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Characterization of endothelial argininosuccinate synthase subcellular localization and protein interactions relevant to the function of the citrulline‐nitric oxide cycle
Author(s) -
Corbin Karen Davidowitz,
Pendleton Laura C,
Shriver Sandra K,
Eichler Duane C
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.852.1
Subject(s) - argininosuccinate synthase , microbiology and biotechnology , citrulline , nitric oxide , enos , nitric oxide synthase , nitric oxide synthase type iii , biochemistry , hsp90 , chemistry , biology , argininosuccinate lyase , endothelial nos , arginine , arginase , heat shock protein , amino acid , gene , organic chemistry
Argininosuccinate synthase (AS) is an essential mediator of endothelial health by providing a dedicated source of arginine for nitric oxide (NO) production and promoting endothelial cell viability. Our laboratory has previously demonstrated that AS is present in endothelial caveolar fractions along with endothelial nitric oxide synthase (eNOS) and argininosuccinate lyase (AL), the core components of the citrulline‐NO cycle. To expand on those studies, we utilized immunofluorescence microscopy and showed that AS localized to the Golgi, perinuclear region and plasma membrane in conjunction with eNOS. We also demonstrated that AS co‐localizes with caveolin‐1 and HSP90, key regulators of eNOS function. Co‐immunoprecipitation studies showed that AS interacts with caveolin‐1 and HSP90. In the case of caveolin‐1, a binding motif in the AS protein sequence suggested a direct interaction. To characterize the nitric oxide metabolome from a more global perspective, we utilized co‐immunoprecipitation followed by mass spectrometry to identify several putative AS and eNOS interacting partners that are either novel or understudied in the regulation of NO production. Our work highlights the functional co‐localization and interactions of citrulline‐NO cycle components in vascular endothelial cells, which has important implications for our understanding of vascular biology.