Search

Introduction: Mitochondria serve as critical Ca2+ sinks, regulating the duration and magnitude of cCa2+ transients and cCa2+ entry into the mitochondrion. Mitochondrial calcium uniporter (MCU) channel is activated in conditions of inflammation and oxidative stress.MCU and its activity are potential therapeutic targets for cardiac hypertrophy and heart failure. Hypothesis: Blunting of mCa2+ uptake can contribute to increased cCa2+ transients associated with excitation-contraction coupling and offer cardio protection following sepsis induced stress. Methods: MCUC96AKI generated using CRISPR-Cas9d10 nickase mRNA co-injected with gRNA into wild-type(C57BL6) zygotes and then implanted into pseudo-pregnant female mice.To elucidate mechanistic role of mCa2+ in modulating cardiac contractility and myocardial bioenergetics, adult cardiomyocytes were isolated from mouse model and subjected to hypoxia/re-oxygenation injury.Polymicrobial Sepsis in 6-week-old C57BL/6J mice were created using Cecal ligation and puncture (CLP) and MCU oxidation at 1, 6, 12 and 24 hours post injury was compared with Sham and control mice (n=15). MCU protein quantification was done with western blot, functional assessment of Reactive oxygen species (ROS) by confocal laser microscopy, echocardiography with Vevo2100 imaging system. Mice were pretreated with MCU blocking agent Ru360 1 hr prior to CLP and compared with the control group at 6 hours. Results: Cardiomyocytes exposed to hypoxia/re-oxygenation(h/R) stress exhibited increased MitoSOX red fluorescence.MCU oxidation was ubiquitously induced in all tissues post CLP surgery and was most significant at 6 hrs (Fig 1a). Oxidation of MCU was marked by increase in ROS in tissue sections of small Intestine of CLP versus the Sham group(p<0.001). (Fig 1 b &c). Pretreatment with Ru360 ameliorated MCU oxidation at 6 hours post CLP injury. MCUC96AKI had significantly decreased ejection fraction and fractional shortening compared to the wild type mice. Further the survival in MCUC96AKI was significantly better than the WT or MCU KO mice (p<0.05). Conclusions: Inhibition of mitochondrial calcium uniporter is a potential therapeutic target and protects from sepsis induced cardiomyopathy by mitigating mitophagy. [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]

Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-Syn) oligomerization and amyloid formation are associated with Parkinson's disease (PD) pathogenesis. Studying familial α-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our in vitro study demonstrated that A53E attenuated α-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis. [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]