Mitochondrial Ca2+ (mCa2+) uptake mediated by the mitochondrial calcium uniporter (MCU) plays a critical role in signal transduction, bioenergetics, and cell death, and its dysregulation is linked to several human diseases. In this study, we report a new ruthenium complex Ru265 that is cell-permeable, minimally toxic, and highly potent with respect to MCU inhibition. Cells treated with Ru265 show inhibited MCU activity without any effect on cytosolic Ca2+ dynamics and mitochondrial membrane potential (ΔΨm). Dose-dependent studies reveal that Ru265 is more potent than the currently employed MCU inhibitor Ru360. Site-directed mutagenesis of Cys97 in the N-terminal domain of human MCU ablates the inhibitory activity of Ru265, suggesting that this matrix-residing domain is its target site. Additionally, Ru265 prevented hypoxia/reoxygenation injury and subsequent mitochondrial dysfunction, demonstrating that this new inhibitor is a valuable tool for studying the functional role of the MCU in intact biological models.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

In this mini-review we provide an overview of sex-and gender-dependent issues in both clinical and preclinical sepsis. The increasing recognition for the need to account for sex and gender in biomedical research brings a unique set of challenges and requires researchers to adopt best practices when conducting and communicating sex-and gender-based research. This may be of particular importance in sepsis, given the potential contribution of sex bias in the failures of translational sepsis research in adults and neonates. Clinical evidence of sex-dependent differences in sepsis is equivocal. Since clinical studies are limited to observational data and confounded by a multitude of factors, preclinical studies provide a unique opportunity to investigate sex differences in a controlled, experimental environment. Numerous preclinical studies have suggested that females may experience favorable outcomes in comparison with males. The underlying mechanistic evidence for sex-dependent differences in sepsis and other models of shock (e.g., trauma-hemorrhage) largely centers around the beneficial effects of estrogen. Other mechanisms such as the immunosuppressive role of testosterone and Xlinked mosaicism are also thought to contribute to observed sex-and gender-dependent differences in sepsis. Significant knowledge gaps still exist in this field. Future investigations can address these gaps through careful consideration of sex and gender in clinical studies, and the use of clinically accurate preclinical models that reflect sex differences. A better understanding of sex-and gender-dependent differences may serve to increase translational research success.

Supplemental Digital Content is available in the text.

Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.

Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca 2+) sensing proteins that, together with Orai1-composed Ca 2+ channels on the plasma membrane (PM), regulate intracellular Ca 2+ levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca 2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca 2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca 2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-nitrosylation causes structural perturbations that converge on the face of the ef-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic ca 2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca 2+ and Soce regulation. Stromal-interaction molecules (STIM)s are endoplasmic reticulum (ER) membrane-inserted calcium (Ca 2+) sensors that respond to fluctuations in luminal stored Ca 2+ levels 1,2. In Homo sapiens, two STIM homologs exist: stromal interaction molecule−1 (STIM1) and −2 (STIM2) 3. Upon ER luminal Ca 2+ store depletion, human STIM1 undergoes structural changes that promote oligomerization and translocation to ER-plasma membrane (PM) junctions 4-6. At these junctions, STIM1 interacts with Orai1 proteins, which are the pore-forming subunits of Ca 2+ release activated Ca 2+ (CRAC) channels 7-9. The direct interaction of STIM1 with Orai1 facilitates gating of the CRAC channels to induce the influx of extracellular Ca 2+ into the cytosol 10-12 , otherwise known as store operated Ca 2+ entry (SOCE) 13,14. The human STIM2 paralog similarly regulates SOCE; however, STIM2 is less efficient than STIM1 in this cellular process 15-18. Instead, STIM2 is more intimately involved in the regulation of basal Ca 2+ levels 15. Interestingly, in lower order eukaryotes such as Drosophila melanogaster and Caenorhabditis elegans, only one STIM gene product has been identified 3. The highly conserved EF-hand and sterile α-motif (EF-SAM) domains of STIMs are the core luminal protein machinery that sense ER Ca 2+ changes 17,19,20 , while the cytosolic STIM-Orai1-activating-region (SOAR) coiled-coils are the highly conserved cytosolic domains that couple to and open Orai1 channels 11,12. Compared

No abstract

Maternal cigarette smoking is a risk factor for congenital heart defects (CHDs). Nicotine replacement therapies are often offered to pregnant women following failed attempts of smoking cessation. However, the impact of nicotine on embryonic heart development is not well understood. In the present study, the effects of maternal nicotine exposure (MNE) during pregnancy on foetal heart morphogenesis were studied. Adult female mice were treated with nicotine using subcutaneous osmotic pumps at 0.75 or 1.5 mg/kg/day and subsequently bred with male mice. Our results show that MNE dose‐dependently increased CHDs in foetal mice. CHDs included atrial and ventricular septal defects, double outlet right ventricle, unguarded tricuspid orifice, hypoplastic left ventricle, thickened aortic and pulmonary valves, and ventricular hypertrophy. MNE also significantly reduced coronary artery size and vessel abundance in foetal hearts. Moreover, MNE resulted in higher levels of oxidative stress and altered the expression of key cardiogenic regulators in the developing heart. Nicotine exposure reduced epicardial‐to‐mesenchymal transition in foetal hearts. In conclusion, MNE induces CHDs and coronary artery malformation in mice. These findings provide insight into the adverse outcomes of foetuses by MNE during pregnancy.