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Martin Latterich - One of the best experts on this subject based on the ideXlab platform.

  • identification of caspase 6 mediated processing of the valosin containing protein P97 in alzheimer s disease a novel link to dysfunction in ubiquitin proteasome system mediated protein degradation
    The Journal of Neuroscience, 2010
    Co-Authors: Dalia Halawani, Sylvain Tessier, Dominique Anzellotti, David A. Bennett, Martin Latterich, Andréa C. Leblanc
    Abstract:

    The valosin-containing protein (P97) is a ubiquitin-dependent ATPase that plays central roles in ubiquitin proteasome system (UPS)-mediated protein degradation pathways. P97 has been recently identified as a putative substrate of active Caspase-6 (Casp6) in primary human neurons. Since Casp6 is activated in mild cognitive impairment (MCI) and Alzheimer9s disease (AD) patients9 brains, the targeting of P97 by Casp6 may represent an important step that leads to UPS impairment in AD. Here, we show that P97 is a Casp6 substrate in vitro and in vivo . Casp6 cleavage of recombinant P97 generated two N-terminal fragments of 28 and 20 kDa, which were not generated by the other two effector caspases, Caspase-3 and Caspase-7. ATP binding to the D1 ATPase ring of P97 reduced the susceptibility of the N-domain to caspase-mediated proteolysis. Mass spectrometric analysis identified VAPD 179 as a Casp6 cleavage site within P979s N-domain. An anti-neoepitope serum immunohistochemically detected P97 cleaved at VAPD 179 in the cytoplasm of the cell soma and neurites of hippocampal neurons in MCI and AD. Overexpression of P97 (1-179) fragment, representing P97 cleaved at D179, impaired the degradation of model substrates in the ubiquitin-fusion degradation and the N-end rule pathways, and destabilized endogenous P97. Collectively, these results show that P97 is cleaved by Casp6 in AD and suggest P97 cleavage as an important mechanism for UPS impairment.

  • 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
    Molecular and Cellular Biology, 2009
    Co-Authors: Dalia Halawani, Martin Latterich, Andréa C. Leblanc, Isabelle Rouiller, Stephen W Michnick, Marc J Servant
    Abstract:

    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.

  • 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
    Molecular and Cellular Biology, 2009
    Co-Authors: Dalia Halawani, Martin Latterich, Andréa C. Leblanc, Isabelle Rouiller, Stephen W Michnick, Marc J Servant
    Abstract:

    The ubiquitous valosin-containing protein (P97/VCP) is a prominent member of the highly conserved AAA+ (ATPases associated with diverse cellular activities) proteins, which are known for their oligomeric structure and chaperone-like activities. P97/VCP is an essential biochemical component of a wide range of ubiquitin-linked cell biological reactions, including ubiquitin-proteasome system-mediated protein degradation (8), Golgi and endoplasmic reticulum (ER) membrane fusion (1, 18), transcription factor activation (19), and DNA repair (10, 16). In these processes, P97 acts as a molecular segregase that utilizes ATP-powered conformational changes in the assembly and disassembly of macromolecular machineries (8, 11). P97 ATPase activity is contingent on the assembly of an inherently stable hexamer (24) comprised of two highly homologous D1 and D2 nucleotide-binding rings and regulatory NH2- and COOH-terminal domains (4). ATP hydrolysis in the D2 ring mediates P97 major ATPase activity, while the D1 ring is involved in the regulation of P97 hexamerization (24). Recently, P97 has been linked to a severe degenerative disorder identified as hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD). The pathogenesis of hIBMPFTD is attributed to autosomal-dominant single-amino-acid substitutions in highly conserved residues within the P97 NH2 domain and D1 ring (28). Patients present with hallmark features of a “protein conformational” disorder in which proteinaceous inclusions accumulate in the cytoplasm and nuclei of degenerating myofibrils (13) and dystrophic neurites (7, 15, 23). Transgenic expression of hIBMPFTD P97 mutants in a murine model sufficiently recapitulates these inclusions (31). While the ultrastructure and composition of hIBMPFTD inclusions are poorly defined, several reports have identified P97/VCP and ubiquitin as major constituents (12, 28, 32). Furthermore, overexpression of hIBMPFTD mutants in C2C12 skeletal myoblasts results in P97 localization to cytoplasmic inclusions and fractionation with insoluble proteins (30). One possibility is that misfolding or misassembly directly mediates P97/VCP accumulation in inclusion bodies, which in turn nucleates the entrapment of its interacting proteins. Alternatively, the biogenesis of P97-containing inclusions could represent an indirect consequence of failure in other P97/VCP-dependent pathways upstream of the ubiquitin-proteasome system. The mechanisms by which hIBMPFTD P97 mutants induce the biogenesis of inclusion bodies in cell culture models are not well understood (9, 30). Despite current progress in resolving more refined P97/VCP crystal structures in various nucleotide-binding states (3, 5), clues about how hIBMPFTD mutations impact P97 structure or conformation remain elusive. Cryo-electron microscopy (cryo-EM) (21, 22) and crystallography studies (4, 5) of P97 in various nucleotide-binding states have revealed extensive communication between the tripartite NH2-D1-D2 domains (17). Conformational changes first initiated in the D2 ring are relayed through a D1-D2 linker region to the D1 α-helical subdomain. The D1 α-helical subdomain in turn communicates these motions to the flexibly linked NH2 domain, thereby regulating its conformation (5). Ultimately, these ATP-powered conformational changes are utilized in remodeling macromolecular complexes. One hypothesis is that hIBMPFTD mutations interfere with P97/VCP biochemical activity by altering its conformation or interdomain communication. For example, structural analysis of P97/VCP in the preactivated ATP-occupied state revealed communication between Arg155 and Arg159 within the NH2-terminal domain and the D1 residue Asn387, all of which are now identified as mutated residues in hIBMPFTD (5, 9). However, it remains to be seen whether hIBMPFTD mutations do indeed induce structural or conformational defects in P97/VCP. In this report, we specifically focused on studying hIBMPFTD P97R155P and P97A232E mutants because Arg155 is the most commonly affected amino acid residue and mutation of Ala232 is associated with increased severity of the disease (13, 28). A series of biochemical approaches were employed to characterize the oligomeric assembly, ATPase activity, and solution structure of wild-type (P97WT) and mutant P97. Our results indicate that recombinant P97R155P and P97A232E assembled into ∼600-kDa complexes, similar to P97WT. Both mutants showed a threefold increase in ATPase activity and increased sensitivity to heat-induced upregulation in ATPase activity. Analysis of the intrinsic protein fluorescence and proteolytic susceptibility provided evidence for conformational defects in the D2 ring. These effects were correlated with increased propensity for aggregation in the presence of ATP. Based on these data, we propose that hIBMPFTD P97 mutations induce structural defects, which may underlie the mechanism of pathogenesis in biological systems.

  • P97 adaptor choice regulates organelle biogenesis.
    Developmental cell, 2006
    Co-Authors: Martin Latterich
    Abstract:

    The AAA protein P97 requires adaptor-like cofactors for its numerous cellular functions. In this issue of Developmental Cell , Uchiyama et al. (2006) identify p37 as a P97 adaptor that is required constitutively for ER and Golgi membrane fusion, analogous to the mitotic membrane fusion role of the adaptor p47. Their study suggests that related P97 adaptors involved in similar cellular pathways can be subject to differential regulation.

  • a major conformational change in P97 aaa atpase upon atp binding
    Molecular Cell, 2000
    Co-Authors: Isabelle Rouiller, Martin Latterich, Virginia M Butel, Ronald A Milligan, Elizabeth M Wilsonkubalek
    Abstract:

    AAA ATPases play central roles in cellular activities. The ATPase P97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of P97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped P97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.

Paul S. Freemont - One of the best experts on this subject based on the ideXlab platform.

  • crystal structure of the catalytic d2 domain of the aaa atpase P97 reveals a putative helical split washer type mechanism for substrate unfolding
    FEBS Letters, 2020
    Co-Authors: Lasse Stach, Xiaodong Zhang, Rhodri M L Morgan, Linda Makhlouf, Alice Douangamath, Frank Von Delft, Paul S. Freemont
    Abstract:

    Several pathologies have been associated with the AAA+ ATPase P97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in P97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made P97 an attractive cancer chemotherapy target. The cellular processes reliant on P97 are well described. High-resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which P97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high-resolution structure of the P97 D2 ATPase domain. This crystal system constitutes a valuable tool for P97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P61 symmetry suggests a mechanism for substrate unfolding by P97. DATABASE: The atomic coordinates and structure factors have been deposited in the PDB database under the accession numbers 6G2V, 6G2W, 6G2X, 6G2Y, 6G2Z and 6G30.

  • the aaa atpase P97 a cellular multitool
    Biochemical Journal, 2017
    Co-Authors: Lasse Stach, Paul S. Freemont
    Abstract:

    The AAA+ (ATPases associated with diverse cellular activities) ATPase P97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. P97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of P97 cofactors have evolved which are essential to P97 function. Ubiquitin-interacting domains and P97-binding domains combine to form bi-functional cofactors, whose complexes with P97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in P97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, P97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by P97, how the cofactors interact with both P97 and its ubiquitinated substrates, P97 enzymology and the current status in developing P97 inhibitors for cancer therapy.

  • the n terminal region of the ubiquitin regulatory x ubx domain containing protein 1 ubxd1 modulates interdomain communication within the valosin containing protein P97
    Journal of Biological Chemistry, 2015
    Co-Authors: Franziska Trusch, Anja Matena, Lisa Koerver, Helene Knaevelsrud, Hemmo Meyer, Paul S. Freemont, Peter Bayer
    Abstract:

    Valosin-containing protein/P97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of P97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the P97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/P97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for P97. In reverse titration experiments, we identified two distant epitopes on the P97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of P97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to P97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the P97-ND1 interface, thereby modulating interdomain communication of P97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of P97 cofactors.

  • the P97 faf1 protein complex reveals a common mode of P97 adaptor binding
    Journal of Biological Chemistry, 2014
    Co-Authors: Caroline A Ewens, Patrik Kloppsteck, Xiaodong Zhang, Silvia Panico, Ciaran Mckeown, Imaobong Ebong, Carol V Robinson, Paul S. Freemont
    Abstract:

    P97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. P97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. P97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-κB activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the P97-FAF1 interaction. We show that FAF1 binds P97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of P97-FAF1 yielded a 17 Å reconstruction of the complex with FAF1 above the P97 ring. Characteristics of P97-FAF1 uncovered in this study reveal common features in the interactions of P97, providing mechanistic insight into how P97 mediates diverse functionalities.

  • distinct conformations of the protein complex P97 ufd1 npl4 revealed by electron cryomicroscopy
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Cecilia Bebeacua, Hemmo Meyer, Xiaodong Zhang, Ciaran Mckeown, Andreas Forster, Paul S. Freemont
    Abstract:

    P97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to P97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with P97. Our reconstructions show that P97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the P97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by P97 and also explains how P97-Ufd1-Npl4 could form other complexes in a hierarchical model of P97-cofactor assembly.

Hisao Kondo - One of the best experts on this subject based on the ideXlab platform.

  • P97 and p47 function in membrane tethering in cooperation with ftcd during mitotic golgi reassembly
    The EMBO Journal, 2021
    Co-Authors: Yayoi Kaneko, Xiaodong Zhang, Kyohei Shimoda, Rafael Ayala, Yukina Goto, Silvia Panico, Hisao Kondo
    Abstract:

    P97ATPase-mediated membrane fusion is required for the biogenesis of the Golgi complex. P97 and its cofactor p47 function in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for P97/p47-mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47-binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or P97 via its association with their polyglutamate motifs. FTCD functions in P97/p47-mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to P97. We also showed that FTCD, p47, and P97 form a big FTCD-P97/p47-FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous P97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the P97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD-P97/p47-FTCD complex in P97/p47-mediated Golgi membrane fusion.

  • vcip135 deubiquitinase and its binding protein wac in P97atpase mediated membrane fusion
    The EMBO Journal, 2011
    Co-Authors: Hisao Kondo, Go Totsukawa, Yayoi Kaneko, Keiji Uchiyama, Hiroyuki Toh, Kaori Tamura
    Abstract:

    Two distinct P97 membrane fusion pathways are required for Golgi biogenesis: the P97/p47 and P97/p37 pathways. VCIP135 is necessary for both pathways, while its deubiquitinating activity is required only for the P97/p47 pathway. We have now identified a novel VCIP135-binding protein, WAC. WAC localizes to the Golgi as well as the nucleus. In Golgi membranes, WAC is involved in a complex containing VCIP135 and P97. WAC directly binds to VCIP135 and increases its deubiquitinating activity. siRNA experiments revealed that WAC is required for Golgi biogenesis. In an in vitro Golgi reformation assay, WAC was necessary only for P97/p47-mediated Golgi reassembly, but not for P97/p37-mediated reassembly. WAC is hence thought to function in P97/p47-mediated Golgi membrane fusion by activating the deubiquitinating function of VCIP135. We also showed that the two P97 pathways function in ER membrane fusion as well. An in vitro ER reformation assay revealed that both pathways required VCIP135 but not its deubiquitinating activity for their ER membrane fusion. This was consistent with the finding that WAC is unnecessary for P97-mediated ER membrane fusion.

  • structural basis of the interaction between the aaa atpase P97 vcp and its adaptor protein p47
    The EMBO Journal, 2004
    Co-Authors: Ingrid Dreveny, Hisao Kondo, Keiji Uchiyama, Xiaodong Zhang, Anthony Shaw, Paul S. Freemont
    Abstract:

    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.

  • complete backbone resonance assignments of p47 the 41kda adaptor protein of the aaa atpase P97
    Journal of Biomolecular NMR, 2004
    Co-Authors: Xuemei Yuan, Paul S. Freemont, Hisao Kondo, Xiaodong Zhang, Ingrid Dreveny, Peter J Simpson, Ciaran Mckeown, Stephen Matthews
    Abstract:

    Xuemei Yuana,b, Peter Simpsona,b, Hisao Kondoc, Ciaran Mckeowna,b, Ingrid Drevenya,b, Xiaodong Zhanga,b, Paul S. Freemonta,b & Stephen Matthewsa,b,∗ aDepartment of Biological Sciences, Wolfson Laboratories and bCentre for Structural Biology, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AZ, U.K.; cCambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 2XY, U.K.

  • the localization and phosphorylation of p47 are important for golgi disassembly assembly during the cell cycle
    Journal of Cell Biology, 2003
    Co-Authors: Keiji Uchiyama, Hisao Kondo, Xiaodong Zhang, Eija Jokitalo, Fumi Kano, Masayuki Murata, Mervi Lindman, Mark Jackman
    Abstract:

    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.

Graham Warren - One of the best experts on this subject based on the ideXlab platform.

  • vcip135 acts as a deubiquitinating enzyme during P97 p47 mediated reassembly of mitotic golgi fragments
    Journal of Cell Biology, 2004
    Co-Authors: Yangzhuang Wang, Graham Warren, Ayano Satoh, Hemmo Meyer
    Abstract:

    The AAA-ATPase P97/Cdc48 functions in different cellular pathways using distinct sets of adapters and other cofactors. Together with its adaptor Ufd1–Npl4, it extracts ubiquitylated substrates from the membrane for subsequent delivery to the proteasome during ER-associated degradation. Together with its adaptor p47, on the other hand, it regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis. The finding of a ubiquitin-binding domain in p47 raises the question as to whether the ubiquitin–proteasome system is also involved in membrane fusion events. Here, we show that P97–p47-mediated reassembly of Golgi cisternae requires ubiquitin, but is not dependent on proteasome-mediated proteolysis. Instead, it requires the deubiquitinating activity of one of its cofactors, VCIP135, which reverses a ubiquitylation event that occurs during mitotic disassembly. Together, these data reveal a cycle of ubiquitylation and deubiquitination that regulates Golgi membrane dynamics during mitosis. Furthermore, they represent the first evidence for a proteasome-independent function of P97/Cdc48.

  • direct binding of ubiquitin conjugates by the mammalian P97 adaptor complexes p47 and ufd1 npl4
    The EMBO Journal, 2002
    Co-Authors: Hemmo Meyer, Yanzhuang Wang, Graham Warren
    Abstract:

    The multiple functions of the P97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1–Npl4, both bind ubiquitin conjugates directly and so link P97 to ubiquitylated substrates. In the case of Ufd1–Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1–Npl4, it binds ubiquitin only when complexed with P97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of P97-mediated reactions.

  • Distinct AAA-ATPase P97 complexes function in discrete steps of nuclear assembly
    Nature Cell Biology, 2001
    Co-Authors: Martin Hetzer, Graham Warren, Hemmo H. Meyer, Tobias C. Walther, Daniel Bilbao-cortes, Iain W. Mattaj
    Abstract:

    Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that P97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the P97–Ufd1–Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a P97–p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.

  • a complex of mammalian ufd1 and npl4 links the aaa atpase P97 to ubiquitin and nuclear transport pathways
    The EMBO Journal, 2000
    Co-Authors: Hemmo Meyer, Darryl J C Pappin, James Shorter, Joachim Seemann, Graham Warren
    Abstract:

    The AAA-ATPase, P97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs P97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to P97. Ufd1/Npl4 competes with p47 for binding to P97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by P97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic P97 activity into different cellular pathways.

  • The p47 co-factor regulates the ATPase activity of the membrane fusion protein, P97
    FEBS letters, 1998
    Co-Authors: Hemmo Meyer, Hisao Kondo, Graham Warren
    Abstract:

    The highly conserved ATPase P97, a member of the AAA-ATPases, is found in a complex with its co-factor p47 in rat liver cytosol. Previously it had been shown that P97-mediated reassembly of Golgi cisternae from mitotic Golgi fragments requires p47 which mediates the binding of P97 to a Golgi t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment factor receptor), syntaxin 5. Here we show that it also suppresses the ATPase activity of P97 by up to 85% in a dose-dependent and saturable manner suggesting that it has other roles in the membrane fusion cycle.

Hemmo Meyer - One of the best experts on this subject based on the ideXlab platform.

  • protein phosphatase 1 complex disassembly by P97 is initiated through multivalent recognition of catalytic and regulatory subunits by the P97 sep domain adapters
    Journal of Molecular Biology, 2020
    Co-Authors: Matthias Kracht, Johannes Van Den Boom, Jonas Seiler, Alexander Kroning, Farnusch Kaschani, Markus Kaiser, Hemmo Meyer
    Abstract:

    Abstract The AAA-ATPase VCP/P97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to P97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of P97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of P97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how P97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.

  • structure of the pub domain from ubiquitin regulatory x domain protein 1 ubxd1 and its interaction with the P97 aaa atpase
    Biomolecules, 2019
    Co-Authors: Mike Blueggel, Hemmo Meyer, Peter Bayer, Johannes Van Den Boom, Christine Beuck
    Abstract:

    AAA+ ATPase P97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst P97 cofactors because it interacts with both termini of P97. Its N-domain binds to the N-domain and N/D1 interface of P97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the P97 C-terminus, but how it controls P97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the P97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing.

  • the n terminal region of the ubiquitin regulatory x ubx domain containing protein 1 ubxd1 modulates interdomain communication within the valosin containing protein P97
    Journal of Biological Chemistry, 2015
    Co-Authors: Franziska Trusch, Anja Matena, Lisa Koerver, Helene Knaevelsrud, Hemmo Meyer, Paul S. Freemont, Peter Bayer
    Abstract:

    Valosin-containing protein/P97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of P97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the P97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/P97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for P97. In reverse titration experiments, we identified two distant epitopes on the P97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of P97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to P97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the P97-ND1 interface, thereby modulating interdomain communication of P97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of P97 cofactors.

  • distinct conformations of the protein complex P97 ufd1 npl4 revealed by electron cryomicroscopy
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Cecilia Bebeacua, Hemmo Meyer, Xiaodong Zhang, Ciaran Mckeown, Andreas Forster, Paul S. Freemont
    Abstract:

    P97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to P97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with P97. Our reconstructions show that P97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the P97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by P97 and also explains how P97-Ufd1-Npl4 could form other complexes in a hierarchical model of P97-cofactor assembly.

  • vcip135 acts as a deubiquitinating enzyme during P97 p47 mediated reassembly of mitotic golgi fragments
    Journal of Cell Biology, 2004
    Co-Authors: Yangzhuang Wang, Graham Warren, Ayano Satoh, Hemmo Meyer
    Abstract:

    The AAA-ATPase P97/Cdc48 functions in different cellular pathways using distinct sets of adapters and other cofactors. Together with its adaptor Ufd1–Npl4, it extracts ubiquitylated substrates from the membrane for subsequent delivery to the proteasome during ER-associated degradation. Together with its adaptor p47, on the other hand, it regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis. The finding of a ubiquitin-binding domain in p47 raises the question as to whether the ubiquitin–proteasome system is also involved in membrane fusion events. Here, we show that P97–p47-mediated reassembly of Golgi cisternae requires ubiquitin, but is not dependent on proteasome-mediated proteolysis. Instead, it requires the deubiquitinating activity of one of its cofactors, VCIP135, which reverses a ubiquitylation event that occurs during mitotic disassembly. Together, these data reveal a cycle of ubiquitylation and deubiquitination that regulates Golgi membrane dynamics during mitosis. Furthermore, they represent the first evidence for a proteasome-independent function of P97/Cdc48.