Thesis Format

Monograph

The Effects of S-glutathionylation on the Structure and Function of the Mitochondrial Calcium Uniporter (MCU)

Author

Arine Pawakian, Western UniversityFollow

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Stathopulos, Peter B.

Abstract

Most calcium (Ca²⁺) entry into the mitochondrial matrix is regulated by the mitochondrial calcium uniporter (MCU). The amino (N)-terminal domain (NTD) of MCU is a regulatory component of the channel. S-Glutathionylation of Cys97 on the MCU-NTD leads to robust MCU activation and increased matrix Ca²⁺. Here, I characterized the biophysical and structural changes induced by Cys97 S-glutathionylation by applying optical spectroscopy, light scattering, solution nuclear magnetic resonance (NMR) and live cell functional experiments. S-Glutathionylation increased solvent exposed hydrophobicity, destabilized and caused large structural perturbations in the MCU-NTD. An S-glutathiomimetic mutation was able to closely recapitulate these biophysical and structural effects, but in the absence of oxidative stress. Indeed, HeLa cells expressing MCU with the S-glutathiomimetic mutation, showed increased mitochondrial Ca²⁺ uptake compared to wild-type MCU expressing cells. Thus, my research revealed new insights into the impact of S-glutathionylation on MCU-NTD and identified the S-glutathiomimetic mutation as a valuable research tool.

Summary for Lay Audience

Mitochondrial calcium (Ca²⁺) plays a critical role in adenosine triphosphate (ATP) production and apoptosis in eukaryotic cells.The mitochondrial Ca²⁺ uniporter (MCU) is the protein channel that selectively mediates most of the Ca²⁺ uptake into the mitochondrial matrix, governing these life and death processes. Consequently, MCU activity is carefully controlled by numerous protein regulators and post-translational modifications (PTMs). S-Glutathionylation is a PTM that occurs on the MCU amino terminal domain (NTD) after reactive oxygen species generation. Specifically, the MCU-NTD cysteine residue at position 97 (Cys97) undergoes S-glutathionylation, increasing MCU channel activity and mitochondrial Ca²⁺ uptake. However, the precise molecular and structural mechanisms leading to increased channel activation remain unclear. Using optical spectroscopies, light scattering and solution nuclear magnetic resonance (NMR) approaches, my data revealed that oxidation-induced S-glutathionylation of MCU-NTD largely destabilized and altered the structure of this domain. Moreover, since oxidative stress is known to cause diverse protein modifications, I engineered an S-glutathiomimetic mutation into the MCU-NTD to study how S-glutathionylation specifically regulates MCU in the absence of oxidative conditions. My data showed that the S-glutathiomimetic mutation recapitulated the biophysical, structural, and functional effects of true S-glutathionylation. Importantly, I demonstrated that HeLa cells overexpressing MCU with the S-glutathiomimetic mutation exhibit increased mitochondrial Ca²⁺ uptake compared to wild-type MCU expressing cells. Overall, my research reveals valuable insights into the structural and molecular alterations induced by S-glutathionylation in MCU-NTD and that an S-glutathiomimetic mutation can be effectively used as a surrogate for this modification in the absence of oxidative stress. This research serves as a foundation for the development of MCU modulators and offers a new research tool to study mitochondrial Ca²⁺ handling associated with various conditions characterized by aberrant Ca²⁺ signaling.

Recommended Citation

Pawakian, Arine, "The Effects of S-glutathionylation on the Structure and Function of the Mitochondrial Calcium Uniporter (MCU)" (2024). Electronic Thesis and Dissertation Repository. 9916.
https://ir.lib.uwo.ca/etd/9916