High Throughput Identification of Mitochondrial Calcium Regulated Proteins

Abstract

Mitochondrial calcium plays a well-known regulatory role in mitochondria. Disruption of mitochondrial Ca2+ uptake is implicated in diseases such as cancer, neurodegenerative, and metabolic diseases. Evaluating the relationship between the disease states and mitochondrial Ca2+ uptake has proven difficult due to limited knowledge of the mediators of mitochondrial Ca2+ signaling. Currently, there are 20 known mitochondrial Ca2+-binding proteins, which were identified using targeted biochemical assays or computational detection of EF-hand domains. It is unknown whether other calcium-regulated mitochondrial proteins with non-canonical Ca2+-binding domains, which are resistant to computational detection, exist. We set out to identify novel mitochondrial Ca2+-binding proteins in a high-throughput and unbiased manner and investigate how Ca2+ ions regulate these proteins and the mitochondrial pathways they control.To identify calcium-regulated proteins, I optimized a biochemical assay, PISA, that detects the conformational changes in proteins after they interact with calcium. I performed PISA on multiple samples - human cells, yeast cells, and mouse mitochondrial enriched samples from liver tissue– and showed the cross-species viability of this assay to find Ca2+-regulated proteins. The data of each PISA experiment, along with a table detailing data from the orthologous human and yeast proteins, are attached to this dissertation as supplemental tables. Focusing on the mitochondrial samples, I correctly identify known Ca2+-binding and non-Ca2+-binding proteins in an unbiased manner, as well as covering above 85% of the mouse liver mitochondrial proteome. Towards understanding the calcium-regulation of select hits, I used microscale thermophoresis (MST) to detect calcium-binding in vitro at physiologically relevant free calcium concentrations, successfully identifying a novel mitochondrial Ca2+-binding protein. My results fill a large hole in the field’s knowledge of mitochondrial Ca2+ signaling and provide multiple avenues for further research by highlighting new molecular players through which mitochondrial Ca2+ regulates mitochondrial functions.