Inhibition of p97-dependent Protein Degradation by Eeyarestatin I

Wang, Qiuyan; Li, Lianyun; Ye, Yihong

doi:10.1074/jbc.m708347200

Cited by 176 publications

(222 citation statements)

References 61 publications

Supporting

Mentioning

210

Contrasting

Order By: Relevance

“…Thus, we sought to address the issue using a chemical inhibitor of ERAD. Eer1 interacts with p97 to regulate ERAD negatively, resulting in the accumulation of polyubiquitinated intermediates (43,44). Our results, using KB cells as a model system, failed to confirm an essential role for p97, although our studies were limited in scope and now await an expanded investigation of PE-toxin interactions with the ERAD pathway.…”

Section: Discussioncontrasting

confidence: 45%

“…If ERAD were necessary, one could expect hits near the top of the mitigator list, but no hits related to the ERAD pathway were noted in top mitigators (or sensitizers). Because the participation of ERAD could not be assessed definitively from our siRNA data, we addressed the issue using the chemical inhibitor, Eeyarestatin 1 (Eer1), which interacts with p97 to regulate ERAD negatively, resulting in the accumulation of polyubiquitinated intermediates (43,44). It has also been suggested that Eer1 inhibits Sec61-mediated protein translocation, because it targets a component of the Sec61 complex that forms the membrane pore of the ER translocon (45).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

Pasetto

Antignani

Ormanoglu

et al. 2015

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

View full text Add to dashboard Cite

Immunotoxins (antibody–toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (∼22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas exotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients. High and low concentrations of SS1P were chosen to allow for the identification of both mitigators and sensitizers. As expected, silencing known essential genes in the immunotoxin pathway, such as mesothelin, furin, KDEL receptor 2, or members of the diphthamide pathway, protected cells. Of greater interest was the observation that many RNAi targets increased immunotoxin sensitivity, indicating that these gene products normally contribute to inefficiencies in the killing pathway. Of the top sensitizers, many genes encode proteins that locate to either the endoplasmic reticulum (ER) or Golgi and are annotated as part of the secretory system. Genes related to the ER-associated degradation system were not among high-ranking mitigator or sensitizer candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for chemical intervention that could increase immunotoxin killing of cancer cells and enhance our understanding of toxin trafficking.

show abstract

Section: Discussioncontrasting

confidence: 45%

Section: Resultsmentioning

confidence: 99%

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

Pasetto

Antignani

Ormanoglu

et al. 2015

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

View full text Add to dashboard Cite

show abstract

“…2A). Further validation of these results was obtained by treating cells with Eeyarestatin (Eer)I, an irreversible inhibitor of VCP (25,26). Similar to treatment with DBeQ or knockdown of VCP, treatment with 10 μM EerI before infection potently inhibited neutralization of AdV by mAb 9C12 (Fig.…”

Section: Resultsmentioning

confidence: 98%

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

Hauler

Mallery

McEwan

et al. 2012

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

View full text Add to dashboard Cite

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...

show abstract

“…Protein bands were quantified using the Odyssey 2.1. Astrocytoma or 293T cells stably expressing FLAG-US2 were generated using the pLNCX2-based retroviral system as described previously (29). 293T cell stably expressing YFP tagged T-cell receptor ␣ chain and astrocytoma cells stably expressing US11 were described previously (28,29).…”

Section: Methodsmentioning

confidence: 99%

The p97 ATPase Dislocates MHC Class I Heavy Chain in US2-expressing Cells via a Ufd1-Npl4-independent Mechanism

Soetandyo

2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The human cytomegalovirus (HCMV) protein US2 hijacks the endoplasmic reticulum (ER)-associated degradation machinery to dispose of MHC class I heavy chain (HC) at the ER. This process requires retrotranslocation of newly synthesized HC molecules from the ER membrane into the cytosol, but the mechanism underlying the dislocation reaction has been elusive. Here we establish an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC HC in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97 dislocates MHC class I HC in US2 cells is distinct from that in US11 cells: the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms can employ distinct p97 ATPase complexes to dislocate substrates.

show abstract

Inhibition of p97-dependent Protein Degradation by Eeyarestatin I

Cited by 176 publications

References 61 publications

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

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

The p97 ATPase Dislocates MHC Class I Heavy Chain in US2-expressing Cells via a Ufd1-Npl4-independent Mechanism

Contact Info

Product

Resources

About