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Title: Small molecule inhibitors targeting protein biogenesis at the endoplasmic reticulum
Author: O'Keefe, Sarah
ISNI:       0000 0004 9354 1235
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2019
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Eukaryotic cells are compartmentalised into membrane-enclosed organelles that perform specific functions. Such organelles, together with specialised vesicles, constitute the secretory pathway; an elaborate network dedicated to the synthesis and folding of secretory proteins and transmembrane proteins (TMPs). The vast majority of these proteins are targeted to the endoplasmic reticulum (ER), the first organellar compartment in the secretory pathway, where they may either be translocated across, or inserted into, the membrane. Once in the lumen, the ER-resident protein folding machinery facilitates their folding concomitant with mechanisms of ER quality control (ERQC) which target proteins that fail to acquire their native conformation to selective degradative pathways. Our understanding of the mechanisms of ER-targeting, protein folding and ERQC has been enhanced through the use of small molecule inhibitors which modulate these processes. These compounds also hold promise for therapeutic application in protein folding related diseases and as broad-spectrum anti-infective agents. Herein, I review the mechanisms of protein biogenesis in the early secretory pathway and the current repertoire of small molecule inhibitors which modulate them. I then present three studies which each utilise small molecule inhibitors with an in vitro system, complemented by other techniques, to study the processes of ER translocation and N-linked glycan processing in the ER. Firstly, I investigate the effects of a panel of iminosugars on N-glycan trimming, finding that five compounds selectively inhibit ER luminal α-glucosidase I and/or α-glucosidase II and further define two compounds as promising 'second generation' iminosugar inhibitors. Secondly, I characterise ipomoeassin F (Ipom-F) as a selective inhibitor of Sec61-mediated protein translocation by showing a potent loss in the ER translocation/membrane integration of Sec61-dependent protein substrates. Thirdly, I utilise Ipom-F as a chemical tool to probe the role(s) of the ER membrane complex (EMC), a recently identified ER insertase, in the membrane insertion of single-pass TMPs. My preliminary findings suggest that certain TMPs may be variably inserted by the EMC, further demonstrating the existence of multiple pathways for membrane protein biogenesis at the ER.
Supervisor: Flitsch, Sabine ; High, Stephen ; Swanton, Eileithyia ; Leonori, Daniele Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Small molecule inhibitors ; Protein biogenesis ; Ipomoeassin-F ; Iminosugars ; ER membrane protein complex ; N-glycan trimming ; Sec61-mediated protein translocation ; Endoplasmic reticulum