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dc.contributor.authorValenzuela, Karla
dc.date.accessioned2016-09-12T13:44:33Z
dc.date.available2016-09-12T13:44:33Z
dc.identifier.urihttp://hdl.handle.net/10222/72217
dc.description.abstractChaperonin 60s (Cpn60s) and their cognate co-chaperonin 10s (Cpn10s) are highly conserved housekeeping proteins that provide favorable conditions for the correct folding of other proteins. The Cpn60/10 of Escherichia coli (known as GroEL/GroES) has been widely studied. Based on these studies it is known that this molecular protein folding machinery is constituted by two GroEL heptameric rings each forming a folding chamber, and a GroES heptameric cap that keeps unfolded proteins inside the chamber. However, bacterial Cpn60s have also evolved additional functions independent of protein folding. The biochemical term recently adopted to describe multifunctional proteins that have a primary well-known function is “moonlighting”, which colloquially refers to having a secondary job (usually at night) in addition to one’s main day job. Some known moonlighting functions of Cpn60s are cell-signaling, proteolytic and toxigenic activity. Unlike GroEL, the Cpn60 of the intracellular bacterial pathogen L. pneumophila (HtpB) reaches the cytoplasm of infected host cells and has been implicated in host cell invasion, microfilament reorganization, mitochondria recruitment and cell-signaling. I hypothesized that HtpB must interact with a cytoplasmic protein in the host cell to exert its unique moonlighting functions, and that functional gains are due to substitutions in key amino acid positions. Therefore, a yeast two hybrid (Y2H) screening using a human cDNA library was performed to find an interaction partner specific for HtpB. In so doing, the human homolog of protein ECM29 (hECM29), which does not interact with GroEL, was found. In addition, putative key amino acids involved in HtpB moonlighting functions were predicted using the ET bioinformatics method. Mutational analysis of the predicted moonlighting-related amino acids led to the identification of 4 residues involved in the HtpB-hECM29 interaction, namely K298, N507, H473 and K474. Since hECM29 couples the 26S proteasome to molecular motors, endocytic vesicles and the endoplasmic reticulum, I propose that exploitation of the HtpB-hECM29 interaction is a previously undescribed strategy used by L. pneumophila to alter protein degradation and vesicular trafficking in the host cell. Additionally, I suggest that although HtpB is a conserved essential protein, it has substitution-prone amino acid positions that have accumulated mutations resulting in the acquisition of novel functions that support the intracellular lifestyle of Legionella pneumophila.en_US
dc.language.isoen_USen_US
dc.subjectHtpBen_US
dc.subjectLegionella pneumophilaen_US
dc.subjectChaperonin 60en_US
dc.titleIDENTIFICATION OF AMINO ACIDS INVOLVED IN THE MOONLIGHTING FUNCTIONS OF HTPB, THE LEGIONELLA PNEUMOPHILA CHAPERONINen_US
dc.date.defence2015-08-18
dc.contributor.departmentDepartment of Microbiology & Immunologyen_US
dc.contributor.degreeMaster of Scienceen_US
dc.contributor.external-examinerAndrew Rogeren_US
dc.contributor.graduate-coordinatorBrent Johnstonen_US
dc.contributor.thesis-readerJohn Rohdeen_US
dc.contributor.thesis-readerJohn Archibalden_US
dc.contributor.thesis-readerChris Barnesen_US
dc.contributor.thesis-supervisorRafael Gardunoen_US
dc.contributor.ethics-approvalNot Applicableen_US
dc.contributor.manuscriptsNot Applicableen_US
dc.contributor.copyright-releaseNot Applicableen_US
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