Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Zong, Guanghui; Hu, Zhijian; O’Keefe, Sarah; Tranter, Dale; Iannotti, Michael J.; Baron, Ludivine; Hall, Belinda S.; Corfield, Katherine; Paatero, Anja O.; Henderson, Mark J.; Roboti, Peristera; Zhou, Jianhong; Sun, Xianwei; Govindarajan, Mugunthan; Rohde, Jason M.; Blanchard, Nicolas; Simmonds, Rachel E.; Inglese, James; Du, Yong; Demangel, Caroline; High, Stephen; Paavilainen, Ville O.; Shi, Wei

doi:10.1021/jacs.8b13506

Cited by 78 publications

(176 citation statements)

References 63 publications

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“…This issue was first analyzed by using the glycoresin Ipomoeassin F (IpomF). IpomF has been shown to inhibit the protein transport activity of the eukaryotic Sec61 complex [49], but whether it also inhibits the homologous SecYEG complex has not been studied so far. The effect of IpomF was first tested on the strictly SecYEG-dependent secretory protein OmpA.…”

Section: Yohp Is Inserted Into the Bacterial Membrane By The Secyeg Tmentioning

confidence: 99%

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

et al. 2020

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Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro-and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries.

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Section: Yohp Is Inserted Into the Bacterial Membrane By The Secyeg Tmentioning

confidence: 99%

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

et al. 2020

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“…17 As we reported earlier, all of the tested mutations conferred strong resistance to AprA, 14 but only moderate resistance was observed for CbA ( Figure 5B). This finding is surprising especially for the R66I mutation that confers essentially complete resistance for all known Sec61 inhibitors, [14][15][16]24 yet only causes moderate (∼7-fold) desensitization to CbA ( Figure 5B). The Sec61α mutations S82P and T86M that confer resistance to other Sec61 inhibitors, but not CbA, are clustered on the lumenal end of the Sec61 lateral gate, whereas mutations R66I and S71P are located in different parts of the Sec61 plug domain ( Figure 6).…”

Section: ■ Results and Discussionmentioning

confidence: 95%

“…Recent work has identified many structurally distinct natural product small molecules that appear to have evolved independently in distinct microorganisms to target Sec61 as a way to modulate or prevent biogenesis of secreted or integral membrane proteins. Intriguingly, all of these inhibitors appear to target Sec61 at its lumenal cavity near the Sec61 lateral gate and plug domains ( Figure 6) whether they inhibit production of Sec61 substrate proteins in a substrate-selective (cotransins) 19,20 or substratenonselective (AprA, mycolactone, IpoF, decatransin) [14][15][16]22,24 manner.…”

Section: ■ Summarymentioning

confidence: 99%

“…18 The first step in protein secretion is entry into the endoplasmic reticulum (ER), after which newly synthesized secretory polypeptides undergo distinct maturation steps that enable correctly folded proteins to exit the ER and be targeted to their correct final destinations. Previously reported natural products that prevent protein entry into the secretory pathway include, in addition to AprA, 14 HUN-7293 (pestahivin) 19,20 and related synthetic cotransins, 19−21 mycolactone A/B, 15,22,23 decatransin, 16 ipomoeassin F (IpoF), 24 and eeyarestatin I compounds. 25 However, the critical step inhibited by CbA during biogenesis of VEGFR-2 and VEGF-A and the direct cellular target of CbA remain unknown.…”

Section: ■ Introductionmentioning

confidence: 99%

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Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins

Tranter¹,

Paatero²,

Kawaguchi³

et al. 2019

Preprint

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Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the trimeric Sec61 translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61α that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms non-identical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A andipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines.Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

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“…Through a partnership with the National Center for Advancing Translational Sciences (NCATS), ipomoeassin F was included within a collection of natural products for screening against a panel of assays to interrogate a variety of biological pathways and assess cell-type-dependent toxicity. 44 Ipomoeassin F was identified as an inhibitor of protein secretion in two distinct quantitative high-throughput screening (qHTS) assays using different cell lines and secretory reporters. 45 In U2-OS human osteosarcoma cells, a protein reporter consisting of secreted NanoLuc (secNLuc) fused to the Z mutant of alpha-1 antitrypsin (secNLuc-ATZ) was designed to monitor the extracellular accumulation of this protein.…”

Section: Journal Of the American Chemical Societymentioning

confidence: 99%

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Shi

Zong²,

Hu³

et al. 2019

Preprint

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The ipomoeassin family of natural resin glycosides is underexplored chemical space with potent antitumor activity revealed in the NCI-60 cell lines screen; however, its mode of action has so far remained unexplored. In this manuscript, we report our chemical proteomics and subsequent biology studies that transform our collective knowledge of the ipomoeassin glycolipids from Organic Synthesis and Medicinal Chemistry to biological mechanism and provide a step change in our understanding of its action at a cellular level. Hence, we created an ipomoeassin F-based biotin affinity probe and used it in live cells to isolate the ER membrane protein Sec61α as its presumptive molecular target. A direct interaction between Sec61α and ipomoeassin F was confirmed by cell imaging, pulldown from purified ER membranes and competition studies using a photo-crosslinking analogue of the cyclodepsipeptide cotransin, a known Sec61α inhibitor. Crucially, we then showed that ipomoeassin F binding has a profound effect on Sec61 function, using both in vitro and in vivo assays for protein translocation and protein secretion respectively. Although structurally quite distinct, the potency of ipomoeassin F is comparable to that of mycolactone, a recently identified and intensely studied inhibitor of Sec61. The ~1,000 fold increase in the ipomoeassin F resistance of two cell lines expressing mutant forms of Sec61α strongly supports our conclusion that the effect of the compound on Sec61α is the primary basis for its potent cytotoxicity. However, we also provide evidence that ipomoeassin F is mechanistically distinct from known Sec61α inhibitors, suggesting that it is a novel structural class that may offer new opportunities to explore the Sec61 protein translocation complex as a therapeutic target for drug discovery.

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Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Cited by 78 publications

References 63 publications

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

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