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The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress‐induced cardiac injury remain unclear. Here, the role of proline‐rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H2O2 resulted in 10‐ to 21‐fold increases in Pyk2 phosphorylation in wild‐type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2‐mediated Ca2+ increase with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca2+ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca2+ uptake and enhance adenosine triphosphate generation. Although Trpm2‐mediated Ca2+ influx phosphorylated Ca2+‐calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H2O2‐treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO‐I/R when compared with WT‐I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria‐targeted superoxide scavenger MitoTempo. Our results suggested that Ca2+ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury. [ABSTRACT FROM AUTHOR]

Mitochondria house evolutionarily conserved pathways of carbon and nitrogen metabolism that drive cellular energy production. Mitochondrial bioenergetics is regulated by calcium uptake through the mitochondrial calcium uniporter (MCU), a multi-protein complex whose assembly in the inner mitochondrial membrane is facilitated by the scaffold factor MCUR1. Intriguingly, many fungi that lack MCU contain MCUR1 homologs, suggesting alternate functions. Herein, we characterize Saccharomyces cerevisiae homologs Put6 and Put7 of MCUR1 as regulators of mitochondrial proline metabolism. Put6 and Put7 are tethered to the inner mitochondrial membrane in a large hetero-oligomeric complex, whose abundance is regulated by proline. Loss of this complex perturbs mitochondrial proline homeostasis and cellular redox balance. Yeast cells lacking either Put6 or Put7 exhibit a pronounced defect in proline utilization, which can be corrected by the heterologous expression of human MCUR1. Our work uncovers an unexpected role of MCUR1 homologs in mitochondrial proline metabolism. Although some fungal mitochondria lack the calcium uniporter, many intriguingly encode homologs of the uniporter assembly factor MCUR1. Here, the authors show that in budding yeast, the MCUR1 homologs Put6 and Put7 regulate mitochondrial proline metabolism, a function also conserved in human MCUR1. [ABSTRACT FROM AUTHOR]

The discovery of MICU1 as gatekeeper of mitochondrial calcium (m Ca2+) entry has transformed our understanding of m Ca2+ flux. Recent studies revealed an additional role of MICU1 as a Ca2+ sensor at MICOS (mitochondrial contact site and cristae organizing system). MICU1's presence at MICOS suggests its involvement in coordinating Ca2+ signaling and mitochondrial ultrastructure. Besides its role in Ca2+ regulation, MICU1 influences cellular signaling pathways including transcription, epigenetic regulation, metabolism, and cell death, thereby affecting human health. Here, we summarize recent findings on MICU1's canonical and noncanonical functions, and its relevance to human health and diseases. • MICU1, a mitochondrial Ca2+ sensor, plays a key role in decoding cytoplasmic Ca2+ signals. • Through the mtCU, MICU1 modulates mitochondrial Ca2+ influx. • MICU1 functions extend beyond mtCU, impacting mitochondrial ultrastructure via MICOS. • Loss of MICU1 leads to bioenergetics failure and increased susceptibility to various cell death pathways. • MICU1 mutations are linked to neuromuscular, cognitive, and cardiac impairments. [ABSTRACT FROM AUTHOR]

Summary Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (m Ca2+) uptake, delays cytosolic Ca2+ (c Ca2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid m Ca2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism. Graphical Abstract Highlights • Mitochondrial Ca2+ powers FAO-dependent hepatocyte mitochondrial respiration • Hepatic MCU deletion promotes lipid accumulation and lowers ketone bodies • Blockade of m Ca2+ buffering enhances AMPK dephosphorylation through PP4 • Restoration of AMPK activity in MCUΔhep model improves lipid clearance Hepatic mitochondrial Ca2+ shapes bioenergetics and lipid homeostasis. Tomar et al. demonstrate that MCU-mediated c Ca2+ buffering serves as a crucial step in controlling hepatic fuel metabolism through an MCU/PP4/AMPK molecular cascade. Identification of these molecular signaling events aids in understanding how perturbation of mitochondrial ion homeostasis may contribute to the etiology of metabolic disorders. [ABSTRACT FROM AUTHOR]

Summary Ca 2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca 2+ ] m uptake rate, elevated mROS, and enhanced [Ca 2+ ] m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity. [ABSTRACT FROM AUTHOR]

Summary Calcium (Ca 2+ ) flux into the matrix is tightly controlled by the mitochondrial Ca 2+ uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca 2+ or Mg 2+ destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg 2+ loading or blockade of mitochondrial Ca 2+ extrusion suppresses MCU Ca 2+ -uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg 2+ and Ca 2+ binding. [ABSTRACT FROM AUTHOR]