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

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.     

Mutations in p97/VCP induce unfolding activity

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.     

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

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.     

COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.     

Recent advances in p97/VCP/Cdc48 cellular functions

p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases.     

The determination of the relationship between p97/VCP,small VCP-interacting protein,two ERAD proteins and steroidogenesis in Leydig cell lines

Background

In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.

Methods and results

HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.

Conclusion

We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

     

VAT, the thermoplasma homolog of mammalian p97/VCP, is an N domain-regulated protein unfoldase

The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATDeltaN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.     

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.     

NSF and p97/VCP: similar at first, different at last

N-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein (VCP) are distantly related members of the ATPases associated with a variety of cellular activities (AAA) family of proteins. While both proteins have been implied in cellular morphology changes involving membrane compartments or vesicles, more recent evidence seems to imply that NSF is primarily involved in the soluble NSF attachment receptor (SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereas p97/VCP is primarily involved in the extraction of membrane proteins. These functional differences are now corroborated by major structural differences based on recent crystallographic and cryo-electron microscopy studies. This review discusses these recent findings.     

Critical role of VCP/p97 in the pathogenesis and progression of non-small cell lung carcinoma

Background

Valosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression.

Methodology/Principal Findings

Our results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05).

Conclusions/Significance

Thus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.     

Nucleotide dependent motion and mechanism of action of p97/VCP

The AAA (ATPases associated with a variety of cellular activities) family of proteins bind, hydrolyze, and release ATP to effect conformational changes, assembly, or disassembly upon their binding partners and substrate molecules. One of the members of this family, the hexameric p97/valosin-containing protein p97/VCP, is essential for the dislocation of misfolded membrane proteins from the endoplasmic reticulum. Here, we observe large motions and dynamic changes of p97/VCP as it proceeds through the ATP hydrolysis cycle. The analysis is based on crystal structures of four representative ATP hydrolysis states: APO, AMP-PNP, hydrolysis transition state ADP x AlF3, and ADP bound. Two of the structures presented herein, ADP and AMP-PNP bound, are new structures, and the ADP x AlF3 structure was re-refined to higher resolution. The largest motions occur at two stages during the hydrolysis cycle: after, but not upon, nucleotide binding and then following nucleotide release. The motions occur primarily in the D2 domain, the D1 alpha-helical domain, and the N-terminal domain, relative to the relatively stationary and invariant D1alpha/beta domain. In addition to the motions, we observed a transition from a rigid state to a flexible state upon loss of the gamma-phosphate group, and a further increase in flexibility within the D2 domains upon nucleotide release. The domains within each protomer of the hexameric p97/VCP deviate from strict 6-fold symmetry, with the more flexible ADP state exhibiting greater asymmetry compared to the relatively rigid ADP x AlF3 state, suggesting a mechanism of action in which hydrolysis and conformational changes move about the hexamer in a processive fashion.     

Disruption of p97/VCP induces autophagosome accumulation,cell cycle arrest and apoptosis in human choriocarcinoma cells

Molecular Biology Reports - Gestational choriocarcinoma is aggressive trophoblastic disease. The development, progression and the cure of this disease is not well-established. p97/Valosin...     

Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47

The AAA ATPase p97/VCP is involved in many cellular events including ubiquitin-dependent processes and membrane fusion. In the latter, the p97 adaptor protein p47 is of central importance. In order to provide insight into the molecular basis of p97 adaptor binding, we have determined the crystal structure of p97 ND1 domains complexed with p47 C-terminal domain at 2.9 A resolution. The structure reveals that the p47 ubiquitin regulatory X domain (UBX) domain interacts with the p97 N domain via a loop (S3/S4) that is highly conserved in UBX domains, but is absent in ubiquitin, which inserts into a hydrophobic pocket between the two p97 N subdomains. Deletion of this loop and point mutations in the loop significantly reduce p97 binding. This hydrophobic binding site is distinct from the predicted adaptor-binding site for the p97/VCP homologue N-ethylmaleimide sensitive factor (NSF). Together, our data suggest that UBX domains may act as general p97/VCP/CDC48 binding modules and that adaptor binding for NSF and p97 might involve different binding sites. We also propose a classification for ubiquitin-like domains containing or lacking a longer S3/S4 loop.     

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.     

Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system

The ATP-driven chaperone valosin-containing protein (VCP)/p97 governs critical steps in ubiquitin-dependent protein quality control and intracellular signalling pathways. It cooperates with diverse partner proteins to help process ubiquitin-labelled proteins for recycling or degradation by the proteasome in many cellular contexts. Recent studies have uncovered unexpected cellular functions for p97 in autophagy, endosomal sorting and regulating protein degradation at the outer mitochondrial membrane, and elucidated a role for p97 in key chromatin-associated processes. These findings extend the functional relevance of p97 to lysosomal degradation and reveal a surprising dual role in protecting cells from protein stress and ensuring genome stability during proliferation.     

Sequence and characterization of cytoplasmic nuclear protein import factor p97

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein.     

Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation

A class of inherited neurodegenerative diseases including Huntington's disease is caused by polyglutamine (polyQ) expansion in the responsible proteins. Pathology is typically associated with polyQ expansions of greater than 40 residues, and the longer the length of the expansion, the earlier the onset of disease. It has been reported that p97/VCP/Cdc48p, a member of AAA family of proteins, can bind to longer polyQ tracts. In Caenorhabditis elegans, two p97/VCP/Cdc48p homologues, C41C4.8 and C06A1.1, have been identified. Our results indicate that these p97/VCP/Cdc48p homologues have essential but redundant functions in C. elegans. To provide a model system for investigating the molecular basis of pathogenesis, we have expressed polyQ expansions fused to green fluorescent protein in the body wall muscle cells of C. elegans. When the repeats are longer than 40, discrete cytoplasmic aggregates are formed and these appear at an early stage of embryogenesis. The formation of aggregates was partially suppressed by co-expression of either C41C4.8 or C06A1.1. These results suggest that these p97/VCP/Cdc48p homologues, AAA chaperones, may play a protective role in polyQ aggregation.