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Permanent link (DOI): https://doi.org/10.7939/R38023

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Characterization of oligomeric state of prokaryotic rhomboid proteases Open Access

Descriptions

Other title
Subject/Keyword
oligomerization rhomboid protease
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
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
Specialization

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.
Citation for previous publication
Padmapriya Sampathkumar, Michelle W. Mak, Sarah J. Fischer-Witholt, Emmanuel Guigard, Cyril M. Kay, M. Joanne Lemieux. 2012 Biochim Biophys Acta 1818(12): 3090-3097

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