ATPase Activity of p97/Valosin-containing Protein Is Regulated by Oxidative Modification of the Evolutionally Conserved Cysteine 522 Residue in Walker A Motif

Noguchi, Masatoshi; Takata, Takahiro; Kimura, Yoko; Manno, Atsushi; Murakami, Katsuhiro; Koike, Masayuki; Ohizumi, Hiroshi; Hori, Seiji; Kakizuka, Akira

doi:10.1074/jbc.m509700200

Cited by 60 publications

(70 citation statements)

References 67 publications

(93 reference statements)

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“…The D2 ATPase domain of p97 contains a reactive cysteine (Cys522) in the ATP-binding pocket (27). Because most of the top 10 hits shown in Table 1 contained electrophilic moieties, we further evaluated their mechanism of action by assaying their ability to inhibit the ATPase activity of purified C522A-p97.…”

Section: Resultsmentioning

confidence: 99%

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

Chou

Brown²,

Minond³

et al. 2011

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

292

356

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A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N 2 ,N 4 -dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.apoptosis | autophagy | unfolded protein response T he AAA (ATPase associated with diverse cellular activities) ATPase p97 is conserved across all eukaryotes and is essential for life in budding yeast (1) and mice (2). p97 was first linked to the ubiquitin-proteasome system (UPS) through its role in the turnover of ubiquitin−β-galactosidase fusion proteins via the "ubiquitin fusion degradation" (UFD) pathway (3). Since then, p97 has been shown to play a critical role in the degradation of misfolded membrane and secretory proteins (4) and has also been linked to a broad array of cellular processes, including Golgi membrane reassembly (5), membrane transport (6), regulation of myofibril assembly (7), cell division (8), formation of protein aggregates (9), and autophagosome maturation (10, 11). The broad range of cellular functions for p97 is thought to derive from its ability to unfold proteins or disassemble protein complexes, but the detailed mechanism of how p97 works and is linked to specific cellular processes remains largely unknown.The structure of p97 comprises three domains: an N-terminal domain that recruits adaptors/substrate specificity factors, followed by two ATPase domains, D1 and D2 (12, 13). p97 monomers assemble to form a homohexamer that is thought to provide a platform for transduction of chemical activity into mechanical force that is applied to substrate proteins. The D1 domain mediates hexamerization (14) and has very low ATPase activity (15). Most of the ATPase activity is contributed by the D2 domain, which is thought to underlie p97's function as a mechanochemical transducer (16).The mechanochemical activity of p97 is linked to substrate proteins by an array of 13 UBX (ubiquitin regulatory X) domain adapters that bind the N-terminal domain of p97 (17), as well as the non-UBX domain adaptors Ufd1 and Npl4 (18). The functions and mechanisms of action of these different p97-adaptor complexes remain poorly u...

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Section: Resultsmentioning

confidence: 99%

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

Chou

Brown²,

Minond³

et al. 2011

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

292

356

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“…In the presence of oxidants, like diamide and peroxide, the overall ATPase activity of the chaperone is significantly decreased [195]. While C69, C77 and C522 were all shown to be S-glutathionylated in vitro, only the substitution of C522 shielded the ATPase activity of VCP against oxidative stress.…”

Section: S-glutathionylationmentioning

confidence: 99%

Regulation of molecular chaperones through post-translational modifications: Decrypting the chaperone code

Cloutier

Coulombe

2013

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point toward the more ambitious endeavor of deciphering the "chaperone code".

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“…A human ortholog, valosin-containing protein, an ATPase reportedly inhibited under oxidative stress conditions, is induced by degenerative disorders. Inhibition is due to the oxidation of a Cys residue that is conserved only in the enzyme from multicellular organisms (Noguchi et al, 2005). It seems possible, therefore, that the CDC48 is also redox regulated in plants.…”

Section: Degradationmentioning

confidence: 99%

Thioredoxin-Linked Proteins Are Reduced during Germination ofMedicago truncatulaSeeds

et al. 2007

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Germination of cereals is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Thioredoxin (Trx) appears to play a role in this transition in cereals. It is not known, however, whether Trx-linked redox changes are restricted to cereals or whether they take place more broadly in germinating seeds. To gain information on this point, we have investigated a model legume, Medicago truncatula. Two complementary gel-based proteomic approaches were followed to identify Trx targets in seeds: Proteins were (1) labeled with a thiol-specific probe, monobromobimane (mBBr), following in vitro reduction by an NADP/Trx system, or (2) isolated on a mutant Trx affinity column. Altogether, 111 Trx-linked proteins were identified with few differences between axes and cotyledons. Fifty nine were new, 34 found previously in cereal or peanut seeds, and 18 in other plants or photosynthetic organisms. In parallel, the redox state of proteins assessed in germinating seeds using mBBr revealed that a substantial number of proteins that are oxidized or partly reduced in dry seeds became more reduced upon germination. The patterns were similar for proteins reduced in vivo during germination or in vitro by Trx. In contrast, glutathione and glutaredoxin were less effective as reductants in vitro. Overall, more than half of the potential targets identified with the mBBr labeling procedure were reduced during germination. The results provide evidence that Trx functions in the germination of seeds of dicotyledons as well as monocotyledons.

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ATPase Activity of p97/Valosin-containing Protein Is Regulated by Oxidative Modification of the Evolutionally Conserved Cysteine 522 Residue in Walker A Motif

Cited by 60 publications

References 67 publications

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

Regulation of molecular chaperones through post-translational modifications: Decrypting the chaperone code

Thioredoxin-Linked Proteins Are Reduced during Germination ofMedicago truncatulaSeeds

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