Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Peter B. Stathopulos

Abstract

Magnesium (Mg2+) is an essential and versatile ion that regulates countless cellular phenomena, from ATP synthesis to enzyme catalysis, protein and nucleic acid stabilization and ion transport. Not surprisingly, intracellular and subcellular Mg2+ is tightly regulated, and dysfunctional Mg2+ homeostasis is implicated in various diseases including cancer, where it influences metabolic reprogramming, apoptosis and multidrug resistance (MDR). Mitochondrial RNA Splicing Protein 2 (MRS2) forms an ion channel that mediates Mg2+ influx into mitochondrial matrix (MM) and has emerged as a key player in regulating intracellular Mg2+. Nevertheless, the precise molecular mechanisms underlying the regulation of human MRS2 and roles in cancer remain enigmatic. To begin elucidating the structural mechanisms underlying MRS2 regulation, we expressed, isolated and biophysically characterized the large, matrix-oriented amino terminal domain (NTD). Our data reveal that MRS2-NTD self-associates, which can be robustly disrupted by Mg2+. Further, we identified D216 and D220 as key residues within the NTD responsible for Mg2+ binding-induced changes in secondary, tertiary and quaternary structure. Functionally, the mutation-induced abrogation in Mg2+ binding to the NTD led to enhanced mitochondrial Mg2+ uptake in mammalian cells, uncovering an NTD-driven regulation mechanism. Next, we looked more specifically at the role of NTD-dependent feedback regulation in hallmarks of cancer, finding that Mg2+ binding to the inhibitory site decreases the self-association affinity by up to two orders of magnitude. Further, we demonstrate that Mg2+ binding to the NTD inhibitory site causes allosteric changes to the domain far from the Mg2+ binding site, and disruption of this Mg2+ sensitivity leads to enhanced Mg2+ uptake, cell migration and resistance to apoptosis. Finally, we isolated and characterized the matrix-oriented carboxyl-terminal domain (CTD), revealing Mg2+-insensitive higher order oligomerization necessary for the pentameric assembly of full-length MRS2. Further, deleting the CTD impaired Mg2+ transport, suppressed cell migration, enhanced doxorubicin-mediated apoptosis and suppressed metabolic activity. Collectively, the work establishes the NTD as a matrix Mg2+ sensor involved in the negative feedback autoregulation of the MRS2 channel and the CTD as a critical assembly domain that may be a useful therapeutic target in the context of gastric cancer and other diseases associated with dysregulated Mg2+ homeostasis.

Summary for Lay Audience

Magnesium (Mg2+) is a mineral that plays an essential role in a multitude of processes within our cells, including energy production, enzyme function, protein and DNA stability, and the transport of ions. The movement of Mg2+ into mitochondria is particularly important for cell survival. Disruption of the cellular and mitochondrial balance of Mg2+ can lead to a variety of diseases, including cancer. Specifically, gastric cancer (GC), one of the most common cancers worldwide, has been linked to increases in mitochondrial Mg2+ transport, which can result in the resistance to chemotherapy. Mitochondrial RNA Splicing Protein 2 (MRS2) forms a channel in mitochondria that controls the movement of Mg2+ into the organelle. However, the mechanisms underlying MRS2 function are poorly understood. Thus, to better comprehend how MRS2 mediates Mg2+ transport, and its involvement in disease, we first biophysically and structurally characterized the large MRS2 amino terminal domain (NTD) that resides in the mitochondrial matrix. We found that the purified and isolated MRS2 NTD self-associates, and Mg2+ increases its a-helical structure, affects its tertiary structure, and disrupts its self-association. We pinpointed D216 and D220 as key residues in the NTD involved in the Mg2+ binding, where mutation of these residues not only disrupts Mg2+-induced changes in structure but also promotes increased mitochondrial Mg2+ uptake in mammalian cells. Moreover, we show that the enhanced Mg2+ uptake caused by the mutation increases cell movement and decreases cell susceptibility to death. Finally, we characterized the matrix-oriented carboxyl-terminal domain (CTD), revealing that this domain has a tendency for higher order oligomerization and is necessary for the pentameric assembly of the MRS2 channel. Further, channels with deleted CTD demonstrate impaired Mg2+ transport, suppressed cell movement, enhanced drug-induced cell death and suppressed metabolic activity. Together, this thesis provides new insights into how the MRS2-NTD detects matrix Mg2+ to self-regulate channel activity, how MRS2 activity influences several hallmarks of cancer, the importance of the CTD in pentameric channel formation, and a potential drug target on MRS2 for the treatment of cancer.

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Available for download on Monday, March 30, 2026

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