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Summary: Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by a lack of high-throughput, unbiased, and quantitative methods to identify protein-calcium engagement. To address this, we adapted protein thermostability assays in budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2,884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of 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-coenzyme A (CoA) reductase DECR1 binds calcium at physiologically relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These discovery-based proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

Mitochondrial complex I deficiency is frequent in congenital, neurologic and cardiovascular disease. Here the authors demonstrate that Complex I stimulates the turnover of a mitochondrial calcium channel, which becomes stabilized during Complex I deficiency, preserving energetic homeostasis.

Summary: Activation of myosin light chain kinase (MLCK) by calcium ions (Ca2+) and calmodulin (CaM) plays an important role in numerous cellular functions including vascular smooth muscle contraction and cellular motility. Despite extensive biochemical analysis, aspects of the mechanism of activation remain controversial, and competing theoretical models have been proposed for the binding of Ca2+ and CaM to MLCK. The models are analytically solvable for an equilibrium steady state and give rise to distinct predictions that hold regardless of the numerical values assigned to parameters. These predictions form the basis of a recently proposed, multi-part experimental strategy for model discrimination. Here we implement this strategy by measuring CaM-MLCK binding using an in vitro FRET system. Interpretation of binding data in light of the mathematical models suggests a partially ordered mechanism for binding CaM to MLCK. Complementary data collected using orthogonal approaches that assess CaM-MLCK binding further support this conclusion.

Tyrosine hydroxylase (Th) expression has previously been reported in Purkinje cells (PCs) of rodents and humans, but its role in the regulation of behavior is not understood. Catecholamines are well known for facilitating cognitive behaviors and are expressed in many regions of the brain. Here, we investigated a possible role in cognitive behaviors of PC catecholamines, by mapping and testing functional roles of Th positive PCs in mice. Comprehensive mapping analyses revealed a distinct population of Th expressing PCs primarily in the posterior and lateral regions of the cerebellum (comprising about 18% of all PCs). To identify the role of PC catecholamines, we selectively knocked out Th in PCs using a conditional knockout approach, by crossing a Purkinje cell-selective Cre recombinase line, Pcp2-Cre, with a floxed tyrosine hydroxylase mouse line (Thlox/lox) to produce Pcp2-Cre;Thlox/lox mice. This manipulation resulted in approximately 50% reduction of Th protein expression in the cerebellar cortex and lateral cerebellar nucleus, but no reduction of Th in the locus coeruleus, which is known to innervate the cerebellum in mice. Pcp2-Cre;Thlox/lox mice showed impairments in behavioral flexibility, response inhibition, social recognition memory, and associative fear learning relative to littermate controls, but no deficits in gross motor, sensory, instrumental learning, or sensorimotor gating functions. Catecholamines derived from specific populations of PCs appear to support cognitive functions, and their spatial distribution in the cerebellum suggests that they may underlie patterns of activation seen in human studies on the cerebellar role in cognitive function.