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dc.contributor.advisorAnderson, Deborah
dc.creatorMarshall, Jeremy Davin Seiberling
dc.date.accessioned2018-01-04T15:07:44Z
dc.date.available2019-01-04T06:05:10Z
dc.date.created2017-11
dc.date.issued2018-01-04
dc.date.submittedNovember 2017
dc.identifier.urihttp://hdl.handle.net/10388/8321
dc.description.abstractThe phosphatidylinositol 3-kinase (PI3K)/PTEN (phosphatase and tensin homologue deleted on chromosome 10) pathway is activated upon stimulation of receptor tyrosine kinases (RTKs) and regulates downstream pathways involved in cell survival, cell growth, cell cycle progression, and protein expression. The protein p85 is uniquely positioned to both positively and negatively regulate the PI3K/PTEN pathway through its interactions with various protein partners including p110, PTEN, and Rab5. The PI3K/PTEN pathway has been shown to be dysregulated in a variety of cancers, including cancers of the breast, prostate, endometrium, and urothelial tract. A better understanding of the interaction between p85 and its various binding partners can further elucidate the mechanisms through which cancer-associated mutations lead to dysregulation of the PI3K/PTEN pathway. To this end we pursued obtaining crystal structure data for the p85 BH domain, alone and in complex with binding partners PTEN and Rab5. Crystal structures were successfully obtained for the bovine p85 BH domain wild-type, individual cancer-associated mutations (E137K, E217K, R262T, E297K), and the engineered mutation R228E. Protein complexes between the p85 BH domain and PTEN or Rab5 were not purified in suitable concentrations for crystallography experiments, and so no structural data was collected for these complexes. Within the p85 BH domain structure a pair of highly coordinated regions of electron density were observed, likely sulfate molecules based on the composition of the crystallization buffer. These regions were coordinated by the p85 residues K224, R228, H234, W237, and Q241. These two highly coordinated regions of electron density were visible for all structures obtained, except the engineered R228E mutant, in which one of these densities was absent. Due to the consist presence of these densities, it was proposed that this region may serve as a potentially uncharacterized binding pocket. Crystallization solutions were prepared exposing crystals of the p85 BH domain to nucleotides, phosphorylated amino acids, or phospholipids, based on the structural similarity between phosphate and sulfate ions. No bound compounds were visible within the determined structures, although high levels of sulfate in the crystallization buffer may have prevented binding of the lower concentration additives. To test p85 for lipid binding in the absence of sulfate, experiments were performed using phosphatidylinositol lipid strips. Direct binding of p85 to lipids present on the lipid strips was detected, with the p85 BH domain alone being sufficient for lipid binding. These results suggest a novel function for the p85 BH domain in direct binding to lipid, which may play a role in regulation of binding partners such as PTEN.
dc.format.mimetypeapplication/pdf
dc.subjectp85
dc.subjectcrystallography
dc.subjectstructural study
dc.subjectcancer
dc.subjectPI3K
dc.titleSTRUCTURAL ANALYSIS OF THE P85 BH DOMAIN IN COMPLEX WITH BINDING PARTNERS AND EFFECT OF MUTATIONS
dc.typeThesis
dc.date.updated2018-01-04T15:07:44Z
thesis.degree.departmentBiochemistry
thesis.degree.disciplineBiochemistry
thesis.degree.grantorUniversity of Saskatchewan
thesis.degree.levelMasters
thesis.degree.nameMaster of Science (M.Sc.)
dc.type.materialtext
dc.contributor.committeeMemberLee, Jeremy
dc.contributor.committeeMemberLukong, Erique
dc.contributor.committeeMemberWu, Yuliang
dc.contributor.committeeMemberKobryn, Kerry
dc.contributor.committeeMemberMoore, Stanley
local.embargo.terms2019-01-04


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