High-Throughput Identification of Calcium Regulated Proteins Across Diverse Proteomes

Abstract

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by lack of high-throughput, unbiased, and quantitative methods to identify proteins-calcium engagement. To address this, we adapted protein thermostability assays in the budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of novel signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-CoA reductase DECR1 binds calcium at physiologically-relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These unbiased, proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)

“…Accordingly, we detected maximal thermal stabilization of CR-1-31-B and CMLD012824 targets in lysates replenished with both an ATP analog and a purine RNA substrate. A recent study noted divergence in calcium-induced thermal shift measurements for 2,4-dienoyl-CoA reductase (DECR1) between crude lysate and intact mitochondria, attributing the difference to insufficient levels of DECR1 substrate and cofactor in lysate (48). Our study supports a similar conclusion and reiterates the need to account for common cofactors and representative substrates for successful capture of complex binding modes.…”

“…changes in protein abundance) in addition to compound-induced thermal stabilization, which makes identification of direct protein targets more challenging (47). Lysate-based formats are useful in identifying direct targets but are performed under non-physiological conditions in which co-factors or co-substrates necessary for target engagement may become diluted or disrupted (43, 48).…”