The road less travelled: membrane protein biogenesis at the ER

Why are a group of membrane proteins resistant to the effects of the small molecule Sec61 inhibitor Ipomoeassin F? Turns out it's because they use an alternative pathway for their ER biogenesis.

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Meet the Researchers

   

    Sarah O'Keefe      Guanghui Zong      Kwabena Duah    Lauren Andrews        Wei Shi        Stephen High

Behind the Paper

30% of the proteins in our cells must either be fully translocated across (secretory proteins) or inserted into (membrane proteins) the membrane of the endoplasmic reticulum (ER) during an early stage of their biogenesis. Given the broad structural and functional diversity of these secretory and membrane proteins, our cells have developed several specialised routes or 'highways' that facilitate protein translocation and/or membrane insertion at the ER concomitant with their ribosomal synthesis1.

Central to the majority of these routes is the Sec61 complex, the predominant protein conducting channel in the ER membrane which acts as a dynamic hub for co-translational protein translocation at the ER1. Recent work has revealed an array of structurally diverse small molecules that inhibit Sec61-mediated ER protein translocation2. Hence, given the many protein clients of the Sec61 complex, Sec61 inhibitors offer potential for therapeutic application in several areas of unmet medical need, including anti-infectives and oncology.

Amongst this Sec61 inhibitor toolbox of potential therapeutics, is ipomoeassin F (Ipom-F); a natural product first isolated from the morning glory family of plants in the Suriname rainforest. After developing an elegant total synthesis (2015) and embarking upon a comprehensive structural-activity-relationship (SAR) programme, Dr Wei Shi (Ball State University) initiated a highly collaborative and forward-thinking research initiative involving multiple lab groups in order to define Ipom-F’s mechanism of action. The principle molecular basis for its cytotoxicity against multiple cancer cell lines proved to be its ability to inhibit ER protein translocation through its strong, yet reversible, binding to the Sec61 complex3.


In this study, we used Ipom-F (chemical structure, left) as a biochemical tool and cells depleted of the Sec61 complex (schematic, middle) and the ER membrane complex (schematic, right) to investigate membrane protein insertion at the ER.

Having initially joined this ‘Ipom-F consortium’ in 20193, the characterisation and exploitation of the inhibitory effects of this small molecule have dominated our research focus over the past few years; a real rags-to-riches tale for a ‘side project’. Having further characterised the effects of Ipom-F on the biogenesis of secretory proteins in cells4, established that it is well-tolerated in mice5 and highlighted its potential as a broad-spectrum antiviral agent (an in vitro study using SARS-CoV-2 proteins)6, we returned our attention to tackling the question that arose when we first began working with Ipom-F: why are a small but functionally important group of membrane proteins, known as type III transmembrane proteins (TMPs), resistant to the effects of this, and other, Sec61 inhibitors?3

By combining Ipom-F-mediated inhibition with siRNA-mediated knockdowns of ER membrane components we have established that type III TMPs are inherently resistant to the effects of small molecule Sec61 inhibitors because, in line with the title of our paper, they utilise 'an alternative pathway for membrane protein biogenesis' at the ER.



This alternative route requires the concerted actions of the Sec61 complex and the ER membrane complex (EMC). Hence, over 30 years since the Sec61 complex was first discovered, new components, routes and, thus, opportunities for targeting therapeutic interventions continue to emerge1. We hope that advancing our understanding of ER membrane protein biogenesis will, ultimately, contribute to its selective modulation for the benefit of human health.


Read the paper here: https://www.nature.com/articles/s42003-021-02363-z


Sarah O’Keefe and Stephen High wrote this blog.


References

  1. O’Keefe, S., Pool, M. R. & High, S. Membrane protein biogenesis at the ER: the highways and byways. FEBS J. doi 10.1111/febs.15905 (2021).
  2. Luesch, H. & Paavilainen, V. O. Natural products as modulators of eukaryotic protein secretion. Nat. Prod. Rep. 37, 717-736 (2020).
  3. Zong, G., Hu, Z., O’Keefe, S., Tranter, D., Ioannotti, M.J., Baron, L., Hall, B., Corfield, K., Paatero, A., Henderson M. et al. Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation. J. Am. Chem. Soc. 141, 8450-8461 (2019).
  4. Roboti, P., O’Keefe, S., Duah, K. B., Shi, W. Q. & High, S. Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis. Sci. Rep. 11, 11562 (2021).
  5. Zong, G., Hu, Z., Duah, K. B., Andrews, L. E., Zhou, J., O’Keefe, S., Whisenhunt, L. H., Shim, J. S., Du, Y., High, S. & Shi, W. Q. Ring Expansion Leads to a More Potent Analogue of Ipomoeassin F. J. Org. Chem. 85, 16226-16235 (2020).
  6. O’Keefe, S., Roboti, P., Duah, K. B., Zong, G., Scheider, H., Shi, W. Q. & High, S. Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor. J. Cell. Sci. 134, jcs.257758 (2021).

Sarah O'Keefe

Postdoctoral Research Associate, University of Manchester