Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU-/- mice have no apparent capacity to rapidly uptake calcium. While basal metabolism appears unaffected, the skeletal muscle of MCU-/- mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU-/- mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU-/- mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not appear to protect MCU-/- cells and tissues from cell death, although MCU-/- hearts fail to respond to the PTP inhibitor cyclosporin A (CsA). Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.

National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust.

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the gene. Finally, we found that the loss of WhiB6 resulted in a virulent strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in are fine-tuned by negative feedback control, linking the expression of the gene to the presence, not the functionality, of the ESX-1 membrane complex.

No abstract

BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5–8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1–4. Furthermore, BRC5–8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5–8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage.

Mitochondrial calcium is thought to play an important role in the regulation of cardiac bioenergetics and function. The entry of calcium into the mitochondrial matrix requires that the divalent cation pass through the inner mitochondrial membrane via a specialized pore known as the mitochondrial calcium uniporter (MCU). Here, we use mice deficient for MCU expression to rigorously assess the role of mitochondrial calcium in cardiac function. Mitochondria isolated from MCU-/- mice have reduced matrix calcium levels, impaired calcium uptake and a defect in calcium-stimulated respiration. Nonetheless, we find that the absence of MCU expression does not affect basal cardiac function at either 12 or 20 months of age. Moreover, the physiological response of MCU-/- mice to isoproterenol challenge or transverse aortic constriction appears similar to control mice. Thus, while mitochondria derived from MCU-/- mice have markedly impaired mitochondrial calcium handling, the hearts of these animals surprisingly appear to function relatively normally under basal conditions and during stress.

Entry of mitochondrial calcium is believed to play an essential role in regulating bioenergetics and initiating cell death pathways. We have recently described a mouse model lacking MCU expression. Surprisingly, these mice are viable and the cells and tissues from these animals do not exhibit any marked protection from cell death. Here, we discuss our findings as well as potential explanations for some of the more unexpected results.

Aims: Nitric oxide (NO) and protein S-nitrosylation (SNO) play important roles in ischemic preconditioning (IPC)-induced cardioprotection. Mitochondria are key regulators of preconditioning, and most proteins showing an increase in SNO with IPC are mitochondrial. The aim of this study was to address how IPC transduces NO/ SNO signaling to mitochondria in the heart. Results: In this study using Langendorff perfused mouse hearts, we found that IPC-induced cardioprotection was blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor), ascorbic acid (a reducing agent to decompose SNO), or methylb-cyclodextrin (MbCD, a cholesterol sequestering agent to disrupt caveolae). IPC not only activated AKT/eNOS signaling but also led to translocation of eNOS to mitochondria. MbCD treatment disrupted caveolar structure, leading to dissociation of eNOS from caveolin-3 and blockade of IPC-induced activation of the AKT/eNOS signaling pathway. A significant increase in mitochondrial SNO was found in IPC hearts compared to perfusion control, and the disruption of caveolae by MbCD treatment not only abolished IPC-induced cardioprotection, but also blocked the IPC-induced increase in SNO. Innovation: These results provide mechanistic insight into how caveolae/eNOS/NO/SNO signaling mediates cardioprotection induced by IPC. Conclusion: Altogether these results suggest that caveolae transduce eNOS/NO/SNO cardioprotective signaling in the heart. Antioxid. Redox Signal. 16, 45-56.