Molecular Mechanisms of PDE5 Regulation

PDE5 调控的分子机制

• 批准号: 6889205
• 负责人: JACKIE David CORBIN
• 金额: $ 32.28万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6889205
• 关键词: 3' 5' cyclic nucleotide phosphodiesterase; X ray crystallography; allosteric site; animal tissue; conformation; cyclic GMP; enzyme mechanism; enzyme structure; gene mutation; infrared spectrometry; nucleotide analog; phosphodiesterase inhibitors; phosphoprotein phosphatase; phosphorylation; protein kinase; protein sequence; structural biology; tissue /cell culture; vascular smooth muscle

DESCRIPTION (Applicant's abstract): Interest in mammalian cGMP cascades has recently been intense. Much research in this area has centered on the signal receptors and guanylyl cyclases that modulate cGMP formation, as well as on cellular actions of cGMP. Natural signals (natriuretic peptides, guanylins and nitric oxide), in addition to many medications, stimulate cGMP cascades by activating these cyclases. Cyclic nucleotide phosphodiesterases (PDEs), which are also highly modulated enzymes, participate in regulating cellular cAMP and cGMP levels by catalyzing breakdown of cAMP and cGMP. The subject of this application is the cGMP-binding cGMP-specific PDE (PDE5), which is specific for cGMP over cAMP at its allosteric cGMP-binding sites and at its catalytic site. The long-term goal is to determine the mechanism of action and cellular regulation of PDE5, a major determinant of cGMP level in many tissues. Classical effects of cGMP include relaxation of smooth muscle, inhibition of platelet activation, neutrophil degranulation, and mediation of vision. New discoveries have expanded this list to include regulation of gene expression, chloride transport in intestine and kidney, watertransport, bone resorption, melanogenesis in skin, long-term nerve depression, and opioid effects. Therapeutic agents that elevate cGMP include PDE inhibitors (e.g., caffeine, papaverine, and sildenafil) and nitrovasodilators (e.g., nitroglycerin). Certain enterotoxins cause secretory diarrhea by elevating cGMP. Dr. Corbin recently demonstrated that in some instances cGMP acts by "crossactivating" cAMP receptors. However, PDE5 is a specific intracellular receptor for cGMP. The present proposal represents a thorough biochemical and physiological investigation of PDE5 regulation. This enzyme is the specific target of sildenafil (Viagra.), which is used in treatment of male erectile dysfunction associated with diabetes, aging, spinal cord injuries and other pathologies. Effects of phosphorylation of PDE5 by cyclic nucleotide-dependent protein kinases on catalysis and allosteric cGMP binding will be explored, and effects of mutating the phosphorylation site, Ser- 102 (human), to Ala, Glu, and Asp will be examined. The principal investigator will study the PDE5 regulatory domain structure and function by expressing truncation mutants that include various combinations of the two allosteric cUMP-binding sites and the phosphorylation site of the enzyme but exclude the catalytic domain. Small angle x-ray scattering and Fourier transformed infra-red spectroscopy will be used to study effects of cGMP and phosphorylation on conformation of these domains. Crystallography of certain truncated regulatory domains will be attempted. The specificity and efficacy with which phosphoprotein phosphatases of vascular smooth muscle extracts catalyze dephosphorylation of Ser-102 will be investigated. Short-term regulation of PDE5 in intact vascular smooth muscle by cGMP analogs, phosphoprotein phosphatase inhibitors, and agents that elevate cGMP will be studied. The possibility that these agents could alter enzyme activity, subcellular localization, or cGMP levels (feedback mechanism) will be examined. Potential non-covalent modulators of PDE5 other than cGMP will be sought. The possibility that PDE5 allosteric sites represent a sequestration site for cGMP in cells will be explored. Results of these studies will provide a basis for understanding fundamental questions relating to cGMP signaling in many tissues.

描述（申请人摘要）：对哺乳动物cGMP级联反应的兴趣 最近很紧张这一领域的许多研究都集中在信号上 受体和鸟苷酸环化酶，调节cGMP的形成，以及 cGMP的细胞作用。天然信号（利钠肽、鸟苷酸和 一氧化氮），除了许多药物，刺激cGMP级联反应， 激活这些环化酶。环核苷酸磷酸二酯酶（PDE）， 也是高度调节的酶，参与调节细胞cAMP， 通过催化cAMP和cGMP的分解来提高cGMP水平。主题的本 应用是cGMP结合cGMP特异性PDE（PDE 5），其特异于 在其别构cGMP结合位点和催化位点，cGMP超过cAMP。 长期的目标是确定作用机制和细胞 PDE 5是许多组织中cGMP水平的主要决定因素。 cGMP的经典作用包括松弛平滑肌、抑制平滑肌细胞增殖。 血小板活化、中性粒细胞脱粒和调节视力。新 发现已经将该列表扩展到包括基因表达的调节， 肠和肾中的氯化物转运、水转运、骨吸收， 皮肤中的黑素生成、长期神经抑制和阿片样物质作用。 升高cGMP的治疗剂包括PDE抑制剂（例如，咖啡因， 罂粟碱和西地那非）和硝基血管扩张剂（例如，硝化甘油）。 某些肠毒素通过升高cGMP引起分泌性腹泻。Corbin博士 最近证明，在某些情况下，cGMP通过“交叉激活” cAMP受体。然而，PDE 5是cGMP的特异性细胞内受体。 目前的建议代表了一个彻底的生物化学和生理学 研究PDE 5调节。这种酶是 西地那非（伟哥），用于治疗男性勃起功能障碍 与糖尿病、衰老、脊髓损伤和其他病理学相关。 环核苷酸依赖蛋白对磷酸化PDE 5的影响 将探索激酶对催化和变构cGMP结合的影响， 将磷酸化位点Ser- 102（人）突变为Ala、Glu和Asp 将被审查。主要研究者将研究PDE 5监管 结构域的结构和功能，通过表达截短突变体，包括 两个别构cUMP结合位点和两个别构cUMP结合位点的各种组合可以被用于治疗癌症。 酶的磷酸化位点，但不包括催化结构域。小 角X射线散射和傅里叶变换红外光谱将是 用于研究cGMP和磷酸化对这些构象的影响 域.某些截短的调节结构域的晶体学将被 企图。磷蛋白磷酸酶的特异性和功效 的血管平滑肌提取物催化Ser-102的去磷酸化， 追究完整血管平滑肌中PDE 5的短期调节 通过cGMP类似物、磷蛋白磷酸酶抑制剂和提高 将研究cGMP。这些物质改变酶的可能性 活性、亚细胞定位或cGMP水平（反馈机制）将被 考察除了cGMP之外的潜在的PDE 5非共价调节剂将是 寻找。PDE 5变构位点代表螯合的可能性 将探索细胞中cGMP的位点。这些研究的结果将提供 理解与cGMP信号传导有关的基本问题的基础， 许多组织。