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dc.contributor.authorYun, Sohee
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.date.accessioned2018-01-19T15:54:25Z
dc.date.available2018-01-19T15:54:25Z
dc.identifier.urihttp://hdl.handle.net/1974/23838
dc.description.abstractAarf domain-containing kinase 3, ADCK3, is a human atypical protein kinase located in the inner membrane of the mitochondria that is required for the biosynthesis of coenzyme Q (CoQ). Stable supply of CoQ is essential to human health as it plays a critical role as an electron transporter in the electron transport chain which is required for proper ATP production. Although the exact role of ADCK3 is unknown, patients possessing mutations within this gene are often diagnosed with autosomal recessive cerebellar ataxia type 2, which results from CoQ deficiency. To better understand the involvement of ADCK3 and other proteins in CoQ biosynthesis, their structure, function, and their interactions with each other were investigated. ADCK3 is hypothesized to regulate the CoQ biosynthesis using its kinase activity to activate several proteins found in the CoQ biosynthetic complex; however, previous work has failed to show direct evidence of its kinase activity and its functional interaction with the other Coq proteins. In this work, using a soluble construct of ADCK3, we solved its structure by X-ray crystallography. Various constructs of ADCK3 were transfected into Human Embryonic Kidney cells in an attempt to extract and identify its substrates. Further, the genes of the potential substrates, Coq3, Coq5, and Coq7 were cloned into various expression vectors and transformed into Escherichia coli expression cell lines. After the extensive expression trials, the combination of N-terminal fusions with maltose-binding proteins and the RIPL E. coli cell expression line was found to facilitate the expression of Coq5 and Coq7. However, using radiometric kinase assays, we show that ADCK3 exhibited no kinase activity towards Coq5 and Coq7. Finally, this work also shows the methyltransferase activity of Coq5 using its substrate analog. It is anticipated that our results will serve as a foundation for further characterization of the proteins involved in the human CoQ10 biosynthesis.en_US
dc.language.isoenen_US
dc.relation.ispartofseriesCanadian thesesen
dc.rightsQueen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada*
dc.rightsProQuest PhD and Master's Theses International Dissemination Agreement*
dc.rightsIntellectual Property Guidelines at Queen's University*
dc.rightsCopying and Preserving Your Thesis*
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.*
dc.rightsCC0 1.0 Universal*
dc.rights.urihttp://creativecommons.org/publicdomain/zero/1.0/*
dc.subjectADCK3en_US
dc.subjectCoq8en_US
dc.subjectProtein Expressionen_US
dc.subjectProtein Purificationen_US
dc.subjectX-ray Crystallographyen_US
dc.subjectCoenzyme Qen_US
dc.subjectCoq5en_US
dc.subjectCoq7en_US
dc.subjectMethyltransferaseen_US
dc.subjectATPase activity assayen_US
dc.subjectKinase Activity Assayen_US
dc.subjectProtein Kinaseen_US
dc.subjectAtypical kinaseen_US
dc.subjectCo-Immunoprecipitationen_US
dc.titleCharacterization of the Human Atypical Kinase, aarf Domain-Containing Kinase 3, and its Substrates Required for Coenzyme Q biosynthesisen_US
dc.typeThesisen
dc.description.degreeMaster of Scienceen_US
dc.contributor.supervisorJia, Zongchao
dc.contributor.departmentBiomedical and Molecular Sciencesen_US


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Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada
Except where otherwise noted, this item's license is described as Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada