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Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium.

ABSTRACT: BACKGROUND:Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, from cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. This calcium buffering system involves the participation of inorganic phosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how calcium-orthophosphate interactions, which normally lead to formation of insoluble precipitates, are capable to dynamically regulate free calcium concentration. Here we test the hypothesis that inorganic polyphosphate, which is a polymerized form of orthophosphate, is capable to from soluble complexes with calcium, playing a significant role in the regulation of the mitochondrial free calcium concentration. METHODS:We used confocal fluorescence microscopy to measure the relative levels of mitochondrial free calcium in cultured hepatoma cells (HepG2) with variable levels of inorganic polyphosphate (polyP). RESULTS:The depletion of polyP leads to the significantly lower levels of mitochondrial free calcium concentration under conditions of pathological calcium overload. These results are coherent with previous observations showing that inorganic polyphosphate (polyP) can inhibit calcium-phosphate precipitation and, thus, increase the amount of free calcium. CONCLUSIONS:Inorganic polyphosphate plays an important role in the regulation of mitochondrial free calcium, leading to its significant increase. GENERAL SIGNIFICANCE:Inorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system.

SUBMITTER: Solesio ME

PROVIDER: S-EPMC7155424 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium.

Solesio M E ME Demirkhanyan L L Zakharian E E Pavlov E V EV

Biochimica et biophysica acta 20160316 6

<h4>Background</h4>Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, from cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. This calcium buffering system involves the ...[more]

PMID: 26994920

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Project description:The Mycobacterium tuberculosis gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, MIC of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for M. tuberculosis virulence was assessed in the murine model. The ppk2::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT. Naive ppk2::Tn mutant-infected macrophages showed increased expression of interleukin 2 (IL-2), IL-9, IL-10, IL-12p70, and gamma interferon (IFN-?) relative to WT-infected macrophages. The ppk2::Tn mutant exhibited significantly lower lung CFU during acute murine infection compared to the control groups. ppk2 is required for control of intrabacillary poly P levels and optimal M. tuberculosis growth and survival in macrophages and mouse lungs. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a highly successful human pathogen because it has developed mechanisms to multiply and survive in the lungs by circumventing the immune system. Identification of virulence factors responsible for M. tuberculosis growth and persistence in host tissues may assist in the development of novel strategies to treat TB. In this study, we found that the mycobacterial enzyme polyphosphate kinase 2 (PPK2) is required for controlling intracellular levels of important regulatory molecules and for maintaining susceptibility to the first-line anti-TB drug isoniazid. In addition, PPK2 was found to be required for M. tuberculosis growth in the lungs of mice, at least in part by suppressing the expression of certain key cytokines and chemokines by inactivated lung macrophages.

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Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium.

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Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium.

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