Journal Staff

Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s) underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system

Structural complexity of the co-chaperone SGTA:a conserved C-terminal region is implicated in dimerization and substrate quality control

BACKGROUND: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo.RESULTS: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain.CONCLUSION: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region.</p

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Funding Information: We thank the Arkansas Nano & Bio Materials Characterization Facility at the Institute for Nano Sciences & Engineering for our imaging studies, and Prof Yoshito Kishi (Harvard University) for the kind gift of synthetic mycolactone A/B used by S.H. and R.S. W.S. is supported by Grant No. R15GM116032 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) and startup funds from the University of Arkansas. This work was also supported in part by Grant No. P30 GM103450 from the National Institute of General Medical Sciences of the NIH and by seed money from the Arkansas Biosciences Institute (ABI). S.O’K. is the recipient of a Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme Award (BB/J014478/ 1), and S.H. holds a Welcome Trust Investigator Award in Science (204957/Z/16/Z). The alpha-1 antitrypsin work was supported by the Alpha-1 Foundation (J.I. and M.J.I.). J.I. and M.J.H. were supported by the intramural program of NCATS, National Institutes of Health, projects 1ZIATR000048-03 (J.I.) and ZIATR000063-04 (M.J.H.). R.S. holds a Welcome Trust Investigator Award in Science (202843/Z/16/Z). C.D. received funding from the Institut Pasteur, the Institut National de la Santé et de la Recherche Med́ icale, and the Fondation Raoul Follereau. N.B.’s synthesis and chemical biology studies of mycolactone were supported by CNRS, Université de Strasbourg, Fondations Potier et Follereau, and the Investisse-ment d’Avenir (Idex Unistra). V.O.P. is supported by the Academy of Finland (Grants 289737 and 314672) and the Sigrid Juselius Foundation. Funding Information: We thank the Arkansas Nano & Bio Materials Characterization Facility at the Institute for Nano Sciences & Engineering for our imaging studies, and Prof Yoshito Kishi (Harvard University) for the kind gift of synthetic mycolactone A/B used by S.H. and R.S. W.S. is supported by Grant No. R15GM116032 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) and startup funds from the University of Arkansas. This work was also supported in part by Grant No. P30 GM103450 from the National Institute of General Medical Sciences of the NIH and by seed money from the Arkansas Biosciences Institute (ABI). S.O'K. is the recipient of a Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme Award (BB/J014478/1), and S.H. holds a Welcome Trust Investigator Award in Science (204957/Z/16/Z). The alpha-1 antitrypsin work was supported by the Alpha-1 Foundation (J.I. and M.J.I.). J.I. and M.J.H. were supported by the intramural program of NCATS, National Institutes of Health, projects 1ZIATR000048-03 (J.I.) and ZIATR000063-04 (M.J.H.). R.S. holds a Welcome Trust Investigator Award in Science (202843/Z/16/Z). C.D. received funding from the Institut Pasteur, the Institut National de la Sante et de la Recherche Medicale, and the Fondation Raoul Follereau. N.B.'s synthesis and chemical biology studies of mycolactone were supported by CNRS, Universite de Strasbourg, Fondations Potier et Follereau and the Investissement d'Avenir (Idex Unistra). V.O.P. is supported by the Academy of Finland (Grants 289737 and 314672) and the Sigrid Juselius Foundation. Publisher Copyright: © 2019 American Chemical Society.Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61 alpha (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61 alpha from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61 alpha forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61 alpha provides compelling evidence that Sec61 alpha is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61 alpha is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61 alpha function and to further investigate its potential as a therapeutic target for drug discovery.Peer reviewe

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ESI causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation

SGTA: a player in the cytosolic quality control of mislocalised proteins

SGTA: a player inn the cytosolic quality control of mislocalised proteins</p

Emergency First Aid at Work_Peristera Roboti_e-certificate

Emergency First Aid at Work</p

Illuminating the functions of selected subunits of the mammalian oligosaccharyltransferase

Illuminating the functions of selected subunits of the mammalian oligosaccharyltransferase</p

Molecular pathology of disease-associated mutations of the proteolipid protein [Poster]

Dysmyelinating Pelizaeus-Merzbacher disease (PMD) is caused by mutations in the gene encoding myelin proteolipid protein (PLP). Different missense mutations in PLP result in PMD of varying severity. Identifying variations in the ability of the ER quality control machinery to deal with different mutants of PLP has helped to explain such phenotypic heterogeneity. We have used a stable inducible HeLa cell system to describe the fundamental cellular events associated with expression of three different transmembrane missense mutants of PLP associated with disease of varying severity.</p

Disease-related misfolding of the myelin proteolipid protein [Thesis]

A wide range of mutations in the myelin integral plasma membrane proteolipid protein (PLP) are associated with dysmyelinating diseases of varying severity, and whilst missense mutations in PLP transmembrane domains cause severe disease, few such mutants result in a mild phenotype. The molecular pathology of such diseases has generally been attributed to endoplasmic reticulum (ER) retention of misfolded PLP, resulting in the induction of ER stress. However, the cellular mechanism(s) that control the observed phenotypic variations have not yet been elucidated. The work documented in this thesis established that the cellular fate of three distinct transmembrane missense mutants of PLP is differentially regulated by the ER quality control process upon stable inducible expression in HeLa cells. The novel, mild disease-associated G245A and W162L variants of PLP and the comparatively well- characterised, severe disease-causing A242V (msd) PLP mutant display distinct levels of ER retention and ubiquitin-proteasome-mediated degradation. Hence, the G245A variant of PLP shows a relatively tight ER retention phenotype and is quickly degraded by the proteasome, whilst W162L PLP is degraded more slowly but rather efficiently via both a proteasomal and a lysosomal pathway. In contrast to the mild disease-associated mutants, the more ‘aggressive’ msd variant is strictly retained in the ER and is significantly more stable. Notably, the expression of msd PLP is associated with rapid formation of mutant PLP oligomers in the ER, probably as the combined result of its ER retention and slow degradation. This premature oligomerisation of msd PLP appears to confer a toxic ‘gain-of-function’ to the misfolded protein, resulting in a prolonged induction of ER stress. These findings suggest a causal link between the accumulation of mutant PLP in the ER, premature PLP oligomerisation, ER stress, and disease severity. Hence, the rate of degradation of mutant PLP appears to provide the basis for the mechanisms that modulate the apparent phenotypic heterogeneity conferred by missense mutations in the encoding gene.</div

Managing at Manchester for Researchers_Peristera Roboti

Managing at Manchester for Researchers is aimed at colleagues on a  research contract who are line managing in their current role or who wish to do so in the future either inside or outside academia. The programme runs over a six month period and is 'recognised' by the Institute of Leadership & Management. The programme builds researchers confidence and skills as managers so they can enhance their personal impact and work effectively with their people or future teams. The programme includes management theory, case studies, practical  exercises, group discussions, and pre and post learning to prompt discussion and reflection around best practice. It also offer the opportunity to work and make contact with other researchers to help to  share experiences, practise skills and gain feedback in a risk-free  environment.</p