Photoreceptor Phosphodiesterase Regulation

光感受器磷酸二酯酶调节

• 批准号: 10346165
• 负责人: Rick H Cote
• 金额: $ 37.23万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10346165
• 关键词: Allosteric Regulation; Architecture; Binding; Blindness; Catalytic Domain; Code; Communication; Complex; Cone dystrophy; Coupled; DNA Sequence Alteration; Defect; Development; Disease; Docking; Enzyme Activation; Enzymes; Etiology; Event; GTP Binding; GTP-Binding Proteins; Genes; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Inherited; Light; Lighting; Link; Mediating; Mission; Modeling; Molecular; Molecular Conformation; Molecular Target; Multienzyme Complexes; Mutation; National Eye Institute; Night Blindness; Outcomes Research; Pathogenicity; Pathway interactions; Photoreceptors; Phototransduction; Process; Proteins; Recovery; Regulation; Research; Retina; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinal Dystrophy; Retinitis Pigmentosa; Rod; Signal Pathway; Signal Transduction; Site; Speed; Therapeutic Intervention; Transducin; Vertebrate Photoreceptors; Vision Disorders; Visual; Work; achromatopsia; alpha Subunit Transducin; base; clinically significant; dimer; disease-causing mutation; enzyme activity; next generation sequencing; personalized therapeutic; phosphoric diester hydrolase; photoactivation; programs; protein B; retinal rods

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.

项目总结/摘要 光感受器磷酸二酯酶（PDE 6）是视觉信号通路的中心酶。精确 调节其激活和去激活对于杆和锥的速度、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和视锥细胞PDE 6基因的遗传突变与多种 视网膜疾病，包括视网膜色素变性，先天性静止性夜盲症和视锥细胞营养不良。 下一代测序正在识别PDE 6基因中越来越多的突变， 其临床意义仍不确定。甚至对视网膜病变的分子病因学知之甚少， 致病突变杆PDE 6由两个催化亚基组成，其活性在黑暗中被抑制- 通过结合两个相同的γ-亚基（Pγ）的适应状态。在光诱导的视觉信号激活后， 在PDE 6通路中，PDE 6活性通过异源三聚体G蛋白转导素的结合来刺激。光的寿命- 激活的PDE 6是由转导蛋白水解其结合的GTP的速率精确控制的， RGS9-1（Regulator of G-protein Signaling9-1）虽然参与调节PDE 6的蛋白质 在光转导过程中，已经确定了PDE 6动态变化的分子序列， 在PDE 6激活和失活过程中， 人们对它了解仍然很少。直到我们了解正常情况下PDE 6调节的机制基础， 光转导，我们将受到阻碍，在开发治疗干预措施，为这些疾病引起的 PDE 6或其结合配偶体的缺陷导致视网膜变性疾病和视觉障碍。 本申请的总体目标是了解伴随PDE 6的事件序列 在恢复过程中，通过转导蛋白激活并随后通过RGS 9 -1和其他蛋白失活， PDE 6进入暗适应状态。我们的实验计划是基于这样的假设，即抑制性Pγ亚基 PDE 6是负责介导多种变构相互作用的“主调节因子”， PDE 6催化二聚体，以及与转导素α-亚基和RGS 9 -1。我们提出两个具体目标， 将（1）描述转导素α亚基和PDE 6催化剂之间结合相互作用的顺序， 抑制性Pγ亚基，以提供杆PDE 6活化的综合模型，以及（2）确定 RGS 9 -1结合后PDE 6失活复合物的分子结构和结构 Pγ亚基的重排加速了活化的PDE 6的终止。这一过程的结果 研究通过提高我们的视网膜疾病计划的目标在国家眼科研究所 预测光转导蛋白突变致病性的能力，从而使开发 针对由视觉信号失调引起的视网膜疾病的个性化治疗干预 视网膜的视杆和视锥感光细胞中的信号通路。