Photoreceptor Phosphodiesterase Regulation

项目摘要/摘要 光感受器磷酸二酯酶(PDE6)是视觉信号通路的中枢酶。精确 对其激活和失活的调节对于杆和锥的速度、灵敏度和恢复是至关重要的 光感受器对光照的反应。视杆细胞PDE6基因和视锥细胞PDE6基因的遗传突变与多种 视网膜疾病，包括视网膜色素变性、先天性静止性夜盲和视锥细胞营养不良。 下一代测序正在识别越来越多的PDE6基因突变，其中绝大多数 其临床意义仍不确定。关于视网膜的分子病因更是知之甚少。 致病突变。Rod PDE6由两个催化亚基组成，它们的活性在黑暗中被抑制- 通过结合两个相同的γ亚基而适应状态(Pγ)。在光诱导的视觉信号激活时 途径，PDE6的活性是通过结合异源三聚体G蛋白，转导蛋白来刺激的。光的寿命- 激活的PDE6由转导蛋白对其结合的GTP的水解率精确控制，这是一个过程 受RGS9-1(G蛋白信号转导调节因子9-1)控制。而参与PDE6调控的蛋白质 在光转导过程中，已经确定了PDE6动态发生的分子序列 在PDE6激活和失活过程中与其结合伙伴相互作用--以及它的变构调节 人们对此仍然知之甚少。直到我们了解了正常情况下PDE6调节的机制基础 光转导，我们将在开发治疗措施方面受到阻碍，这些疾病是由 PDE6或其结合伙伴的缺陷会导致视网膜退行性疾病和视力障碍。 本应用程序的总体目标是了解伴随PDE6的事件的顺序 复苏过程中转导蛋白的激活和RGS9-1等蛋白的失活 PDE6的暗适应状态。我们的实验计划是基于这样的假设：抑制性P-γ亚单位 是“主调节器”，负责调节体内发生的多种变构相互作用。 PdE6催化二聚体，以及转导蛋白α亚基和RGS9-1。我们提出了两个具体目标， 将(1)描述转导蛋白α亚基与PDE6催化和 抑制P-γ亚基，以提供一个全面的杆状PDE6激活模型，以及(2)确定 RGS9-1结合的PDE6失活复合体的分子结构和结构 Pγ亚基的重排加速了激活的PDE6的终止。这样做的结果是 通过加强我们的研究来推进国家眼科研究所视网膜疾病计划的目标 预测光转导蛋白突变的致病性的能力，从而使 视信号失调性视网膜疾病的个体化治疗干预 视网膜视杆细胞和视锥感光细胞的通路。

PROJECT SUMMARY/ABSTRACT Photoreceptor phosphodiesterase (PDE6) is the central enzyme of the visual signaling pathway. Precise regulation of its activation and deactivation is essential for the speed, sensitivity, and recovery of rod and cone photoreceptors to illumination. Inherited mutations in rod and cone PDE6 genes have been linked in a variety of retinal diseases, including retinitis pigmentosa, congenital stationary night blindness, and cone dystrophy. Next-generation sequencing is identifying a growing number of mutations in PDE6 genes, the large majority of which remain of uncertain clinical significance. Even less is known about the molecular etiology of retinal disease-causing mutations. Rod PDE6 consists of two catalytic subunits whose activity is inhibited in the dark- adapted state by binding of two identical γ-subunits (Pγ). Upon light-induced activation of the visual signaling pathway, PDE6 activity is stimulated by binding of the heterotrimeric G-protein, transducin. The lifetime of light- activated PDE6 is precisely controlled by the rate at which the transducin hydrolyzes its bound GTP, a process controlled by RGS9-1 (Regulator of G-protein Signaling9-1). While the proteins involved in regulation of PDE6 during phototransduction have been identified, the molecular sequence of events in which PDE6 dynamically interacts with its binding partners--as well as its allosteric regulation--during PDE6 activation and deactivation remain poorly understood. Until we understand the mechanistic basis of PDE6 regulation during normal phototransduction, we will be hampered in developing therapeutic interventions for those diseases arising from defects in PDE6 or its binding partners that result in retinal degenerative diseases and visual disorders. The overall objective of this application is to understand the sequence of events accompanying PDE6 activation by transducin and its subsequent inactivation by RGS9-1 and other proteins during recovery of PDE6 to its dark-adapted state. Our experimental plan is based on the hypothesis that the inhibitory Pγ subunit of PDE6 is the “master regulator” responsible for mediating multiple allosteric interactions that occur within the PDE6 catalytic dimer, as well as with the transducin α-subunit and RGS9-1. We propose two specific aims that will (1) delineate the sequence of binding interactions between transducin α-subunits and PDE6 catalytic and inhibitory Pγ subunits to provide a comprehensive model of rod PDE6 activation, and (2) determine the molecular architecture of the PDE6 inactivation complex upon RGS9-1 binding and the structural rearrangements of the Pγ subunit that accelerate termination of activated PDE6. The outcomes of this research advance the goals of the Retinal Diseases Program at the National Eye Institute by enhancing our ability to predict the pathogenicity of mutations in phototransduction proteins, thereby enabling development of personalized therapeutic interventions for retinal diseases resulting from dysregulation of the visual signaling pathway in rod and cone photoreceptor cells of the retina.