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Elucidating the mitochondrial architecture of branched-chain amino acid metabolism enzymes: implications for treatment

McCalley, Stephen (2018) Elucidating the mitochondrial architecture of branched-chain amino acid metabolism enzymes: implications for treatment. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Branched chain amino acid (BCAA) metabolism occurs within the mitochondrial matrix and is comprised of 17 enzymes, some shared, organized into three pathways for the catabolism of leucine, isoleucine, and valine (LEU, ILE, and VAL respectively). However, the physical relationships of the various enzymes in the pathways are unknown. Diseases such as isovaleric academia, propionic and methylmalonic acidemias, and maple syrup urine disease are among the disorders caused by genetic deficiencies of BCAA metabolism.
I examined the BCAA pathways and their physical interactions in vitro through proteomics analysis and in situ using stimulated emission depletion microscopy. The functional interactions of the pathways were measured by flux analysis with labeled LEU, ILE, and VAL in patient and wild type cell lines, with quantification of the labeled metabolic end-products of each pathway. I examined novel potential treatments for propionic acidemia patient derived cell lines, quantitating protein, ROS production, and mitochondrial mass response to therapeutic agents.
My proteomic and imaging studies are consistent with the existence of one or more energetically favorable, metabolite-channeling BCAA super-complex(es). Flux studies demonstrate that the end products of LEU, ILE and VAL metabolism are generated in both wild type and patient derived cells lines, implying cross-talk of BCAA pathways, and a close proximity of shared enzymes. Additionally, I found that the end-product of ILE and VAL, propionyl-CoA, does not readily enter the TCA cycle, as previously thought, while propionyl-CoA from odd chains fats does. Finally, propionyl-CoA carboxylase (PCC) deficient cell lines showed improved mitochondrial function when treated with a cardiolipin binding protein that stabilizes the inner mitochondrial membrane in cell cultures. PCC protein amount was slightly increased in treated patient cells, and ROS and mitochondrial mass was significantly decreased.
These results provide novel insight into BCAA metabolism and offer new opportunities for the development of therapeutic agents for their defects. Furthermore, because propionyl-CoA derived from odd-chain fatty acids does, in fact, readily enter the TCA cycle, results from the metabolic flux studies will impact public health by changing potential therapies used to replenish the TCA cycle intermediates for disorders of fatty acid oxidation as well as BCAA metabolism.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
McCalley, Stephenspm42@pitt.eduspm42
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairVockley, Jerryvockleyg@upmc.edugev1
Committee MemberKammerer, Candacecmk3@pitt.educmk3
Committee MemberGoetzman, Ericesg5@pitt.eduesg5
Committee MemberUrban, Zsolturbanz@pitt.eduurbanz
Date: 28 June 2018
Date Type: Publication
Defense Date: 23 February 2018
Approval Date: 28 June 2018
Submission Date: 22 April 2018
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 93
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: BCAA metabolism, organic acidurias, metabolon, mitochondria, genetics
Date Deposited: 28 Jun 2018 20:38
Last Modified: 01 May 2020 05:15


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