DOI

10.17077/etd.d7o1-said

Document Type

Dissertation

Date of Degree

Summer 2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Biochemistry

First Advisor

Taylor, Eric

First Committee Member

Rubenstein, Peter

Second Committee Member

Baker, Sheila

Third Committee Member

Brenner, Charles

Fourth Committee Member

Wallrath, Lori

Fifth Committee Member

Wright, Michael

Abstract

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix, thereby linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient c.C289T and c.T236A MPC1 alleles disrupt MPC function. MPC1 c.C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and full-length point mutant (R97W). MPC1 c.T236A encodes a full-length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from human patient MPC1 mutations and inform fundamental MPC structure-function relationships. These results also demonstrate an efficient molecular genetic system using the mouse C2C12 cell line to mechanistically investigate human inborn errors in pyruvate metabolism.

Keywords

Carbohydrate metabolism, Cell Biology, Inborn error, Metabolism, Mitochondria, Transport

Pages

xvii, 133 pages

Bibliography

Includes bibliographical references (pages 110-133).

Copyright

Copyright © 2019 Lalita Oonthonpan

Included in

Biochemistry Commons

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