Characterization of AS Regulatory Protein, a Novel Protein Implicated in Endothelial Stress Response

Abstract

The citrulline‐nitric oxide cycle is a tightly regulated system for the production of nitric oxide (NO) in endothelial cells. The cycle consists of the proteins endothelial nitric oxide synthase (eNOS), argininosuccinate synthase (AS), and argininosuccinate lyase (AL). AS and AL serve to recycle citrulline, a product of the production of NO, to arginine, the principle substrate for eNOS. AS regulatory protein (ARP) is a small protein encoded by an upstream open reading frame in minor expressed AS mRNAs. Overexpression of ARP suppressed AS expression and NO production in endothelial cells while selective knockdown of the longer AS transcripts that produce ARP resulted in increased AS protein expression and NO production. To investigate the subcellular localization of ARP, a custom polyclonal antibody was generated. Fluorescence microscopy studies demonstrated that ARP is expressed throughout the cytoplasm but that expression seems predominant in the perinuclear endoplasmic reticulum and golgi, co‐localizing with both eNOS and AS. Co‐immunostaining for ARP and TIA‐1, a marker for stress granule formation, demonstrated that ARP moves to stress granules when stress is induced artificially by sodium arsenite in endothelial cells. This data demonstrates that ARP may play a role in the sequestration of mRNAs under conditions of stress. (Supported by the James & Esther King Biomedical Research Program Grant).