Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum

Cross, Benedict C. S.; McKibbin, Craig; Callan, Anna C.; Roboti, Peristera; Piacenti, Michela; Rabu, Catherine; Wilson, Cornelia M.; Whitehead, Roger C.; Flitsch, Sabine L.; Pool, Martin; High, Stephen; Swanton, Eileithyia

doi:10.1242/jcs.054494

Cited by 91 publications

(151 citation statements)

References 40 publications

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“…Each of these steps could presumably prevent the completion of HC translation, thus resulting in rapidly degraded translation intermediates. Recently, the small molecule inhibitor eeyarestatin 1 (ES 1 ) was shown to efficiently inhibit protein translocation and prevent transfer of the RNC from the SR to SEC61 (10). In contrast, ES 1 did not prevent docking of the SRP/RNC to the SR.…”

Section: Resultsmentioning

confidence: 99%

“…Eeyarestatin 1 (ES 1 ) and eeyarestatin R35 (ES R35 ) were kind gifts from Stephen High (University of Manchester, Manchester, United Kingdom). Eeyarestatin treatments were based on methods previously described (10). TRFs in culture were treated with dimethyl sulfoxide (DMSO) or 10 M of the proteasome inhibitor MG132 for 4 h, followed by treatment with DMSO, 10 M ES 1 , or 10 M ES R35 for 1 h in the continuing presence of DMSO or MG132.…”

Section: Methodsmentioning

confidence: 99%

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The Cytoplasmic Domain of Rhesus Cytomegalovirus Rh178 Interrupts Translation of Major Histocompatibility Class I Leader Peptide-Containing Proteins prior to Translocation

Richards

Scholz

Powers

et al. 2011

J Virol

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Cytomegalovirus (CMV) efficiently evades many host immune defenses and encodes a number of proteins that prevent antigen presentation by major histocompatibility complex class I (MHC-I) molecules in order to evade recognition and killing of infected cells by cytotoxic CD8؉ T cells. We recently showed that rhesus CMV-specific Rh178 intercepts MHC-I protein translation before interference of MHC-I maturation by homologues of the human CMV US6 family. Here, we demonstrate that Rh178 localizes to the membrane of the endoplasmic reticulum, displaying a short luminal and large cytosolic domain, and that the membraneproximal cytosolic portion is essential for inhibition of MHC-I expression. We further observed that Rh178 does not require synthesis of full-length MHC-I heavy chains but is capable of inhibiting the translation of short, unstable amino-terminal fragments of MHC-I. Moreover, the transfer of amino-terminal fragments containing the MHC-I signal peptide renders recipient proteins susceptible to targeting by Rh178. The cytosolic orientation of Rh178 and its ability to target protein fragments carrying the MHC-I signal peptide are consistent with Rh178 intercepting partially translated MHC-I heavy chains after signal recognition particledependent transfer to the endoplasmic reticulum membrane. However, interference with MHC-I translation by Rh178 seems to occur prior to SEC61-dependent protein translocation, since inhibition of MHC-I translocation by eeyarestatin 1 resulted in a full-length degradation intermediate that can be stabilized by proteasome inhibitors. These data are consistent with Rh178 blocking protein translation of MHC-I heavy chains at a step prior to the start of translocation, thereby downregulating MHC-I at a very early stage of translation.Cytomegaloviruses (CMV), members of the betaherpesviridae, are masters at evading the host immune system. All CMV genomes dedicate many of their open reading frames (ORFs) to escaping various mechanisms of immune defense. CMVencoded immunomodulators function to circumvent cellautonomous defenses such as apoptosis and the interferon (IFN) response, as well as to prevent innate and adaptive immune responses by natural killer (NK) cells and T cells (5,13,34,61). These proteins allow the virus to establish primary infection, maintain persistent infection, and support repeated superinfection of chronically infected hosts. The study of cytomegaloviral immunomodulatory proteins not only has underscored the important and delicate relationship between virus and host but also has revealed novel proteins of the host immune system like the UL16-protein binding family of NKG2D ligands and the UL18-binding NK cell inhibitory receptor LIR-1 (7, 8).In addition, CMV immunomodulators have been employed to decipher basic cell biological principles such as protein quality control. Glycoproteins within the US6 family of human CMV (HCMV) (25) block endoplasmic reticulum (ER)-associated degradation of major histocompatibility complex class I (MHC-I) proteins and thereby prevent...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Cytoplasmic Domain of Rhesus Cytomegalovirus Rh178 Interrupts Translation of Major Histocompatibility Class I Leader Peptide-Containing Proteins prior to Translocation

Richards

Scholz

Powers

et al. 2011

J Virol

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“…A general translocation inhibitor is eeyarestatin, a chemical most likely binding to the translocon and preventing the transfer of the nascent-chain-ribosome complex from the SRP-SRP receptor targeting complex to the translocon in mammalian cells (Cross et al, 2009). Apratoxin A, a cyanobacterial metabolite, has been shown to inhibit cotranslational translocation in vitro (Liu et al, 2009), but the blocked step is unknown.…”

Section: Introductionmentioning

confidence: 99%

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

Junne

Wong

Studer

et al. 2015

Journal of Cell Science

108

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A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61a1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co-and posttranslationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.

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“…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). KB cells incubated with Eer1 and A B Fig.…”

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.

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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.

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Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum

Abstract: Results ESSupplementary material available online at

Cited by 91 publications

References 40 publications

The Cytoplasmic Domain of Rhesus Cytomegalovirus Rh178 Interrupts Translation of Major Histocompatibility Class I Leader Peptide-Containing Proteins prior to Translocation

The Cytoplasmic Domain of Rhesus Cytomegalovirus Rh178 Interrupts Translation of Major Histocompatibility Class I Leader Peptide-Containing Proteins prior to Translocation

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

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

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