Investigating the early events in proteasome assembly

dc.contributor.advisorKusmierczyk, Andrew
dc.contributor.authorRamamurthy, Aishwarya
dc.contributor.otherAtkinson, Simon
dc.contributor.otherRandall, Stephen Karl, 1953-
dc.date.accessioned2015-03-10T15:34:01Z
dc.date.available2015-08-02T09:30:41Z
dc.date.issued2014
dc.degree.date2014en_US
dc.degree.disciplineDepartment of Biologyen
dc.degree.grantorPurdue Universityen_US
dc.degree.levelM.S.en_US
dc.descriptionIndiana University-Purdue University Indianapolis (IUPUI)en_US
dc.description.abstractProteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.en_US
dc.identifier.urihttps://hdl.handle.net/1805/6023
dc.identifier.urihttp://dx.doi.org/10.7912/C2/2183
dc.language.isoen_USen_US
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 United States
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/us/
dc.subjectself assemblyen_US
dc.subjectproteasome assemblyen_US
dc.subjectcrosslinkingen_US
dc.subject.lcshProteolytic enzymes -- Research -- Analysis -- Methodologyen_US
dc.subject.lcshProteins -- Metabolism -- Researchen_US
dc.subject.lcshProteins -- Crosslinking -- Researchen_US
dc.subject.lcshSelf-assembly (Chemistry)en_US
dc.subject.lcshEscherichia colien_US
dc.subject.lcshEscherichia coli -- Physiologyen_US
dc.subject.lcshProteins -- Conformationen_US
dc.subject.lcshUbiquitinen_US
dc.subject.lcshSaccharomyces cerevisiaeen_US
dc.subject.lcshProtein bindingen_US
dc.subject.lcshCellular control mechanismsen_US
dc.subject.lcshGene expressionen_US
dc.subject.lcshArchaebacteriaen_US
dc.subject.lcshArchaebacteria -- Metabolismen_US
dc.titleInvestigating the early events in proteasome assemblyen_US
dc.typeThesisen
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