Characterization of oligomeric state of prokaryotic rhomboid proteases

• Author / Creator

  Sampath Kumar, Padmapriya

• 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.

• Subjects / Keywords

  • Oligomerization rhomboid protease

• Graduation date

  Fall 2012

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R38023

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.