A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

Beskow, Anne; Grimberg, Kristian Björk; Bott, Laura C.; Salomons, Florian A.; Dantuma, Nico P.; Young, Patrick

doi:10.1016/j.jmb.2009.09.050

Cited by 108 publications

(125 citation statements)

References 63 publications

(77 reference statements)

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“…However, several proteins have been identified that, like IscS, preferentially bind and stabilize a disordered target protein (20)(21)(22). Many of these are chaperone proteins that rescue misfolded proteins (23)(24)(25) or are proteins involved in protein degradation (26)(27)(28)(29)(30)(31)(32).…”

Section: Discussionmentioning

confidence: 99%

Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase

Kim

Tonelli²,

Markley³

2011

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

171

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The scaffold protein for iron-sulfur cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25°C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn (II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S → D reaction, and to decrease the rate of the D → S reaction. In the saturated IscU:IscS complex, IscU is largely disordered. In vitro cluster assembly reactions provided evidence for the functional importance of the S ⇆ D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed cluster assembly; variants that favor the D state were found to assemble less stable clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the cluster once it is assembled.amino acid sequence effects on protein stability | protein order-disorder transition | two-dimensional exchange spectroscopy | biogenesis of Fe-S clusters I ron-sulfur (Fe-S) clusters, which are among the most ancient and ubiquitous protein prosthetic groups, function in electron transport, enzymatic catalysis, and chemical sensing reactions or as structural units (1). Humans and other higher eukaryotes utilize the ISC (iron-sulfur cluster) system as the essential Fe-S cluster assembly mechanism in mitochondria, and defects in this system have been linked to a large number of human diseases (2). The prokaryotic ISC system has served as a useful model for understanding Fe-S cluster assembly and delivery. The bacterial system utilizes several proteins that have eukaryotic homologs: IscU (scaffold protein), IscS (cysteine desulfurase), HscB (cochaperone), HscA (chaperone), and CyaY (regulation or iron delivery, analog of human frataxin) (1). Interactions among these proteins have been shown to be critical for efficient Fe-S cluster biogenesis (3). The IscU protein acts as a scaffold on which the Fe-S clusters are assembled and from which the clusters are transferred to various apoproteins. IscS is a homodimeric pyridoxyl-5′-phosphatedependent cysteine desulfurase (4). Each IscS subunit binds an IscU molecule and transfers sulfane sulfur generated from the conversion of cysteine to alanine to the cluster ligand cysteines of IscU (5). HscA and HscB, the DnaK-like chaperone and the DnaJ-like cochaperone pro...

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Section: Discussionmentioning

confidence: 99%

Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase

Kim

Tonelli²,

Markley³

2011

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

171

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“…Several mechanistic hypotheses have been proposed to explain the role of VCP in proteasomal degradation of cellular substrates. It has been suggested that VCP may be required for initial unfolding of tightly folded substrates that lack an intrinsically unstructured region as initiation site for the proteasome (20). The AdV capsid may qualify as such a substrate (8,30).…”

Section: Discussionmentioning

confidence: 99%

“…VCP forms a homohexameric barrel-shaped ring of about 16 nm in diameter (17), a structure not dissimilar to the proteasome but without any associated protease activity. The N domain of VCP binds directly to multiubiquitin chains (18) and, although substrate ubiquitination does not seem to be a prerequisite for VCP interaction (19), a general role for VCP in unfolding ubiquitin-fusion degradation (UFD) substrates before proteasomal degradation has been suggested (20). Most recently, it has been shown that VCP is recruited to stalled proteasomes and relieves their (experimentally induced) impairment (21).…”

Section: Intracellular Immunity | Cdc48mentioning

confidence: 99%

“…VCP is involved in the proteasomal degradation of a specific set of cellular substrates (13,18,20,27). To investigate whether VCP is required for viral capsid degradation during ADIN, we performed a fate-of-capsid assay.…”

Section: Vcp Is Required For Proteasomal Degradation Of Viral Capsid Andmentioning

confidence: 99%

“…These data suggest that VCP has a crucial role in proteasomal degradation of viral capsid. (20) or when it becomes stalled by challenging amounts of substrate (21). We, therefore, investigated whether VCP is constitutively required for TRIM21-mediated degradation or whether its role is substrate-dependent.…”

Section: Vcp Is Required For Proteasomal Degradation Of Viral Capsid Andmentioning

confidence: 99%

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AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization

Hauler

Mallery

McEwan

et al. 2012

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

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Tripartite motif-containing 21 (TRIM21) is a cytosolic IgG receptor that mediates intracellular virus neutralization by antibody. TRIM21 targets virions for destruction in the proteasome, but it is unclear how a substrate as large as a viral capsid is degraded. Here, we identify the ATPase p97/valosin-containing protein (VCP), an enzyme with segregase and unfoldase activity, as a key player in this process. Depletion or catalytic inhibition of VCP prevents capsid degradation and reduces neutralization. VCP is required concurrently with the proteasome, as addition of inhibitor after proteasomal degradation has no effect. Moreover, our results suggest that it is the challenging nature of virus as a substrate that necessitates involvement of VCP, since intracellularly expressed IgG Fc is degraded in a VCP-independent manner. These results implicate VCP as an important host factor in antiviral immunity.A ntibody neutralization is a key component of the antiviral response and provides protective immunity. Our recent work has shown that neutralization can occur inside cells, in an effector-driven process mediated by tripartite motif-containing 21 (TRIM21). TRIM21, a cytosolic antibody receptor of ultrahigh affinity to IgG Fc (1, 2), is recruited to antibody-bound virus and targets the complex to the proteasome for degradation (3). This process potently neutralizes viral infection and has been termed antibody-dependent intracellular neutralization (ADIN) (4). ADIN is dependent upon the E3 ubiquitin ligase activity of TRIM21 and can be abrogated by chemical inhibition of the proteasome.Although both proteasomal activity and ubiquitination are necessary for ADIN, the exact mechanism of virus degradation is poorly understood. Specifically, it is not clear how the proteasome can degrade a virion, a compact proteinaceous particle much larger than the proteasome itself. The 26S proteasome has a mass of ∼2.5 MDa (5), and the pore through which substrates must pass to access the proteolytic chamber is no greater than 2 nm in diameter (6, 7). In contrast, human adenovirus (AdV), a virus potently neutralized by TRIM21, has a diameter of ∼100 nm and a mass of 150 MDa (8).Although there are ATPases in the 19S regulatory subunit of the 26S proteasome that may unfold substrates and allow them to enter through the pore (5, 6), AdV virions are much larger than any of the proteasome's known cellular substrates. Because ADIN has been shown to be independent of autophagy but dependent on proteasomal degradation (3), we hypothesized that an additional energydependent step of AdV capsid disassembly and/or unfolding might precede proteasomal degradation of the virus.In recent studies, the ATPase p97/VCP of the AAA (ATPases associated with diverse cellular activities) family has been implicated in the proteasomal degradation of certain cytosolic substrates (9-13). VCP is capable of dissociating proteins from large cellular structures such as the endoplasmic reticulum (14), the mitotic spindle (12), the nuclear envelope (15), and chromatin (1...

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The ATPase VCP/p97 functions as a disaggregase against toxic Huntingtin‐exon1 aggregates

Ghosh

Ranjan

2018

FEBS Letters

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Intracellular protein aggregation is characterized by accumulation of misfolded proteins. Chaperones, degradation machineries, and quality-control mechanisms counteract protein aggregation. In this study, we report that the ATPase valosin-containing protein (VCP/p97) acts as a functional disaggregase that disassembles Huntingtin-exon1 aggregates in vitro and in HeLa cells. The N-terminal part of VCP (Cdc48_N domain) interacts with the N-terminal 17-amino acid region of Huntingtin-exon1. We show that VCP has properties of a disaggregase, since it is capable of reducing preformed protein aggregates and displays increased ATPase activity in the presence of protein aggregates. However, VCP shows high divergence/disparity from other disaggregases. Taken together, our studies show the novel function of VCP/p97 as a disaggregase which detangles protein aggregates to probably channelize their degradation.

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A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

Cited by 108 publications

References 63 publications

Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase

Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase

AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization

The ATPase VCP/p97 functions as a disaggregase against toxic Huntingtin‐exon1 aggregates

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