Regulation of Retinal cGMP-Phosphodiesterases

项目概要/摘要 这项研究计划的长期目标是阐明分子机制 在视杆细胞中cGMP-磷酸二酯酶（PDE 6）的功能和调节的基础， 视锥细胞作为光传导级联反应中的中心效应酶， PDE 6对感光细胞生理学和存活至关重要。严重视网膜疾病 包括视网膜色素变性、色盲和先天性利伯黑蒙， 编码PDE 6亚基或芳香烃受体的基因中的致病性突变- 相互作用蛋白样1（AIPL 1），一种专门的PDE 6分子伴侣。尽管我们先进的 由于对PDE 6在光转导中的调节的了解，对PDE 6的折叠知之甚少。 以及这种酶的成熟，而拟议的研究旨在填补这一空白。我们 有证据表明，AIPL 1与普遍存在的伴侣蛋白HSP 90形成复合物， 这是PDE 6成熟为功能酶所必需的。我们将定义 蛋白质-蛋白质界面和溶液中HSP 90 β/AIPL 1复合物的结构， 获得关于这种分子伴侣复合物如何与PDE 6相互作用的机制见解。基于 在我们的初步研究中，我们假设新生的PDE 6假设一个封闭的 非功能状态，以及HSP 90/AIPL 1复合物和PDE 6的调节性β亚基 诱导和/或催化PDE 6从闭合状态转变为功能性开放状态 通过中间状态。我们提出了一个详细的模型，如何HSP 90，AIPL 1 和P β促进PDE 6的成熟，并将通过以下方式严格询问该模型： 利用我们的异源表达系统在HEK 293 T细胞中表达锥体PDE 6C， 在Pde 6 g-/-/Pde 6ccpfl 1小鼠模型中的杆PDEAB。此外，我们将使用HSP 90 敲除小鼠，突出了HSP 90在感光细胞中的重要性 由于其快速视网膜变性表型，为了验证我们的假设， PDE 6成熟是退行性表型的关键原因。阐明 PDE 6成熟的分子机制将对视网膜病变有重要意义。 疾病和设计新的治疗策略。

Project summary/Abstract The long-term goal of this research program is to elucidate the molecular mechanisms underlying the function and regulation of cGMP-phosphodiesterases (PDE6) in rod and cone photoreceptor cells. As a central effector enzyme in the phototransduction cascade, PDE6 is critical to photoreceptor cell physiology and survival. Severe retinal diseases including retinitis pigmentosa, achromatopsia, and Leber congenital amaurosis result from pathogenic mutations in genes that encode PDE6 subunits or aryl hydrocarbon receptor- interacting protein-like 1 (AIPL1), a specialized PDE6 chaperone. Despite our advanced understanding of the PDE6 regulation in phototransduction, little is known about the folding and maturation of this enzyme, and the proposed research aims to fill this gap. Our evidence suggests that AIPL1 forms a complex with ubiquitous chaperone HSP90 that is absolutely required for maturation of PDE6 into a functional enzyme. We will define the protein-protein interfaces and the structure of the HSP90/AIPL1 complex in solution to gain mechanistic insights into how this chaperone complex interacts with PDE6. Based on our preliminary studies, we hypothesize that nascent PDE6 assumes a closed nonfunctional state, and the HSP90/AIPL1 complex and the regulatory P-subunit of PDE6 induce and/or catalyze the transition of PDE6 from the closed state to the functional open state via an intermediate state. We propose a detailed model as to how HSP90, AIPL1 and P facilitate the maturation of PDE6 and will rigorously interrogate this model by leveraging our heterologous expression system for cone PDE6C in HEK293T cells, and rod PDEAB in the Pde6g-/-/Pde6ccpfl1 mouse model. In addition, we will use the HSP90 knockout mouse, which highlights the importance of HSP90 in photoreceptor cells because of its rapid retinal degeneration phenotype, to test our hypothesis that defects in PDE6 maturation are the key cause of the degenerative phenotype. Elucidation of the molecular mechanisms of PDE6 maturation will have important implications for retinal diseases and the design of new therapeutic strategies.