Arginase II protein regulates Parkin-dependent p32 degradation that contributes to Ca2+-dependent eNOS activation in endothelial cells

Abstract

Aims Arginase 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 signaling cascade. Therefore, we investigated a novel function of ArgII in the regulation of p32 stability. Methods and Results mRNA levels were measured by qRT-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 signaling 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 signaling cascade activation. Conclusion The 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. Translational Perspective In many vascular disorders, the downregulation of ArgII has been shown to be beneficial. This enzyme plays a crucial role in the regulation Ca2+ concentrations in a p32-dependent manner and activates the eNOS activation signaling cascade. In this study, we discovered that ArgII downregulation, inhibition of its activity, and gene knockout/down, induced the activation of Parkin (an E3-ubiquitin ligase) through a CaMKII-dependent mechanism. ArgII protein, as a p32 binding partner, prevented Parkin-dependent p32 ubiquitination, but inhibition of ArgII activity had no effect on ubiquitination. These novel findings have the potential to be translated into future therapeutic strategies to treat vascular diseases.

Arginase: shedding light on the mechanisms and opportunities in cardiovascular diseases

“…Moreover, decreased bioavailability of L-arginine also induces NOS-uncoupling, which produces superoxide anion instead of NO, and the increased oxidative stress further exacerbates NOS-uncoupling [ 5 ], and aggravates NO inactivation. Furthermore, ARG2 promotes Ca 2+ -dependent eNOS phosphorylation through parkin-dependent p32 degradation [ 30 ]. As a result, an imbalanced increase in arginase in the cardiovascular system, particularly in ECs, will limit NO production and promote superoxide formation, eventually leading to vascular endothelial dysfunction.…”

Regulation of Glucose, Fatty Acid and Amino Acid Metabolism by Ubiquitination and SUMOylation for Cancer Progression

“…Under hypoxic conditions, SENP1-mediated deSUMOylation of KLF15 leads to the translocation of KLF15 from the nucleus to the cytoplasm, which induces the expression of Arg2 ( Pandey et al, 2018 ). Arg2 was recently shown to regulate Parkin-dependent p32 degradation, promoting Ca 2+ -dependent eNOS activation ( Koo et al, 2021 ).…”

CD62E- and ROS-Responsive ETS Improves Cartilage Repair by Inhibiting Endothelial Cell Activation through OPA1-Mediated Mitochondrial Homeostasis

“…Studies have shown that various intracellular signaling pathways, including nuclear factor κB (NF-κB), mitogen-activated kinase kinase 1 (MEK1), and mitogen-activated protein kinase (MAPK), regulate VEC activation [ 14 – 16 ]. Recent studies have also indicated that intracellular second messengers, such as reactive oxygen species (ROS) and Ca 2+ , which are regulated by mitochondria, play a role in mediating VEC activation, proliferation, migration, and gene expression under various angiogenic stimuli [ 17 , 18 ]. Opa1 (Optic atrophy protein 1), located in the inner membrane of mitochondria, mediates mitochondrial inner fusion and is critical for promoting the stability of the respiratory chain complex, regulating ROS production and mitochondrial Ca 2+ influx, and maintaining cytoplasmic homeostasis [ 19 , 20 ].…”