Structure and function of the AAA + ATPase p97/Cdc48p

doi:10.1016/j.gene.2016.02.042

Gene

Volume 583, Issue 1, 25 May 2016, Pages 64-77

https://doi.org/10.1016/j.gene.2016.02.042 Get rights and content

Highlights

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p97/Cdc48p participates in various cellular pathways by functioning as a “segregase”.
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Mutations in p97 have been associated with various human diseases.
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Significant progress has been made in obtaining atomic resolution structures of p97 in different conformations.
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Controversy remains as to the mechanism underlying force generation by p97.

Abstract

p97 (also known as valosin-containing protein (VCP) in mammals or Cdc48p in Saccharomyces cerevisiae) is an evolutionarily conserved ATPase present in all eukaryotes and archaebacteria. In conjunction with a collection of cofactors and adaptors, p97/Cdc48p performs an array of biological functions mostly through modulating the stability of ‘client’ proteins. Using energy from ATP hydrolysis, p97/Cdc48p segregates these molecules from immobile cellular structures such as protein assemblies, membrane organelles, and chromatin. Consequently, the released polypeptides can be efficiently degraded by the ubiquitin proteasome system or recycled. This review summarizes our current understanding of the structure and function of this essential cellular chaperoning system.

Section snippets

Structural features of p97

Structural information on p97 was initially obtained with negatively stained samples examined by electron microscopy (EM). The results provided the basic shape of a ring-like hexameric molecule (Peters et al., 1990, Zhang et al., 1994). Higher resolution structures were later obtained by cryo-EM (Rouiller et al., 2000) and by crystallization of a N–D1 fragment (Zhang et al., 2000). These studies confirmed the hexameric assembly of p97, but also showed that unlike many bacterial AAA + proteins,

p97-interacting proteins

A large collection of p97/Cdc48p-interacting proteins has been identified through proteomic studies. These proteins either function as adaptors that link p97/Cdc48p to a specific subcellular compartment or substrate, or serve as cofactors that help to process substrates. Cofactors usually have enzymatic activities that can process protein modifiers such as N-glycan or ubiquitin conjugates that are appended to p97 substrate (e.g. N-glycanase, ubiquitin ligase, and deubiquitinase).

Although a few

Biological functions of p97/Cdc48p

The diverse biological functions of p97 have been extensively reviewed (Dantuma and Hoppe, 2012, Franz et al., 2014, Meyer et al., 2012, Meyer and Weihl, 2014, Yamanaka et al., 2012). Therefore, we only highlight a few key established functions in this review. In general, p97 uses ATP hydrolysis to segregate polypeptides from large protein assemblies or immobile cellular structures such as membranes or chromatin, and therefore, facilitates the degradation of the released polypeptides by the 26S

Molecular basis of force generation

Although intensely studied, the molecular mechanism underlying the “segregase” activity of p97/Cdc48p remains poorly defined. Major unresolved issues are whether or not p97/Cdc48p unfolds its client proteins, and therefore disrupting their interactions with protein assemblies, membranes, or chromatin; if so, whether or not it can act as a ‘translocase’ that moves substrates through the central pore.

p97 inhibitors and cancer therapy

By screening and characterizing compounds that inhibit the degradation of a fluorescence-labeled ERAD substrate, the first p97 inhibitor Eeyarestatin (EerI) was reported a few years ago (Fiebiger et al., 2004, Wang et al., 2008, Wang et al., 2010). Structure–activity relationship studies suggested that EerI contains two functional modules: a nitrofuran ring that binds the D1 domain of p97 and an aromatic ring-containing module that recruits EerI to cellular membranes including the ER. Once

Relevance to human diseases

p97 has received much attention recently also because genetic studies have linked mutations in p97 to pathogenesis of several human diseases including Inclusion Body Myopathy associated with Paget's disease of the bone and Frontotemporal Dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS) (Johnson et al., 2010, Watts et al., 2004).

IBMPFD is an autosomal dominant, progressive, and ultimately fatal disorder with initial symptoms typically appearing in adulthood (Kimonis et al., 2000). The

Conclusions and perspective

Through years of studies, we have significantly deepened our understanding on the structure and function of p97/Cdc48p. Most noticeable is the rapid expansion in our knowledge on cofactors and adaptors and the corresponding new functions of this essential chaperone system. Nonetheless, several fundamental questions regarding the mechanistic action of p97/Cdc48p remain unanswered. The most important one is whether the conformational changes observed in the reported studies are truly

Acknowledgments

This review and the corresponding Gene Wiki article are written as part of the Gene Wiki Review series—a series resulting from a collaboration between the journal GENE and the Gene Wiki Initiative ( https://en.wikipedia.org/wiki/Valosin-containing_protein). The Gene Wiki Initiative is supported by National Institutes of Health (GM089820). The research in the laboratories of D. Xia and Y. Ye is supported by the Intramural Research Program of the National Cancer Institute and of the National

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p97 is a human AAA+ (ATPase associated with diverse cellular activities, also known as valosin-containing protein [VCP]) ATPase, which is involved in diverse cellular processes such as membrane fusion and proteolysis. Lysine-specific methyltransferase of p97 (METTL21D) was identified as a class I methyltransferase that catalyzes the trimethylation of Lys315 of p97, a so-called VCP lysine methyltransferase (VCPKMT). Interestingly, VCPKMT disassembles a single hexamer ring consisting of p97-D1 domain and methylates Lys315 residue. Herein, the structures of S-adenosyl-L-methionine-bound VCPKMT and S-adenosyl-L-homocysteine-bound VCPKMT in complex with p97 N/D1 (N21-Q458) were reported at a resolution of 1.8 Å and 2.8 Å, respectively. The structures revealed the molecular details for the recognition and methylation of monomeric p97 by VCPKMT. Using biochemical analysis, we also investigated whether the methylation of full-length p97 could be sufficiently enhanced through cooperation between VCPKMT and the C terminus of alveolar soft part sarcoma locus (ASPL). Our study provides the groundwork for future structural and mechanistic studies of p97 and inhibitors.
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Metabolomics analysis of an AAA-ATPase Cdc48-deficient yeast strain
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The ubiquitin-specific chaperone AAA-ATPase Cdc48 and its orthologs p97/valosin-containing protein (VCP) in mammals play crucial roles in regulating numerous intracellular pathways via segregase activity, which separates polyubiquitinated targets from membranes or binding partners. Interestingly, high-throughput experiments show that a vast number of metabolic enzymes are modified with ubiquitin. Therefore, Cdc48 may regulate metabolic pathways, for example by acting on the polyubiquitin chains of metabolic enzymes; however, the role of Cdc48 in metabolic regulation remains largely unknown. To begin to analyze the role of Cdc48 in metabolic regulation in yeast, we performed a metabolomics analysis of temperature-sensitive cdc48-3 mutant cells. We found that the amount of metabolites in the glycolytic pathway was altered. Moreover, the pool of nucleotides, as well as the levels of metabolites involved in the tricarboxylic acid cycle and oxidative phosphorylation, increased, whereas the pool of amino acids decreased. These results suggest the involvement of Cdc48 in metabolic regulation in yeast. In addition, because of the roles of p97/VCP in regulating multiple cellular pathways, its inhibition is being considered as a promising anticancer drug target. We propose that the metabolomics study of Cdc48-deficient yeast will be useful as a complement to p97/VCP-related pathological and therapeutic studies.
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Citation Excerpt :
p97 adenosine triphosphatase (ATPase) in metazoans and its Cdc48 orthologs in Saccharomyces cerevisiae segregate polyubiquitinated proteins into proteasomes for degradation (Erzberger and Berger, 2006; Meyer and Weihl, 2014; Xia et al., 2016).
The heterodimer of human ubiquitin fusion degradation 1 (hUfd1) and human nuclear protein localization 4 (hNpl4) is a major cofactor of human p97 adenosine triphosphatase (ATPase). The p97-Ufd1-Npl4 complex translocates the ubiquitin-conjugated proteins from the endoplasmic reticulum membrane to the cytoplasm. Ubiquitinated proteins are then degraded by the proteasome. The structures of Npl4 and Ufd1-Npl4 (UN) complex in Saccharomyces cerevisiae have been recently reported; however, the structures of hNpl4 and the human UN complex remain unknown. Here, we report the crystal structures of the human UN complex at a resolution of 2.7 Å and hNpl4 at a resolution of 3.0 Å. We also present atomic details and characterization of the human UN complex. Crystallographic studies and site-directed mutagenesis of the hUfd1 residues involved in the interaction with hNpl4 revealed the atomic details of the two proteins.
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Replication of positive-strand RNA viruses depends on usurped cellular membranes and co-opted host proteins. Based on pharmacological inhibition and genetic and biochemical approaches, the authors identified critical roles of the cellular Cdc48 unfoldase/segregase protein in facilitating the replication of tomato bushy stunt virus (TBSV). We show that TBSV infection induces the expression of Cdc48 in Nicotiana benthamiana plants. Cdc48 binds to the TBSV replication proteins through its N-terminal region. In vitro TBSV replicase reconstitution experiments demonstrated that Cdc48 is needed for efficient replicase assembly and activity. Surprisingly, the in vitro replication experiments also showed that excess amount of Cdc48 facilitates the disassembly of the membrane-bound viral replicase-RNA template complex. Cdc48 is also needed for the recruitment of additional host proteins. Because several human viruses, including flaviviruses, utilize Cdc48, also called VCP/p97, for replication, we suggest that Cdc48 might be a common panviral host factor for plant and animal RNA viruses.

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Published by Elsevier B.V.

Gene wiki reviewStructure and function of the AAA + ATPase p97/Cdc48p

Highlights

Abstract

Section snippets

Structural features of p97

p97-interacting proteins

Biological functions of p97/Cdc48p

Molecular basis of force generation

p97 inhibitors and cancer therapy

Relevance to human diseases

Conclusions and perspective

Acknowledgments

Cell

J. Biol. Chem.

Cancer Cell

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Cell

J. Biol. Chem.

Cell

FEBS Lett.

J. Struct. Biol.

Cell

J. Mol. Biol.

Trends Cell Biol.

Exp. Cell Res.

Structure

Structure

J. Mol. Biol.

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Structure

Structure

Structure

Free Radic. Biol. Med.

Mol. Cell

J. Struct. Biol.

J. Biol. Chem.

J. Biol. Chem.

Neuron

Genet. Med.

J. Biol. Chem.

Cell

J. Biol. Chem.

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Struct. Biol.

J. Biol. Chem.

The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks

Nat. Struct. Mol. Biol.

2.3 A resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition

Science

Identification of the Cdc48*20S proteasome as an ancient AAA + proteolytic machine

Science

Bipartite determinants mediate an evolutionarily conserved interaction between Cdc48 and the 20S peptidase

Proc. Natl. Acad. Sci. U. S. A.

HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins

Mol. Biol. Cell

Gene wiki review
Structure and function of the AAA + ATPase p97/Cdc48p