Master of Science
Physiology and Pharmacology
Stathopulos, Peter B.
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.
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.
Available for download on Thursday, January 30, 2025