oda Y, Okada T, Yoshida H, Kaufman RJ, Nagata K and Mori K

    Department of Molecular and Cellular Biology Institute for Frontier Medical Sciences, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

    Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. Components of both productive folding and ER-associated degradation (ERAD) mechanisms are known to be up-regulated by the unfolded protein response (UPR). We describe two novel components of mammalian ERAD, Derlin-2 and -3, which show weak homology to Der1p, a transmembrane protein involved in yeast ERAD. Both Derlin-2 and -3 are up-regulated by the UPR, and at least Derlin-2 is a target of the IRE1 branch of the response, which is known to up-regulate ER degradation enhancing alpha-mannosidase-like protein (EDEM) and EDEM2, receptor-like molecules for misfolded glycoprotein. Overexpression of Derlin-2 or -3 accelerated degradation of misfolded glycoprotein, whereas their knockdown blocked degradation. Derlin-2 and -3 are associated with EDEM and p97, a cytosolic ATPase responsible for extraction of ERAD substrates. These findings indicate that Derlin-2 and -3 provide the missing link between EDEM and p97 in the process of degrading misfolded glycoproteins.

    The Journal of cell biology 2006;172;3;383-93

  • Dominant-negative effect of mutant valosin-containing protein in aggresome formation.

    Kitami MI, Kitami T, Nagahama M, Tagaya M, Hori S, Kakizuka A, Mizuno Y and Hattori N

    Department of Neurology, Juntendo University School of Medicine, Bunkyo, Tokyo 113-0033, Japan.

    Lewy bodies (LBs) are the pathologic hallmark of Parkinson's disease. Recent studies revealed that LBs exhibit several morphologic and molecular similarities to aggresomes. Aggresomes are perinuclear aggregates representing intracellular deposits of misfolded proteins. Recently, valosin-containing protein (VCP) was one of the components of LBs, suggesting its involvement in LB formation. Here, we showed the localization of VCP in aggresomes induced by a proteasome inhibitor in cultured cells. Cells overexpressing mutant VCP (K524M: D2) showed reduced aggresome formation relative to those overexpressing wild-type and mutant (K251M: D1) VCPs. Our findings suggest that the D2 domain is involved in aggresome formation.

    FEBS letters 2006;580;2;474-8

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway.

    Schulze A, Standera S, Buerger E, Kikkert M, van Voorden S, Wiertz E, Koning F, Kloetzel PM and Seeger M

    Charité, Universitaetsmedizin Berlin, Campus Charité Mitte, Institut für Biochemie Monbijoustr. 2, 10117 Berlin, Germany.

    To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.

    Journal of molecular biology 2005;354;5;1021-7

  • ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif.

    Noguchi M, Takata T, Kimura Y, Manno A, Murakami K, Koike M, Ohizumi H, Hori S and Kakizuka A

    Laboratory of Functional Biology, Kyoto University Graduate School of Biostudies and Solution Oriented Research for Science and Technology (JST), Kyoto 606-8501, Japan.

    Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.

    The Journal of biological chemistry 2005;280;50;41332-41

  • Proteomic analysis of SUMO4 substrates in HEK293 cells under serum starvation-induced stress.

    Guo D, Han J, Adam BL, Colburn NH, Wang MH, Dong Z, Eizirik DL, She JX and Wang CY

    Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, GA 30912, USA.

    The substrates of SUMO4, a novel member for the SUMO gene family, were characterized in HEK293 cells cultured under serum starvation by proteomic analysis. We identified 90 SUMO4 substrates including anti-stress proteins such as antioxidant enzymes and molecular chaperones or co-chaperones. The substrates also include proteins involved in the regulation of DNA repair and synthesis, RNA processing, protein degradation, and glucose metabolism. Several SUMO4-associated transcription factors were characterized by Western blot analyses. AP-1 was selected for in vitro conjugation assays to confirm SUMO4 sumoylation of these transcription factors. Further functional analyses of the transcription factors suggested that SUMO4 sumoylation represses AP-1 and AP-2alpha transcriptional activity, but enhances GR DNA binding capacity. These results demonstrate that SUMO4 sumoylation may play an important role in the regulation of intracellular stress.

    Biochemical and biophysical research communications 2005;337;4;1308-18

  • Inclusion body myopathy and Paget disease is linked to a novel mutation in the VCP gene.

    Haubenberger D, Bittner RE, Rauch-Shorny S, Zimprich F, Mannhalter C, Wagner L, Mineva I, Vass K, Auff E and Zimprich A

    Department of Neurology, Medical University of Vienna, Vienna, Austria.

    Mutations in the valosin-containing protein (VCP) on chromosome 9p13-p12 were recently found to be associated with hereditary inclusion body myopathy, Paget disease of the bone, and frontotemporal dementia (IBMPFD). We identified a novel missense mutation in the VCP gene (R159H; 688G>A) segregating with this disease in an Austrian family of four affected siblings, who exhibited progressive proximal myopathy and Paget disease of the bone but without clinical signs of dementia.

    Neurology 2005;65;8;1304-5

  • Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane.

    Lilley BN and Ploegh HL

    Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

    Polypeptides that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated from the ER membrane to the cytosol where they are degraded by the proteasome. Derlin-1, a member of a family of proteins that bears homology to yeast Der1p, was identified as a factor that is required for the human cytomegalovirus US11-mediated dislocation of class I MHC heavy chains from the ER membrane to the cytosol. Derlin-1 acts in concert with the AAA ATPase p97 to remove dislocation substrate proteins from the ER membrane, but it is unknown whether other factors aid Derlin-1 in its function. Mammalian genomes encode two additional, related proteins (Derlin-2 and Derlin-3). The similarity of the mammalian Derlin-2 and Derlin-3 proteins to yeast Der1p suggested that these as-yet-uncharacterized Derlins also may play a role in ER protein degradation. We demonstrate here that Derlin-2 is an ER-resident protein that, similar to Derlin-1, participates in the degradation of proteins from the ER. Furthermore, we show that Derlin-2 forms a robust multiprotein complex with the p97 AAA ATPase as well as the mammalian orthologs of the yeast Hrd1p/Hrd3p ubiquitin-ligase complex. The data presented here define a set of interactions between proteins involved in dislocation of misfolded polypeptides from the ER.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;40;14296-301

  • Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane.

    Ye Y, Shibata Y, Kikkert M, van Voorden S, Wiertz E and Rapoport TA

    Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

    Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by retrotranslocation into the cytosol, a pathway hijacked by certain viruses to destroy MHC class I heavy chains. The translocation of polypeptides across the ER membrane requires their polyubiquitination and subsequent extraction from the membrane by the p97 ATPase [also called valosin-containing protein (VCP) or, in yeast, Cdc48]. In higher eukaryotes, p97 is bound to the ER membrane by a membrane protein complex containing Derlin-1 and VCP-interacting membrane protein (VIMP). How the ubiquitination machinery is recruited to the p97/Derlin/VIMP complex is unclear. Here, we report that p97 interacts directly with several ubiquitin ligases and facilitates their recruitment to Derlin-1. During retrotranslocation, a substrate first interacts with Derlin-1 before p97 and other factors join the complex. These data, together with the fact that Derlin-1 is a multispanning membrane protein forming homo-oligomers, support the idea that Derlin-1 is part of a retrotranslocation channel that is associated with both the polyubiquitination and p97-ATPase machineries.

    Funded by: NIGMS NIH HHS: R01 GM05586-10

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;40;14132-8

  • Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis.

    Bruneel A, Labas V, Mailloux A, Sharma S, Royer N, Vinh J, Pernet P, Vaubourdolle M and Baudin B

    Service de Biochimie A, Hôpital Saint-Antoine, AP-HP, Paris, France. arnaud.bruneel@sat.ap-hop-paris.fr

    We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

    Proteomics 2005;5;15;3876-84

  • Valosin-containing protein phosphorylation at Ser784 in response to DNA damage.

    Livingstone M, Ruan H, Weiner J, Clauser KR, Strack P, Jin S, Williams A, Greulich H, Gardner J, Venere M, Mochan TA, DiTullio RA, Moravcevic K, Gorgoulis VG, Burkhardt A and Halazonetis TD

    Cell Signaling Technology, Inc., Beverly, Massachusetts, USA.

    The response of eukaryotic cells to DNA damage includes the activation of phosphatidylinositol-3 kinase-related kinases (PIKK), such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK). These three kinases have very similar substrate specificities in vitro, but in vivo, their substrates overlap only partially. Several in vivo substrates of ATM and ATR have been identified and almost all of them are involved in DNA damage-induced cell cycle arrest and/or apoptosis. In contrast, few in vivo substrates of DNA-PK have been identified. These include histone H2AX and DNA-PK itself. We identify here valosin-containing protein (VCP) as a novel substrate of DNA-PK and other PIKK family members. VCP is phosphorylated at Ser784 within its COOH terminus, a region previously shown to target VCP to specific intracellular compartments. Furthermore, VCP phosphorylated at Ser784 accumulated at sites of DNA double-strand breaks (DSBs). VCP is a protein chaperone that unfolds and translocates proteins. Its phosphorylation in response to DNA damage and its recruitment to sites of DNA DSBs could indicate a role of VCP in DNA repair.

    Funded by: NCI NIH HHS: CA09171, CA09677

    Cancer research 2005;65;17;7533-40

  • Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC).

    Amanchy R, Kalume DE, Iwahori A, Zhong J and Pandey A

    McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry and Oncology, Johns Hopkins University, 733 N. Broadway, Baltimore, MD 21205, USA.

    Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.

    Funded by: NCI NIH HHS: CA 88843; NHLBI NIH HHS: HV 28180

    Journal of proteome research 2005;4;5;1661-71

  • Role of p97 AAA-ATPase in the retrotranslocation of the cholera toxin A1 chain, a non-ubiquitinated substrate.

    Kothe M, Ye Y, Wagner JS, De Luca HE, Kern E, Rapoport TA and Lencer WI

    GI Cell Biology, Children's Hospital Boston, the Harvard Digestive Diseases Center, Harvard Medical School, Massachusetts 02115, USA.

    The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.

    Funded by: NIDDK NIH HHS: DK34854, DK48106

    The Journal of biological chemistry 2005;280;30;28127-32

  • Evidence for chaperone heterocomplexes containing both Hsp90 and VCP.

    Prince T, Shao J, Matts RL and Hartson SD

    Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035, USA.

    With assistance from co-chaperone partner proteins, Hsp90 plays an essential positive role in supporting the structure and function of numerous client proteins in vivo. Hsp90's co-chaperone partnerships are believed to regulate and/or target its function. Here we describe associations between Hsp90 chaperone machinery and another chaperone, the 97-kDa valosin-containing protein VCP. Coimmunoadsorption assays indicate that VCP occurs in one or more native heterocomplexes containing Hsp90 and the Hsp90 partner proteins Cdc37, FKBP52, and p23. Functional characterizations indicate that VCP is not an Hsp90 substrate, but rather demonstrate the biochemical hallmarks of an Hsp90 co-chaperone. Potential roles for a collaboration between for Hsp90 and VCP are discussed.

    Funded by: NIEHS NIH HHS: ES011992; NIGMS NIH HHS: GM51608

    Biochemical and biophysical research communications 2005;331;4;1331-7

  • Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry.

    Ahmed M, Forsberg J and Bergsten P

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. meftun.khandker@drl.ox.ac.uk

    Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.

    Journal of proteome research 2005;4;3;931-40

  • The p95-100 kDa ligand of the T cell-specific adaptor (TSAd) protein Src-homology-2 (SH2) domain implicated in TSAd nuclear import is p97 Valosin-containing protein (VCP).

    Marti F and King PD

    Department of Microbiology and Immunology, University of Michigan Medical School, 6606 Medical Science Building II, Ann Arbor, Michigan 48109-0620, USA.

    T cell-specific adapter protein (TSAd) is required for normal T cell antigen receptor (TCR)-induced transcription of cytokine genes in T cells. How TSAd controls cytokine transcription is unknown. Previously, we have shown that TSAd is actively transported to the nucleus of T cells suggesting that this adapter may in part function within this cellular compartment. Nuclear translocation of TSAd is dependent upon an intact Src-homology-2 (SH2) domain and a p95-100 kDa ligand of the SH2 domain has been implicated in nuclear import. Here, using microchemical techniques, we identify p95-100 as p97 Valosin-containing protein (VCP) whose homolog in yeast is the cell division control protein, CDC48. Physical interaction between TSAd and VCP can be demonstrated between endogenous proteins in T cells. Interaction is direct and is dependent upon phosphorylation of tyrosine residue 805 of VCP that has been previously recognized as a major target of tyrosine kinase(s) involved in TCR signaling. Significantly, with the use of CDC48 mutant yeast, we demonstrate that VCP/CDC48 is required for transport of TSAd into the eukaryotic nucleus. These findings provide important insights into the mechanism of TSAd nuclear import and the role of TSAd in T cell signal transduction.

    Funded by: NIAID NIH HHS: AI050699

    Immunology letters 2005;97;2;235-43

  • Human Fas-associated factor 1, interacting with ubiquitinated proteins and valosin-containing protein, is involved in the ubiquitin-proteasome pathway.

    Song EJ, Yim SH, Kim E, Kim NS and Lee KJ

    Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea.

    Human Fas-associated factor 1 (hFAF1) is a novel protein having multiubiquitin-related domains. We investigated the cellular functions of hFAF1 and found that valosin-containing protein (VCP), the multiubiquitin chain-targeting factor in the degradation of the ubiquitin-proteasome pathway, is a binding partner of hFAF1. hFAF1 is associated with the ubiquitinated proteins via the newly identified N-terminal UBA domain and with VCP via the C-terminal UBX domain. The overexpression of hFAF1 and a truncated UBA domain inhibited the degradation of ubiquitinated proteins and increased cell death. These results suggest that hFAF1 binding to ubiquitinated protein and VCP is involved in the ubiquitin-proteasome pathway. We hypothesize that hFAF1 may serve as a scaffolding protein that regulates protein degradation in the ubiquitin-proteasome pathway.

    Molecular and cellular biology 2005;25;6;2511-24

  • Mutant valosin-containing protein causes a novel type of frontotemporal dementia.

    Schröder R, Watts GD, Mehta SG, Evert BO, Broich P, Fliessbach K, Pauls K, Hans VH, Kimonis V and Thal DR

    Department of Neurology, University Hospital Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. rolf.schroeder@ukb.uni-bonn.de

    Mutations in the valosin-containing protein (VCP) gene on chromosome 9p13-p12 recently have been shown to cause autosomal dominant inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia. Here, we report the central nervous system autopsy findings in a 55-year-old German patient with inclusion body myopathy and frontotemporal dementia who harbors a heterozygous R155C missense mutation residing in the N-terminal CDC48 domain of VCP, which is involved in ubiquitin binding. We demonstrate that mutant VCP causes a novel type of frontotemporal dementia characterized by neuronal nuclear inclusions containing ubiquitin and VCP.

    Annals of neurology 2005;57;3;457-61

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • Substrate-trapping techniques in the identification of cellular PTP targets.

    Blanchetot C, Chagnon M, Dubé N, Hallé M and Tremblay ML

    McGill Cancer Centre, McGill University, 3655 Sir William-Osler, Room 715, Montreal, Quebec, H3G1Y6 Canada. christophe.blanchetot@mcgill.ca

    Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP "substrate-trapping" mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.

    Methods (San Diego, Calif.) 2005;35;1;44-53

  • Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders.

    Ishigaki S, Hishikawa N, Niwa J, Iemura S, Natsume T, Hori S, Kakizuka A, Tanaka K and Sobue G

    Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya 466-8500, Japan.

    Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.

    The Journal of biological chemistry 2004;279;49;51376-85

  • A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs.

    McNeill H, Knebel A, Arthur JS, Cuenda A and Cohen P

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.

    A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.

    The Biochemical journal 2004;384;Pt 2;391-400

  • AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation.

    Zhong X, Shen Y, Ballar P, Apostolou A, Agami R and Fang S

    Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.

    Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.

    Funded by: NIGMS NIH HHS: R01 GM69967-01A1

    The Journal of biological chemistry 2004;279;44;45676-84

  • A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol.

    Ye Y, Shibata Y, Yun C, Ron D and Rapoport TA

    Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

    Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor.

    Nature 2004;429;6994;841-7

  • Interaction of U-box-type ubiquitin-protein ligases (E3s) with molecular chaperones.

    Hatakeyama S, Matsumoto M, Yada M and Nakayama KI

    Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.

    Members of the U-box family of proteins constitute a class of ubiquitin-protein ligases (E3s) distinct from the HECT-type and RING finger-containing E3 families. Two representative mammalian U-box proteins, UFD2a and CHIP, interact with the molecular chaperones VCP and either Hsp90 or Hsc70, respectively, and are implicated in the degradation of damaged proteins. We have now investigated the roles of mammalian U-box proteins by performing a comprehensive screen for molecules that interact with these proteins in the yeast two-hybrid system. All mammalian U-box proteins tested were found to interact with molecular chaperones or cochaperones, including Hsp90, Hsp70, DnaJc7, EKN1, CRN, and VCP. These observations suggest that the function of U box-type E3s is to mediate the degradation of unfolded or misfolded proteins in conjunction with molecular chaperones as receptors that recognize such abnormal proteins.

    Genes to cells : devoted to molecular & cellular mechanisms 2004;9;6;533-48

  • Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein.

    Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, Pestronk A, Whyte MP and Kimonis VE

    Division of Genetics, Children's Hospital Boston, 300 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) is a dominant progressive disorder that maps to chromosome 9p21.1-p12. We investigated 13 families with IBMPFD linked to chromosome 9 using a candidate-gene approach. We found six missense mutations in the gene encoding valosin-containing protein (VCP, a member of the AAA-ATPase superfamily) exclusively in all 61 affected individuals. Haplotype analysis indicated that descent from two founders in two separate North American kindreds accounted for IBMPFD in approximately 50% of affected families. VCP is associated with a variety of cellular activities, including cell cycle control, membrane fusion and the ubiquitin-proteasome degradation pathway. Identification of VCP as causing IBMPFD has important implications for other inclusion-body diseases, including myopathies, dementias and Paget disease of bone (PDB), as it may define a new common pathological ubiquitin-based pathway.

    Nature genetics 2004;36;4;377-81

  • Werner syndrome protein directly binds to the AAA ATPase p97/VCP in an ATP-dependent fashion.

    Indig FE, Partridge JJ, von Kobbe C, Aladjem MI, Latterich M and Bohr VA

    Laboratory of Molecular Gerontology, NIA, NIH, Baltimore MD 21224, USA. indigfr@grc.nia.nih.gov

    We have previously shown that the Werner syndrome helicase, WRNp, a member of the RecQ helicase family, forms a tight molecular complex with the p97/Valosin containing protein (VCP), a member of the AAA (ATPases associated with diverse cellular activities) family of proteins. This interaction is disrupted by chemical agents that confer DNA damage, suggesting that VCP plays an important role in the signal-dependent release of WRNp from its nucleolar sequestration site. Here, we characterized the structural requirements for interactions between WRNp and VCP and for the nuclear localization of VCP. We discovered that VCP directly binds to the RQC (RecQ conserved) domain of WRNp, which is a highly conserved motif common to the RecQ helicase family. This interaction is ATP-dependent, suggesting that VCP plays a mechanistic role in releasing WRNp from the nucleolus. Immunohistochemical analysis of various VCP domains and mutated proteins expressed in vitro demonstrated that VCP may contain several hierarchical cellular localization motifs within its domain structure.

    Journal of structural biology 2004;146;1-2;251-9

  • Expression of valosin-containing protein in colorectal carcinomas as a predictor for disease recurrence and prognosis.

    Yamamoto S, Tomita Y, Hoshida Y, Sakon M, Kameyama M, Imaoka S, Sekimoto M, Nakamori S, Monden M and Aozasa K

    Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.

    Purpose: Valosin-containing protein (VCP or p97) is associated with antiapoptotic function and metastasis via activation of the nuclear factor-kappaB signaling pathway. The present study was designed to investigate the prognostic significance of VCP expression in colorectal adenocarcinoma.

    We analyzed VCP expression immunohistochemically in 129 patients with colorectal carcinoma ages 35-84 years. The staining intensity of tumor cells was categorized as either weaker-to-equal (low VCP expression) or stronger (high expression) than that in noncancerous colonic mucosa. We also analyzed 8 colorectal adenomas and 10 metastatic foci.

    Results: Low VCP expression was noted in 41 (31.8%) cases and high expression in 88 (68.2%) cases. A low level of VCP expression was noted in all adenomas, whereas a high level was seen in all metastatic tumors. A significant difference was observed in depth of invasion (T(1-2) versus T(3-4), P < 0.05), presence or absence of venous invasion (P < 0.05), and tumor stage (I and II versus III and IV; P < 0.05) between adenocarcinomas with low and high VCP expression. Patients with high VCP-expressing tumors had a higher recurrence rate (P < 0.001) and poorer disease-free and overall survival (P < 0.01 and P < 0.05, respectively) compared with the low expression group. Multivariate analysis revealed VCP expression level to be an independent prognosticator for both disease-free and overall survival. VCP level was an indicator of disease-free survival in both stage II and III (pathological Tumor-Node-Metastasis classification, P < 0.05 and <0.01, respectively).

    Conclusions: A high expression level of VCP in tumors is a poor prognostic marker in patients with colorectal carcinomas.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;2;651-7

  • Valosin-containing protein (p97) and Ki-67 expression is a useful marker in detecting malignant behavior of pancreatic endocrine neoplasms.

    Yamamoto S, Tomita Y, Nakamori S, Hoshida Y, Iizuka N, Okami J, Nagano H, Dono K, Umeshita K, Sakon M, Ishikawa O, Ohigashi H, Aozasa K and Monden M

    Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine, Yamadaoka, Suita City, Japan.

    Objective: To determine the prognostic value of valosin-containing protein (VCP) expression and the Ki-67 labeling index (LI) in pancreatic endocrine neoplasms (PENs), the present analysis was employed.

    Methods: The Ki-67 LI and VCP expression at the mRNA and protein level were evaluated in 32 patients (12 male and 20 female) with PENs aged from 22 to 73 years (median 49 years). VCP staining intensity in tumor cells was categorized as weaker (level 1) or equal to stronger (level 2) compared to nontumorous islet cells. Ki-67 LI was divided into two categories: level 1, Ki-67 LI < 5%, and level 2, > or = 5%.

    Results: Five cases (15.6%) showed level 1 and 25 (84.4%) level 2 VCP expression by immunohistochemistry. A significant association was observed between VCP expression and the malignant behavior of PENs (p < 0.01). All level 1 VCP tumors were benign PENs. Quantitative reverse transcription polymerase chain reaction analysis showed higher VCP mRNA expression in malignant PENs (n = 5) than benign cases (n = 5) (p < 0.05). For Ki-67 LI, 28 cases (87.5%) showed level 1 and 4 (12.5%) level 2 expression. All patients with level 2 Ki-67 LI had metastasis.

    Conclusion: VCP expression analysis and Ki-67 LI are useful prognosticators for PENs.

    Oncology 2004;66;6;468-75

  • Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis.

    Doss-Pepe EW, Stenroos ES, Johnson WG and Madura K

    Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

    Machado-Joseph disease is caused by an expansion of a trinucleotide CAG repeat in the gene encoding the protein ataxin-3. We investigated if ataxin-3 was a proteasome-associated factor that recognized ubiquitinated substrates based on the rationale that (i) it is present with proteasome subunits and ubiquitin in cellular inclusions, (ii) it interacts with human Rad23, a protein that may translocate proteolytic substrates to the proteasome, and (iii) it shares regions of sequence similarity with the proteasome subunit S5a, which can recognize multiubiquitinated proteins. We report that ataxin-3 interacts with ubiquitinated proteins, can bind the proteasome, and, when the gene harbors an expanded repeat length, can interfere with the degradation of a well-characterized test substrate. Additionally, ataxin-3 associates with the ubiquitin- and proteasome-binding factors Rad23 and valosin-containing protein (VCP/p97), findings that support the hypothesis that ataxin-3 is a proteasome-associated factor that mediates the degradation of ubiquitinated proteins.

    Funded by: NCI NIH HHS: CA 83875, R01 CA083875; NIA NIH HHS: AG 01047, K01 AG001047

    Molecular and cellular biology 2003;23;18;6469-83

  • D1 ring is stable and nucleotide-independent, whereas D2 ring undergoes major conformational changes during the ATPase cycle of p97-VCP.

    Wang Q, Song C, Yang X and Li CC

    Basic Research Laboratory, Science Applications International Corporation Frederick, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.

    The 97-kDa valosin-containing protein (p97-VCP) belongs to the AAA (ATPases associated with various cellular activities) family and acts as a molecular chaperone in diverse cellular events, including ubiquitinproteasome-mediated degradation. We previously showed that VCP contains a substrate-binding domain, N, and two conserved ATPase domains, D1 and D2, of which D2 is responsible for the major enzyme activity. VCP has a barrel-like structure containing two stacked homo-hexameric rings made of the D1 and D2 domains, and this structure is essential for its biological functions. During ATPase cycles, VCP undergoes conformational changes that presumably apply tensions to the bound substrate, leading to the disassembly of protein complexes or unfolding of the substrate. How ATPase activity is coupled with the conformational changes in VCP complex and the D1 and D2 rings is not clear. In this report, we took biochemical approaches to study the structure of VCP in different nucleotide conditions to depict the conformational changes in the ATPase cycles. In contrast to many AAA chaperones that require ATP/ADP to form oligomers, both wild type VCP and ATP-binding site mutants can form hexamers without the addition of nucleotide. This nucleotide-independent hexamerization requires an intact D1 and the down-stream linker sequence of VCP. Tryptophan fluorescence and trypsin digestion analyses showed that ATP/ADP binding induces dramatic conformational changes in VCP. These changes do not require the presence of an intact ATP-binding site in D1 and is thus mainly attributed to the D2 domain. We propose a model whereby D1, although undergoing minor conformational changes, remains as a relatively trypsin-resistant hexameric ring throughout the ATPase cycle, whereas D2 only does so when it binds to ATP or ADP. After ADP is released at the end of the ATP hydrolysis, D2 ring is destabilized and adopts a relatively flexible and open structure.

    Funded by: NCI NIH HHS: N01-CO-56000

    The Journal of biological chemistry 2003;278;35;32784-93

  • The localization and phosphorylation of p47 are important for Golgi disassembly-assembly during the cell cycle.

    Uchiyama K, Jokitalo E, Lindman M, Jackman M, Kano F, Murata M, Zhang X and Kondo H

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, UK. hk228@cam.ac.uk

    In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.

    The Journal of cell biology 2003;161;6;1067-79

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties.

    Chevalier MS and Johnson DC

    Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97239, USA.

    Human cytomegalovirus (HCMV) glycoprotein US2 increases the proteasome-mediated degradation of major histocompatibility complex (MHC) class I heavy chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. US2-initiated degradation of MHC proteins apparently involves the recruitment of cellular proteins that participate in a process known as endoplasmic reticulum (ER)-associated degradation. ER-associated degradation is a normal process by which misfolded proteins are recognized and translocated into the cytoplasm for degradation by proteasomes. It has been demonstrated that truncated forms of US2, especially those lacking the cytoplasmic domain (CT), can bind MHC proteins but do not cause their degradation. To further assess how the US2 CT domain interacts with the cellular components of the ER-associated degradation pathway, we constructed chimeric proteins in which the US2 CT domain or the CT and transmembrane (TM) domains replaced those of the HCMV glycoprotein US3. US3 also binds both class I and II proteins but does not cause their degradation. Remarkably, chimeras containing the US2 CT domain caused the degradation of both MHC class I and II proteins although this degradation was less than that by wild-type US2. Therefore, the US2 CT and TM domains can confer on US3 the capacity to degrade MHC proteins. We also analyzed complexes containing MHC proteins and US2, US3, US11, or US3/US2 chimeras for the presence of cdc48/p97 ATPase, a protein that binds polyubiquitinated proteins and likely functions in the extraction of substrates from the ER membrane before the substrates meet proteasomes. p97 ATPase was present in immunoprecipitates containing US2, US11, and two chimeras that included the US2 CT domain, but not in US3 complexes. Therefore, it appears that the CT domain of US2 participates in recruiting p97 ATPase into ER-associated degradation complexes.

    Funded by: NCI NIH HHS: CA73996, R01 CA073996; NEI NIH HHS: EY11245, R01 EY011245

    Journal of virology 2003;77;8;4731-8

  • Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation.

    Ficarro S, Chertihin O, Westbrook VA, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr JC, Hunt DF and Visconti PE

    Department of Chmeistry, University of Virginia, Charlottesville, Virginia 22908, USA.

    Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

    Funded by: NICHD NIH HHS: HD 38082; NIGMS NIH HHS: GM 37537; PHS HHS: U54 29099

    The Journal of biological chemistry 2003;278;13;11579-89

  • Elevated expression of valosin-containing protein (p97) in hepatocellular carcinoma is correlated with increased incidence of tumor recurrence.

    Yamamoto S, Tomita Y, Nakamori S, Hoshida Y, Nagano H, Dono K, Umeshita K, Sakon M, Monden M and Aozasa K

    Departments of Surgery and Clinical Oncology, and Pathology, Osaka University Graduate School of Medicine, Osaka, Japan.

    Purpose: Valosin-containing protein (VCP; also known as p97) has been shown to be associated with antiapoptotic function and metastasis via activation of the nuclear factor-kappaB signaling pathway. In this study, association of VCP expression with recurrence of hepatocellular carcinoma (HCC) and patient survival was examined.

    VCP expression in 170 patients (139 male and 31 female) with ages ranging from 31 to 81 years (median, 61 years) was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells.

    Results: Immunohistochemically, 57 patients (35.2%) showed level 1, and 105 patients (64.8%) showed level 2, VCP expression. Quantitative RT-PCR analysis revealed higher VCP mRNA expression in level 2 patients (n = 7) than level 1 (n = 4) (P <.05). Patients with VCP-level 2 HCC showed higher rate of portal vein invasion in the tumor (P <.01) and poorer disease-free and overall survival (P <.0001 and P <.05, respectively) compared with level 1 patients. Multivariate analysis revealed VCP expression level, tumor multiplicity, and degree of fibrosis in the noncancerous liver tissue to be independent prognosticators for disease-free and overall survival. VCP level was an indicator for disease-free survival in each early- (I and II) and advanced- (III and IV) stage group of pathologic tumor-node-metastasis classification (P <.001 and P <.01, respectively).

    Conclusion: VCP expression level has prognostic significance for disease-free and overall survival of patients with HCC.

    Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2003;21;3;447-52

  • Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a.

    Kaneko C, Hatakeyama S, Matsumoto M, Yada M, Nakayama K and Nakayama KI

    Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka, 812-8582, Japan.

    UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (approximately 5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5(') flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway.

    Biochemical and biophysical research communications 2003;300;2;297-304

  • SVIP is a novel VCP/p97-interacting protein whose expression causes cell vacuolation.

    Nagahama M, Suzuki M, Hamada Y, Hatsuzawa K, Tani K, Yamamoto A and Tagaya M

    School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan.

    VCP/p97 is involved in a variety of cellular processes, including membrane fusion and ubiquitin-dependent protein degradation. It has been suggested that adaptor proteins such as p47 and Ufd1p confer functional versatility to VCP/p97. To identify novel adaptors, we searched for proteins that interact specifically with VCP/p97 by using the yeast two-hybrid system, and discovered a novel VCP/p97-interacting protein named small VCP/p97-interacting protein (SVIP). Rat SVIP is a 76-amino acid protein that contains two putative coiled-coil regions, and potential myristoylation and palmitoylation sites at the N terminus. Binding experiments revealed that the N-terminal coiled-coil region of SVIP, and the N-terminal and subsequent ATP-binding regions (ND1 domain) of VCP/p97, interact with each other. SVIP and previously identified adaptors p47 and ufd1p interact with VCP/p97 in a mutually exclusive manner. Overexpression of full-length SVIP or a truncated mutant did not markedly affect the structure of the Golgi apparatus, but caused extensive cell vacuolation reminiscent of that seen upon the expression of VCP/p97 mutants or polyglutamine proteins in neuronal cells. The vacuoles seemed to be derived from endoplasmic reticulum membranes. These results together suggest that SVIP is a novel VCP/p97 adaptor whose function is related to the integrity of the endoplasmic reticulum.

    Molecular biology of the cell 2003;14;1;262-73

  • VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo.

    Uchiyama K, Jokitalo E, Kano F, Murata M, Zhang X, Canas B, Newman R, Rabouille C, Pappin D, Freemont P and Kondo H

    Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, United Kingdom.

    NSF and p97 are ATPases required for the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of organelles. NSF uses ATP hydrolysis to dissociate NSF/SNAPs/SNAREs complexes, separating the v- and t-SNAREs, which are then primed for subsequent rounds of fusion. In contrast, p97 does not dissociate the p97/p47/SNARE complex even in the presence of ATP. Now we have identified a novel essential factor for p97/p47-mediated membrane fusion, named VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135), and show that it binds to the p97/p47/syntaxin5 complex and dissociates it via p97 catalyzed ATP hydrolysis. In living cells, VCIP135 and p47 are shown to function in Golgi and ER assembly.

    Funded by: Wellcome Trust

    The Journal of cell biology 2002;159;5;855-66

  • Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells.

    Kobayashi T, Tanaka K, Inoue K and Kakizuka A

    Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan.

    Abnormal protein accumulation and cell death with cytoplasmic vacuoles are hallmarks of several neurodegenerative disorders. We previously identified p97/valosin-containing protein (VCP), an AAA ATPase with two conserved ATPase domains (D1 and D2), as an interacting partner of the Machado-Joseph disease (MJD) protein with expanded polyglutamines that causes Machado-Joseph disease. To reveal its pathophysiological roles in neuronal cells, we focused on its ATPase activity. We constructed and characterized PC12 cells expressing wild-type p97/VCP and p97(K524A), a D2 domain mutant. The expression level, localization, and complex formation of both proteins were indistinguishable, but the ATPase activity of p97(K524A) was much lower than that of the wild type. p97(K524A) induced cytoplasmic vacuoles that stained with an endoplasmic reticulum (ER) marker, and accumulation of polyubiquitinated proteins in the nuclear and membrane but not cytoplasmic fractions was observed, together with the elevation of ER stress markers. These results show that p97/VCP is essential for degrading membrane-associated ubiquitinated proteins and that profound deficits in its ATPase activity severely affect ER quality control, leading to abnormal ER expansion and cell death. Excessive accumulation of misfolded proteins may inactivate p97/VCP in several neurodegenerative disorders, eventually leading to the neurodegenerations.

    The Journal of biological chemistry 2002;277;49;47358-65

  • Yeast two-hybrid screening identifies binding partners of human Tom34 that have ATPase activity and form a complex with Tom34 in the cytosol.

    Yang CS and Weiner H

    Biochemistry Department, Purdue University, West Lafayette, Indiana 47907, USA.

    In the accompany paper (Mukhopadhyay, A., Avramova, L. V. and Weiner, H., Arch. Biochem. Biophys.), it was shown that Tom34, a previously proposed putative translocase of the mitochondrial outer membrane, binds to the mature region of a precursor protein and appears to be a cytosol protein. Here Tom34 was used as bait in a yeast two-hybrid screening to search for its potential binding partners. Two of the identified proteins were the ATPase-related valosin-containing protein (VCP) and the lysosomal H(+)-transporting ATPase member M (ATP6M). Tom34 was found primarily in the cytosol while VCP and ATP6M were found in the cytosol as well as in nonmitochondrial organelles. Tom34 formed a approximately 400-kDa complex with them in the cytosol. Tom34 was found to possess a weak ATPase activity that did not change when associated with VCP. The tetratricopeptide repeat (TPR) motif region of Tom34 (residue 201-256) was responsible for binding to the other proteins. Tom34 appears not to be a member of the mitochondrial outer membrane translocase family but might function as a chaperone-like protein during protein translocation.

    Funded by: NIAAA NIH HHS: AA10795; NIGMS NIH HHS: GM53269

    Archives of biochemistry and biophysics 2002;400;1;105-10

  • VCP (p97) regulates NFkappaB signaling pathway, which is important for metastasis of osteosarcoma cell line.

    Asai T, Tomita Y, Nakatsuka S, Hoshida Y, Myoui A, Yoshikawa H and Aozasa K

    Department of Pathology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. yt@molpath.med.osaka-u.ac.jp

    In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin-containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor-kappaBalpha (IkappaBalpha), an inhibitor of nuclear factor-kappaB (NFkappaB), whether VCP influences NFkappaB activation or not was examined by using VCP-transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor-alpha (TNFalpha), Dunn/VCPs showed constantly activated NFkappaB, although in the original Dunn cells and control vector transfectant (Dunn/Dunn-c) NFkappaB activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated IkappaBalpha (p-IkappaBalpha) in the cytoplasm of confluent Dunn/Dunn-c cells compared to that of Dunn/VCPs. Therefore, decrease of p-IkappaBalpha degrading activity might be responsible for the decrease in NFkappaB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn-c cells after TNFalpha stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP-1 inoculated male C3H mice compared to those in Dunn/Dunn-c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti-apoptotic potential in these cells owing to constant NFkappaB activation via efficient cytoplasmic p-IkappaBalpha degrading activity may explain the increased metastatic potential of these cells.

    Japanese journal of cancer research : Gann 2002;93;3;296-304

  • Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

    Seigneurin-Berny D, Verdel A, Curtet S, Lemercier C, Garin J, Rousseaux S and Khochbin S

    Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U309, Equipe Chromatine et Expression des Gènes, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, 38706 La Tronche Cedex, France.

    The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

    Molecular and cellular biology 2001;21;23;8035-44

  • Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation.

    Dai RM and Li CC

    Intramural Research Support Program, SAIC Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.

    The ubiquitin-proteasome (Ub-Pr) degradation pathway regulates many cellular activities, but how ubiquitinated substrates are targeted to the proteasome is not understood. We have shown previously that valosin-containing protein (VCP) physically and functionally targets the ubiquitinated nuclear factor kappaB inhibitor, IkappaBalpha to the proteasome for degradation. VCP is an abundant and a highly conserved member of the AAA (ATPases associated with a variety of cellular activities) family. Besides acting as a chaperone in membrane fusions, VCP has been shown to have a role in a number of seemingly unrelated cellular activities. Here we report that loss of VCP function results in an inhibition of Ub-Pr-mediated degradation and an accumulation of ubiquitinated proteins. VCP associates with ubiquitinated proteins through the direct binding of its amino-terminal domain to the multi-ubiquitin chains of substrates. Furthermore, its N-terminal domain is required in Ub-Pr-mediated degradation. We conclude that VCP is a multi-ubiquitin chain-targeting factor that is required in the degradation of many Ub-Pr pathway substrates, and provide a common mechanism that underlies many of the functions of VCP.

    Nature cell biology 2001;3;8;740-4

  • Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro.

    Lavoie C, Chevet E, Roy L, Tonks NK, Fazel A, Posner BI, Paiement J and Bergeron JJ

    Department of Anatomy and Cell Biology, McGill University, Montreal, QC, H3A 2B2, Canada.

    The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;25;13637-42

  • Gene expression profiling in the human hypothalamus-pituitary-adrenal axis and full-length cDNA cloning.

    Hu RM, Han ZG, Song HD, Peng YD, Huang QH, Ren SX, Gu YJ, Huang CH, Li YB, Jiang CL, Fu G, Zhang QH, Gu BW, Dai M, Mao YF, Gao GF, Rong R, Ye M, Zhou J, Xu SH, Gu J, Shi JX, Jin WR, Zhang CK, Wu TM, Huang GY, Chen Z, Chen MD and Chen JL

    Rui-Jin Hospital, Shanghai Institute of Endocrinology, Shanghai Second Medical University, China.

    The primary neuroendocrine interface, hypothalamus and pituitary, together with adrenals, constitute the major axis responsible for the maintenance of homeostasis and the response to the perturbations in the environment. The gene expression profiling in the human hypothalamus-pituitary-adrenal axis was catalogued by generating a large amount of expressed sequence tags (ESTs), followed by bioinformatics analysis (http://www.chgc.sh.cn/ database). Totally, 25,973 sequences of good quality were obtained from 31,130 clones (83.4%) from cDNA libraries of the hypothalamus, pituitary, and adrenal glands. After eliminating 5,347 sequences corresponding to repetitive elements and mtDNA, 20,626 ESTs could be assembled into 9, 175 clusters (3,979, 3,074, and 4,116 clusters in hypothalamus, pituitary, and adrenal glands, respectively) when overlapping ESTs were integrated. Of these clusters, 2,777 (30.3%) corresponded to known genes, 4,165 (44.8%) to dbESTs, and 2,233 (24.3%) to novel ESTs. The gene expression profiles reflected well the functional characteristics of the three levels in the hypothalamus-pituitary-adrenal axis, because most of the 20 genes with highest expression showed statistical difference in terms of tissue distribution, including a group of tissue-specific functional markers. Meanwhile, some findings were made with regard to the physiology of the axis, and 200 full-length cDNAs of novel genes were cloned and sequenced. All of these data may contribute to the understanding of the neuroendocrine regulation of human life.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;17;9543-8

  • Clinical and molecular studies in a unique family with autosomal dominant limb-girdle muscular dystrophy and Paget disease of bone.

    Kimonis VE, Kovach MJ, Waggoner B, Leal S, Salam A, Rimer L, Davis K, Khardori R and Gelber D

    Department of Pediatircs, Southern Illinois University-School of Medicine, Springfield 62794-9658, USA.

    Purpose: To characterize the clinical features and perform linkage analysis of candidate loci in a large Illinois family with autosomal dominant limb-girdle muscular dystrophy (LGMD) and Paget disease of bone (PDB).

    Methods: The family includes 11 affected individuals (8 M, 3 F). Clinical, biochemical and radiologic evaluations were performed to delineate clinical features of the disorder. Linkage analysis with polymorphic markers was performed for previously identified LGMD, PDB and cardiomyopathy loci.

    Results: Onset of PDB is early, at a mean age of 35 y, with classic distribution involving the spine, pelvis, and skull. Muscle weakness and atrophy is progressive with mildly elevated to normal creatine phosphokinase levels. Muscle biopsy in the oldest male revealed vacuolated fibers, however, in others revealed nonspecific myopathy. Affected individuals die from progressive muscle weakness, and respiratory and cardiac failure in their 40s-60s. Linkage analysis excluded autosomal dominant and recessive LGMD, PDB, and cardiomyopathy loci.

    Conclusion: Autosomal dominant LGMD associated with PDB is an unusual disorder. Linkage analysis indicates a unique locus in this family.

    Funded by: NHGRI NIH HHS: T15 HG000016

    Genetics in medicine : official journal of the American College of Medical Genetics 2000;2;4;232-41

  • VCP, a weak ATPase involved in multiple cellular events, interacts physically with BRCA1 in the nucleus of living cells.

    Zhang H, Wang Q, Kajino K and Greene MI

    Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104-6082, USA.

    BRCA1, a breast/ovarian cancer susceptibility gene, undergoes mutations in as many as 50% of familial breast tumors. Recent studies indicate that BRCA1 may be involved in DNA damage repair. Here, we demonstrate that the BRCA1 protein physically associates with valosin-containing protein (VCP), a member of the ATPases associated with a variety of cellular activities (AAA) superfamily. In vitro studies revealed that VCP, via its N- terminal region, binds to amino acid residues 303-625 in the BRCA1 protein. Although found predominantly in the cytoplasm and, less abundantly, in the nucleus, VCP can be translocated from the nucleus after stimulation with epidermal growth factor. Collectively, our results suggest that VCP, by binding to BRCA1, participates in the DNA damage-repair function as an ATP transporter, possibly facilitating the transcription-coupled repair.

    Funded by: NCI NIH HHS: CA-16520; NIDDK NIH HHS: DK-19525

    DNA and cell biology 2000;19;5;253-63

  • Identification of the cell cycle regulator VCP (p97/CDC48) as a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1.

    Zhang SH, Liu J, Kobayashi R and Tonks NK

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

    The human band 4.1-related protein-tyrosine phosphatase PTPH1 was introduced into NIH3T3 cells under the control of a tetracycline-repressible promoter. Ectopic expression of wild type PTPH1 dramatically inhibited cell growth, whereas a catalytically impaired mutant showed no effect. To identify the direct target of PTPH1 in the cell, we generated a substrate-trapping mutant, in which an invariant aspartate residue was changed to alanine (D811A in PTPH1). The PTPH1-D811A mutant trapped primarily a 97-kDa tyrosine-phosphorylated protein, which was determined to be VCP (also named p97 or yeast CDC48), from various cell lysates in vitro. However, when expressed in mammalian cells, the D811A mutant was observed to contain high levels of phosphotyrosine and did not trap substrates. Mutation of tyrosine 676 to phenylalanine (Y676F) in the PTPH1-D811A mutant led to a marked reduction in phosphotyrosine content. Furthermore, this double mutant specifically trapped VCP in vivo and recognized the C-terminal tyrosines of VCP, whose phosphorylation is important for cell cycle progression in yeast. Like wild type PTPH1, this double mutant also inhibited cell proliferation. Moreover, induction of wild type PTPH1 resulted in specific dephosphorylation of VCP without changing the overall phosphotyrosine profile of the cells. VCP has been implicated in control of a variety of membrane functions, including membrane fusions, and is a regulator of the cell cycle. Our results suggest that PTPH1 may exert its effects on cell growth through dephosphorylation of VCP, thus implicating tyrosine phosphorylation as an important regulator of VCP function.

    Funded by: NCI NIH HHS: CA53840

    The Journal of biological chemistry 1999;274;25;17806-12

  • Syntaxin 5 is a common component of the NSF- and p97-mediated reassembly pathways of Golgi cisternae from mitotic Golgi fragments in vitro.

    Rabouille C, Kondo H, Newman R, Hui N, Freemont P and Warren G

    Cell Biology Laboratory, Imperial Cancer Research Fund, London, United Kingdom.

    A cell-free system that mimics the reassembly of Golgi stacks at the end of mitosis requires two ATPases, NSF and p97, to rebuild Golgi cisternae. Morphological studies now show that alpha-SNAP, a component of the NSF pathway, can inhibit the p97 pathway, whereas p47, a component of the p97 pathway, can inhibit the NSF pathway. Anti-syntaxin 5 antibodies and a soluble, recombinant syntaxin 5 inhibited both pathways, suggesting that this t-SNARE is a common component. Biochemical studies confirmed this, showing that p47 binds directly to syntaxin 5 and competes for binding with alpha-SNAP. p47 also mediates the binding of p97 to syntaxin 5 and so plays an analogous role to alpha-SNAP, which mediates the binding of NSF.

    Cell 1998;92;5;603-10

  • Involvement of valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha.

    Dai RM, Chen E, Longo DL, Gorbea CM and Li CC

    Intramural Research Support Program, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.

    The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappaB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IkappaBalpha complexes sediment in the 19 S fractions, while the unmodified IkappaBalpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IkappaBalpha are critical for VCP binding, which in turn is necessary but not sufficient for IkappaBalpha degradation; while the N-terminal domain of IkappaBalpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IkappaBalpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IkappaBalpha.

    The Journal of biological chemistry 1998;273;6;3562-73

  • Mapping the valosin-containing protein (VCP) gene on human chromosome 9 and mouse chromosome 4, and a likely pseudogene on the mouse X chromosome.

    Hoyle J, Tan KH and Fisher EM

    Neurogenetics Unit, Imperial College of Medicine at St. Mary's, London, UK.

    Funded by: NCRR NIH HHS: 2P20RR22 583-02

    Mammalian genome : official journal of the International Mammalian Genome Society 1997;8;10;778-80

  • NVL: a new member of the AAA family of ATPases localized to the nucleus.

    Germain-Lee EL, Obie C and Valle D

    Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    We report the cloning of NVL, a newly recognized human gene that encodes an approximately 110-kDa nuclear protein designated NVLp (nuclear VCP-like protein), which is a member of a rapidly growing family of ATP-binding proteins recently denoted the AAA family (ATPases associated with diverse cellular activities) (W. H. Kunau et al., 1993, Biochimie 75:209-224). NVL was isolated by degenerate PCR using oligonucleotides corresponding to the yeast PEX1 gene, which is necessary for peroxisomal biogenesis. Two cDNAs, designated NVL.1 and NVL.2, may represent alternatively spliced forms of a single gene that maps to chromosome 1q41-q42.2. NVL has greatest similarity to the VCP subfamily of AAA proteins, is widely expressed, and encodes a nuclear protein with two highly similar ATP-binding domains. We speculate that NVLp is involved in an ATP-dependent nuclear process.

    Funded by: NICHD NIH HHS: HD00989, HD10981

    Genomics 1997;44;1;22-34

  • Chromosome localization of human genes for clathrin adaptor polypeptides AP2 beta and AP50 and the clathrin-binding protein, VCP.

    Druck T, Gu Y, Prabhala G, Cannizzaro LA, Park SH, Huebner K and Keen JH

    Jefferson Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

    Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed "assembly proteins," or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2 alpha and AP2 beta, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2 beta (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with APs. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34.

    Funded by: NCI NIH HHS: CA51083; NIGMS NIH HHS: GM-28526, GM49217

    Genomics 1995;30;1;94-7

  • An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments.

    Rabouille C, Levine TP, Peters JM and Warren G

    Cell Biology Laboratory, Imperial Cancer Research Fund, London, England.

    Golgi cisternae regrew in a cell-free system from mitotic Golgi fragments incubated with buffer alone. Pretreatment with NEM or salt washing inhibited regrowth, but this could be restored either by p97, an NSF-like ATPase, or by NSF together with SNAPs and p115, a vesicle docking protein. The morphology of cisternae regrown with p97 and NSF-SNAPs-p115 differed, suggesting that they play distinct roles in rebuilding Golgi cisternae after mitosis.

    Cell 1995;82;6;905-14

  • Valosin-containing protein, VCP, is a ubiquitous clathrin-binding protein.

    Pleasure IT, Black MM and Keen JH

    Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

    Clathrin is the structural protein of coated membranes involved in receptor-mediated endocytosis and aspects of Golgi sorting in eukaryotic cells. We have now detected a stoichiometric complex of clathrin with a novel protein of M(r) approximately 100,000 (100K) in lysates of different mammalian cells. Formation of the complex, which also includes the 70K heat-shock protein Hsc70, occurs within 15 min of synthesis. The 100K protein has been identified as valosin-containing protein (VCP; ref. 1), an early substrate for tyrosine phosphorylation on T-cell receptor activation. Further, VCP is the mammalian homologue of yeast Cdc48p (ref. 3) and is a member of a larger gene family that includes putative ATP-binding proteins involved in vesicle transport and fusion, 26S proteasome function, regulation of the expression of human immunodeficiency virus, and assembly of peroxisomes. The association with clathrin and the morphological and catalytic similarity to the chaperonin proteins indicate that VCP may modulate protein-protein interactions in membrane transport processes.

    Nature 1993;365;6445;459-62

  • Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin.

    Dawson SJ and White LA

    Department of Microbiology, Southampton General Hospital, U.K.

    A patient with Haemophilus aphrophilus endocarditis was successfully treated with ciprofloxacin. The response to treatment with cefotaxime and netilmicin for 12 days was poor but was satisfactory to a 6 weeks' course of ciprofloxacin.

    The Journal of infection 1992;24;3;317-20

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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