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KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II.

ABSTRACT: Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150?mM potassium chloride (KCl). It is presumed that inclusion of 150?mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II.

SUBMITTER: Suman M

PROVIDER: S-EPMC4884806 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II.

Suman Mishra M Rajnikant Mishra M

Scientifica 20160516

Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of ...[more]

PMID: 27293971

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Project description:AimsArginase II (ArgII) plays a key role in the regulation of Ca2+ between the cytosol and mitochondria in a p32-dependent manner. p32 contributes to endothelial nitric oxide synthase (eNOS) activation through the Ca2+/CaMKII/AMPK/p38MAPK/Akt signalling cascade. Therefore, we investigated a novel function of ArgII in the regulation of p32 stability.Methods and resultsmRNA levels were measured by quantitative reverse transcription-PCR, and protein levels and activation were confirmed by western blot analysis. Ca2+ concentrations were measured by FACS analysis and a vascular tension assay was performed. ArgII bound to p32, and ArgII protein knockdown using siArgII facilitated the ubiquitin-dependent proteasomal degradation of p32. β-lactone, a proteasome inhibitor, inhibited the p32 degradation associated with endothelial dysfunction in a Ca2+-dependent manner. The amino acids Lys154, Lys 180, and Lys220 of the p32 protein were identified as putative ubiquitination sites. When these sites were mutated, p32 was resistant to degradation in the presence of siArgII, and endothelial function was impaired. Knockdown of Pink/Parkin as an E3-ubiquitin ligase with siRNAs resulted in increased p32, decreased [Ca2+]c, and attenuated CaMKII-dependent eNOS activation by siArgII. siArgII-dependent Parkin activation was attenuated by KN93, a CaMKII inhibitor. Knockdown of ArgII mRNA and its gene, but not inhibition of its activity, accelerated the interaction between p32 and Parkin and reduced p32 levels. In aortas of ArgII-/- mice, p32 levels were reduced by activated Parkin and inhibition of CaMKII attenuated Parkin-dependent p32 lysis. siParkin blunted the phosphorylation of the activated CaMKII/AMPK/p38MAPK/Akt/eNOS signalling cascade. However, ApoE-/- mice fed a high-cholesterol diet had greater ArgII activity, significantly attenuated phosphorylation of Parkin, and increased p32 levels. Incubation with siArgII augmented p32 ubiquitination through Parkin activation, and induced signalling cascade activation.ConclusionThe results suggest a novel function for ArgII protein in Parkin-dependent ubiquitination of p32 that is associated with Ca2+-mediated eNOS activation in endothelial cells.

Dataset Information

KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II.

Publications

KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II.

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