Cotransin is a cyclodepsipeptide inhibitor of protein translocation and has been demonstrated to inhibit cotranslational translocation of a variety of proteins by targeting the mammalian ER translocation channel Sec61 (Besemer, Harant et al. 2005, Garrison, Kunkel et al. 2005). Genetic screens in cancer cells found out the mutations near the luminal plug domain of Sec61 confer resistance to cotransin inhibition and thus outline the proposed binding site for cotransin (MacKinnon, Paavilainen et al. 2014). However molecular details of cotransin interactions remain unknown.
Purpose of my first project was to express the human Sec61 translocation channel in correct stoichiometric ratios. To our knowledge, heterotrimeric expression of Sec61 has not been achieved previously. Baculovirus system was chosen express the Sec61 heterotrimeric complex. To vary expression levels of Sec61α and Sec61γ relative to Sec61β, separate baculovirus constructs were prepared. Proper co-transfection ratios between these viruses to express Sec61subunits in correct stoichiometric ratios were calculated during my pro gradu and insect cell expression was then scaled up using the determined virus ratios. Results from Sec61 expression have returned sufficient quantities of the translocation channel for biochemical analyses. Final expression seems to contain high lipid/protein ratio, which may have been caused due to insect cells not-fully adapted to the media. The expression should be repeated in well-adapted healthy insect cells and then accessed for protein quality. For its isolation for crystallization studies, co-immunoprecipitation may be a preferred way to pull down the Sec61 complex. Detergent based solubilization may be alternatively used to isolate Sec61.
The second part of my pro gradu work included expression of SecYEβ translocation channel from Pyrococcus furiosus, along with its various humanized mutants, whose DNA constructs had been provided by Dr. Ville Paavilainen. A major goal of this work was to express the mutants which bind to cotransin. Via photo-crosslinking and click chemistry analyses, a mutant binding to cotransin was identified and scaled-up. Our photo-crosslinking studies were able to demonstrate that the wild-type SecYEβ does not bind to cotransin in vitro. Results from photo-crosslinking assays during this project also demonstrated that other known translocation inhibitors Mycolactone and Apratoxin A can bind to mammalian Sec61 channel. These results are consistent with unpublished work from Paavilainen lab (Paatero et al).
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:nbnfioulu-201510142073 |
| Date | 14 October 2015 |
| Creators | Syed, S. (Shahan) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © Shahan Syed, 2015 |
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