Characterization of oligomeric state of prokaryotic rhomboid proteasesReport as inadecuate

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oligomerization rhomboid protease

Sampath Kumar, Padmapriya

Supervisor and department: Dr. Joanne Lemieux, Dept. of Biochemistry

Examining committee member and department: Dr. Joel Weiner, Dept. of Biochemistry Dr. Richard Fahlman, Dept. of Biochemistry Dr. Emmanuelle Cordat, Dept. of Physiology

Department: Department of Biochemistry


Date accepted: 2012-09-27T08:45:38Z

Graduation date: 2012-09

Degree: Master of Science

Degree level: Master's

Abstract: Rhomboid proteases are a remarkable class of intramembrane enzymes that carry out cleavage of transmembrane substrates within or proximal to the lipid bilayer. These proteases have been linked to several human diseases such as cancer, diabetes and early-onset blindness. They are also involved in diverse processes including quorum sensing and cell differentiation in bacteria. To better understand the mechanisms underlying the proteolytic action and function of these proteases, we have focussed on investigating its regulation. In this thesis, the concept of oligomerization as a possible mode of regulation is examined. To assess the oligomeric state of three prokaryotic rhomboid proteases from Haemophilus influenza hiGlpG, Escherichia coli ecGlpG and Bacillus subtilis YqgP, sedimentation equilibrium analysis was carried out. The predominant species for the three rhomboid proteases was observed to be dimeric. To examine the effect of the membrane domain alone on dimerization, hiGlpG, the simplest form of rhomboid representing the core of six transmembrane domains, was studied further. Gel filtration, crosslinking and functional assay demonstrate that hiGlpG is dimeric and functional in dodecylmaltoside detergent solution. More importantly, co-immunoprecipitation studies establish that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. This thesis also investigates the physiological role of ecGlpG rhomboid from E. coli. The potential of E. coli TatA as a substrate for ecGlpG is examined using an in vitro functional assay. Additionally, affinity pull-down and co-immunoprecipitation techniques are performed to identify possible substrates for this rhomboid.

Language: English

DOI: doi:10.7939-R38023

Rights: Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.

Author: Sampath Kumar, Padmapriya



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