| Summary: | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010. === "June 2010." Cataloged from PDF version of thesis. === Includes bibliographical references (p. 96-102). === Proteolysis plays a vital role in cellular processes including regulatory pathways and protein quality control in prokaryotes and eukaryotes. ATP-dependent protein degradation is mediated by multimeric protease complexes, each consisting of a AAA+ ATPase and a peptidase component. Substrate selection by the proteases is a highly regulated process to ensure minimal errant protein degradation. Substrates are usually recognized by proteases through degradation tags or degrons. Accessory proteins called adaptors can also modulate substrate selection by proteases. These adaptors have the potential to affect substrate specificity as well as expand the repertoire of substrates that can be degraded by proteases. Understanding how proteases interact with a wide range of adaptors and substrates can provide valuable insight into the complex process of substrate selection. In this thesis, I have investigated the interaction between the AAA+ protease CIpXP and the adaptor protein SspB. The highly conserved N-terminal domain of the unfoldase CIpX interacts with SspB and other specific adaptor proteins and substrates. However, these binding partners do not use one simple sequence motif to mediate the protein-protein interaction. This diversity in protein-binding was further demonstrated by the cross-species CIpX-SspB interactions in Caulobacter crescentus and Escherichia coli. Despite little sequence homology, C. crescentus SspB (ccSspBa) and E. coli SspB (EcSspB) are able to interact with CIpX from either species. We analyzed these interactions to understand how the N-terminal domain of CIpX is able to recognize diverse adaptors and substrates while still retaining specificity. We identified the region important for interaction of ccSspBa with CIpX. Mutagenesis studies of the C-terminal region of the adaptor were conducted and the variants were tested for their ability to functionally interact with the CIpXP protease. Using these data and the results of peptide-binding experiments, we identified residues within the C-terminal region of ccSspBa that are important for tethering to CIpX. We also conducted functional and peptide-binding studies on the EcSspB CIpX-binding (XB) region. Interestingly, the two XB regions are very different in both length and sequence. However, despite this dissimilarity, competition studies argue that the two XB peptides bind to identical or overlapping sites of the CIpX N domains of C. crescentus and E. coli. This cross-species interaction between SspB and CIpX highlights how the CIpX N domain provides a versatile platform for binding a variety of adaptors and substrates. We also performed a proteomic-screen to investigate the effect of SspB on CIpXP substrate profile in E. coli. The preliminary data has provided a list of candidate SspB-interacting substrates, further analyses of which will contribute to the understanding of the biological impact of SspB on substrate selection by CIpXP. === by Tahmeena Chowdhury. === Ph.D.
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