Post-translational modifications and protein quality control of mitochondrial channels and transporters

Mitochondria play a critical role in energy metabolism and signal transduction, which is tightly regulated by proteins, metabolites, and ion fluxes. Metabolites and ion homeostasis are mainly mediated by channels and transporters present on mitochondrial membranes. Mitochondria comprise two distinct compartments, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM), which have differing permeabilities to ions and metabolites. The OMM is semipermeable due to the presence of non-selective molecular pores, while the IMM is highly selective and impermeable due to the presence of specialized channels and transporters which regulate ion and metabolite fluxes. These channels and transporters are modulated by various post-translational modifications (PTMs), including phosphorylation, oxidative modifications, ions, and metabolites binding, glycosylation, acetylation, and others. Additionally, the mitochondrial protein quality control (MPQC) system plays a crucial role in ensuring efficient molecular flux through the mitochondrial membranes by selectively removing mistargeted or defective proteins. Inefficient functioning of the transporters and channels in mitochondria can disrupt cellular homeostasis, leading to the onset of various pathological conditions. In this review, we provide a comprehensive overview of the current understanding of mitochondrial channels and transporters in terms of their functions, PTMs, and quality control mechanisms

The leucine aminopeptidase of Staphylococcus aureus is secreted and contributes to biofilm formation

SummaryBackgroundStaphylococcus aureus has emerged as a major drug-resistant pathogen in hospital- and community-acquired infections. Leucine aminopeptidase (LAP) is known to be essential for survival of the bacteria; however the LAP of S. aureus has not been extensively characterized. In this study, we report a detailed characterization of the S. aureus LAP.MethodsLAP from S. aureus was cloned, purified, and further biochemically characterized. The expression of LAP was analyzed by Western blotting. Growth and biofilm formation were analyzed spectrophotometrically.ResultsLAP was cloned from S. aureus and expressed as a 55 kDa protein, whereas the molecular weight of the native protein is approximately 600 kDa. LAP showed amidolytic activity against l-leucine p-nitroanilide. Optimal activity was observed at pH 8.5 and 37°C with a Vmax of 2500μmol/min/mg protein. LAP enzymatic activity was inhibited by ion chelators and enhanced by divalent metal ions, specifically Ni. LAP is secreted by laboratory as well as clinical strains. Bestatin, an inhibitor of LAP, inhibits S. aureus growth and biofilm formation.ConclusionsTo our knowledge, this is the first detailed characterization of LAP from S. aureus and suggests its importance in survival and pathogenesis

Mitochondrial calcium exchange links metabolism with the epigenome to control cellular differentiation.

Fibroblast to myofibroblast differentiation is crucial for the initial healing response but excessive myofibroblast activation leads to pathological fibrosis. Therefore, it is imperative to understand the mechanisms underlying myofibroblast formation. Here we report that mitochondrial calcium (mCa2+) signaling is a regulatory mechanism in myofibroblast differentiation and fibrosis. We demonstrate that fibrotic signaling alters gating of the mitochondrial calcium uniporter (mtCU) in a MICU1-dependent fashion to reduce mCa2+ uptake and induce coordinated changes in metabolism, i.e., increased glycolysis feeding anabolic pathways and glutaminolysis yielding increased α-ketoglutarate (αKG) bioavailability. mCa2+-dependent metabolic reprogramming leads to the activation of αKG-dependent histone demethylases, enhancing chromatin accessibility in loci specific to the myofibroblast gene program, resulting in differentiation. Our results uncover an important role for the mtCU beyond metabolic regulation and cell death and demonstrate that mCa2+ signaling regulates the epigenome to influence cellular differentiation

Small conductance calcium-activated potassium current and the mechanism of atrial arrhythmia in mice with dysfunctional melanocyte-like cells

BACKGROUND: The melanin synthesis enzyme dopachrome tautomerase (Dct) regulates intracellular Ca(2+) in melanocytes. Homozygous Dct knockout (Dct(-/-)) adult mice are vulnerable to atrial arrhythmias (AA). OBJECTIVE: The purpose of this study was to determine whether apamin-sensitive small conductance Ca(2+)-activated K(+) (SK) currents are upregulated in Dct(-/-) mice and contribute to AA. METHODS: Optical mapping was used to study the membrane potential of the right atrium in Langendorff perfused Dct(-/-) (n = 9) and Dct(+/-) (n = 9) mice. RESULTS: Apamin prolonged action potential duration (APD) by 18.8 ms (95% confidence interval [CI] 13.4-24.1 ms) in Dct(-/-) mice and by 11.5 ms (95% CI 5.4-17.6 ms) in Dct(+/-) mice at a pacing cycle length of 150 ms (P = .047). The pacing cycle length threshold to induce APD alternans was 48 ms (95% CI 34-62 ms) for Dct(-/-) mice and 21 ms (95% CI 12-29 ms) for Dct(+/-) mice (P = .002) at baseline, and it was 35 ms (95% CI 21-49 ms) for Dct(-/-) mice and 22 ms (95% CI 11-32 ms) for Dct(+/-) mice (P = .025) after apamin administration. Apamin prolonged post-burst pacing APD by 8.9 ms (95% CI 3.9-14.0 ms) in Dct(-/-) mice and by 1.5 ms (95% CI 0.7-2.3 ms) in Dct(+/-) mice (P = .005). Immunoblot and quantitative polymerase chain reaction analyses showed that protein and transcripts levels of SK1 and SK3 were increased in the right atrium of Dct(-/-) mice. AA inducibility (89% vs 11%; P = .003) and duration (281 seconds vs 66 seconds; P = .008) were greater in Dct(-/-) mice than in Dct(+/-) mice at baseline, but not different (22% vs 11%; P = 1.00) after apamin administration. Five of 8 (63%) induced atrial fibrillation episodes in Dct(-/-) mice had focal drivers. CONCLUSION: Apamin-sensitive SK current upregulation in Dct(-/-) mice plays an important role in the mechanism of AA

Inhibition of NADPH oxidase 2 (NOX2) prevents sepsis-induced cardiomyopathy by improving calcium handling and mitochondrial function

Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increased cardiac oxidative stress and mitochondrial dysfunction have been observed during sepsis, but the mechanisms responsible for these abnormalities have not been determined. We hypothesized that NADPH oxidase 2 (NOX2) activation could be responsible for sepsis-induced oxidative stress and cardiomyopathy. Treatment of isolated adult mouse cardiomyocytes with low concentrations of the endotoxin lipopolysaccharide (LPS) increased total cellular reactive oxygen species (ROS) and mitochondrial superoxide. Elevated mitochondrial superoxide was accompanied by depolarization of the mitochondrial inner membrane potential, an indication of mitochondrial dysfunction, and mitochondrial calcium overload. NOX2 inhibition decreased LPS-induced superoxide and prevented mitochondrial dysfunction. Further, cardiomyocytes from mice with genetic ablation of NOX2 did not have LPS-induced superoxide or mitochondrial dysfunction. LPS decreased contractility and calcium transient amplitude in isolated cardiomyocytes, and these abnormalities were prevented by inhibition of NOX2. LPS decreased systolic function in mice, measured by echocardiography. NOX2 inhibition was cardioprotective in 2 mouse models of sepsis, preserving systolic function after LPS injection or cecal ligation and puncture (CLP). These data show that inhibition of NOX2 decreases oxidative stress, preserves intracellular calcium handling and mitochondrial function, and alleviates sepsis-induced systolic dysfunction in vivo. Thus, NOX2 is a potential target for pharmacotherapy of sepsis-induced cardiomyopathy

MIRO-1 Determines Mitochondrial Shape Transition upon GPCR Activation and Ca^(2+) Stress

Mitochondria shape cytosolic calcium ([Ca^(2+)]_c) transients and utilize the mitochondrial Ca_2^+ ([Ca^(2+)]_m) in exchange for bioenergetics output. Conversely, dysregulated [Ca^(2+)]_c causes [Ca^(2+)]_m overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca^(2+) uptake exhibited elevated [Ca^(2+)]_c and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca^(2+)-induced shape change that is distinct from mitochondrial fission and swelling. [Ca^(2+)]_c elevation, but not MCU-mediated Ca^(2+) uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca^(2+)-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro1 is a cytosolic Ca^(2+) sensor that decodes metazoan Ca^(2+) signals as MiST

MIRO-1 Determines Mitochondrial Shape Transition upon GPCR Activation and Ca^(2+) Stress

Mitochondria shape cytosolic calcium ([Ca^(2+)]_c) transients and utilize the mitochondrial Ca_2^+ ([Ca^(2+)]_m) in exchange for bioenergetics output. Conversely, dysregulated [Ca^(2+)]_c causes [Ca^(2+)]_m overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca^(2+) uptake exhibited elevated [Ca^(2+)]_c and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca^(2+)-induced shape change that is distinct from mitochondrial fission and swelling. [Ca^(2+)]_c elevation, but not MCU-mediated Ca^(2+) uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca^(2+)-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro1 is a cytosolic Ca^(2+) sensor that decodes metazoan Ca^(2+) signals as MiST

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery