REGULATION OF sGC IN sGC/Hsp90/NOS COMPLEXES

说明(申请人提供)：鸟苷环化酶(SGC)的可溶性异构体是一种在大多数组织中大量存在的异二聚体蛋白质，是主要的“一氧化氮(NO)受体”，负责合成cGMP以响应NO的激活。由于NO的不稳定性质、生物活性NO的数量以及因此，NO-sGC相互作用的程度与sGC和NO合成酶(NOS)产生的NO之间的分子间距离以及竞争配体的存在，特别是超氧化物的存在呈负相关。NO-超氧化物生成的过氧亚硝酸盐是一种高度活性的有毒自由基，负责许多蛋白质的硝化和失活。蛋白质之间的相互作用是一氧化氮合酶活性的重要决定因素，尤其是一氧化氮合酶与普遍存在的热休克蛋白90(Hsp90)的结合大大增强了它的活性。到目前为止，人们一直认为sGC存在于细胞质中，在那里它与其他非配体竞争。与这一假设相反，我们最近发现，sGC存在于内皮细胞和血管内皮细胞中至少包含Hsp90和一氧化氮合酶(eNOS或iNOS)的多蛋白复合体中，并且这种联系在体外和体内都能增强其对内源性NO或硝基血管扩张剂来源的NO的反应活性。这些新发现的Hsp90-sGC复合体为理解NO的多细胞效应提供了新的模型。在拟议的研究中，我们将研究这些相互作用的分子机制，并检验这些新模型代表旨在改善sGC激活、最大化NO-sGC相互作用和减少超氧化物清除NO的生理机制的假设。

DESCRIPTION (provided by applicant): The soluble isoform of guanylate cyclase (sGC) is a heterodimer protein abundant in most tissues and the principal "nitric oxide (NO) receptor", responsible for the synthesis of cGMP in response to activation by NO. Because of the labile nature of NO, the amount of biologically active NO and consequently, the degree of the NO-sGC interaction are inversely related to both the intermolecular distance between sGC and the NO synthase (NOS)-generated NO, as well as the presence of competing ligands, especially superoxide. The NO-superoxide-generated peroxynitrite is a highly reactive, toxic radical responsible for the nitration and deactivation of numerous proteins. Protein-protein interactions are important determinants of NOS activity; in particular, the association of NOS with the ubiquitous heat shock protein 90 (Hsp90) greatly enhances its activity. To date, it has been assumed that sGC exists free in the cytoplasm where it competes with other NO ligands. Contrary to this assumption, we recently discovered that sGC exists in a multi-protein complex involving at least Hsp90 and NOS (eNOS or iNOS), in both endothelial and smooth muscle cells, and that this association enhances its activity in response to either endogenous NO or nitrovasodilator-derived NO, both in vitro and in vivo. This newly discovered Hsp90-sGC complexes provide novel models for understanding the multicellular effects of NO. In the proposed studies, we will investigate the molecular mechanisms of these interactions and test the hypothesis that these new models represent physiological mechanisms aimed at improving sGC activation, maximizing the NO-sGC interaction and at reducing NO scavenging by superoxide.