Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox” led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

尽管细胞内l-精氨酸的浓度达到饱和，但内皮细胞 (EC) 中一氧化氮 (NO) 的产生可以被外源性精氨酸刺激。这种被称为“精氨酸悖论”的现象导致了精氨酸回收途径的发现，其中l-瓜氨酸被回收到l-精氨酸通过利用两种重要的尿素循环酶精氨琥珀酸合成酶 (ASS) 和精氨琥珀酸裂解酶 (ASL)。先前的工作表明，ASL 存在于含有 hsp90 和内皮 NO 合酶 (eNOS) 的 NO 合成复合物中。然而，尚不清楚 hsp90 是否与 ASS 和 ASL 形成功能性复合物，以及它是否参与调节它们的活性。因此，阐明 hsp90 在精氨酸再循环途径中的作用是本研究的目标。我们的数据表明 ASS 和 ASL 都由 hsp90 陪伴。用格尔德霉素 (GA) 抑制 hsp90 活性，降低 ASS 和 ASL 的活性并降低细胞l-牛主动脉内皮细胞（BAEC）中的精氨酸水平。尽管 mRNA 水平没有变化，hsp90 抑制导致 ASS 和 ASL 蛋白的时间依赖性降低。我们通过 E3 泛素连接酶、Hsc70 相互作用蛋白 (CHIP) 的 C 末端和热休克蛋白 hsp70 进一步将这种蛋白质损失与 ASS 和 ASL 的蛋白酶体依赖性降解联系起来。CHIP 的瞬时过表达足以刺激 ASS 和 ASL 降解，而 CHIP 突变蛋白的过表达将 TPR 和 U-box 域识别为 ASS 和 ASL 降解所必需的。该研究提供了对 EC 中l-精氨酸再循环的分子调控的新见解，并将蛋白酶体途径作为刺激 NO 信号传导的可能治疗靶点。