Regulation of Retinal cGMP phosphodiesterases

描述（由申请人提供）：本研究项目的长期目标是阐明棒状和锥状cgmp -磷酸二酯酶（PDE6）的功能和调控的分子机制。pde6是脊椎动物视觉传导级联的关键效应酶。转导蛋白通过解除对PDE6催化二聚体的抑制来激活PDE6。子单元(P3)。PDE6棒在环核苷酸PDEs中是独特的，因为它是由PDE6A和PDE6B催化亚基组成的催化异源二聚体。我们假设PDE6棒的异源二聚化在杆光传导中起重要作用。PDE6A和PDE6B的单独性质和杆状PDE6异二聚化的功能意义将在目标1中进行研究。我们的目标是通过转导蛋白对杆状细胞PDE6激活的机制有一个新的认识，并推进杆状细胞光传导级联的现有模型。基于现有证据和我们的初步研究，我们假设P？PDE6催化亚基对全酶在光感受器细胞中的正常运输至关重要。目的2是验证这一假设，并探讨P？PDE6的表达和转运目标1和目标2将通过广泛使用转基因非洲爪蟾作为表达突变体PDE6的新途径来实现。人类PDE6A和PDE6B基因突变分别导致3-4%的隐性视网膜色素变性（RP）病例。作为目的3，我们将研究小鼠PDE6B亚基中N605S隐性突变引起小鼠非典型视网膜变性（atrd3）的机制。基于初步证据，我们假设atrd3是由PDE6的功能改变触发的，可能反映了人类RP的某些形式和机制。atrd3的机制将使用突变小鼠模型与转基因非洲爪蟾的锥形PDE6C突变体模型进行比较。其抑制PDE6的分子细节-亚基将在第4章用蛋白质晶体学的方法加以阐明。PDE5/6催化结构域与P？-63-87和西地那非（伟哥）将首次提供PDE6和PDE6之间关键相互作用的结构见解。并揭示了伟哥对视力副作用的结构基础。研究人员将研究嵌合atrd3突变体的结构，以揭示与PDE6突变相关的视网膜疾病的分子机制。总的来说，这些研究将促进我们对PDE6的调控的理解，并有助于阐明PDE6基因突变导致RP的机制。公共卫生相关性：棒状和锥状cGMP磷酸二酯酶（PDE6家族）是视力的关键效应酶。PDE6的突变导致人类视网膜疾病，如视网膜色素变性、先天性静止性夜盲症和色盲。这项研究将使人们对PDE6的结构、功能和调控的理解达到一个新的水平，这对于揭示病变视网膜中PDE6突变的机制是必要的。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to elucidate the molecular mechanisms of the function and regulation of rod and cone cGMP-phosphodiesterases (PDE6). PDE6s serve as key effector enzymes in the vertebrate visual transduction cascade. Transducin activates PDE6 by relieving the inhibition imposed on the PDE6 catalytic dimer by two ?-subunits (P3). Rod PDE6 is unique among cyclic nucleotide PDEs in that it is a catalytic heterodimer composed of the PDE6A and PDE6B catalytic subunitsWe hypothesize that the heterodimerization of rod PDE6 plays an important role in rod phototransduction. The individual properties of PDE6A and PDE6B and the functional significance of rod PDE6 heterodimerization will be examined in aim 1. The goal is to bring a new level of understanding to the mechanism of rod PDE6 activation by transducin and advance the current models of the rod phototransduction cascade. Based on the existing evidence and our preliminary studies, we hypothesize that the interaction of P? with the PDE6 catalytic subunits is critical to the proper transport of the holoenzyme in photoreceptor cells. Aim 2 is to test this hypothesis and probe the mechanisms of P? in expression and transport of PDE6. Aims 1 and 2 will be achieved through extensive use of transgenic Xenopus laevis as a novel approach for the expression of mutant PDE6. Mutations in the human PDE6A and PDE6B genes each are responsible for 3-4% of cases of recessive retinitis pigmentosa (RP). As aim 3 we will investigate the mechanism of the recessive mutation N605S in the mouse PDE6B subunit causing atypical retinal degeneration (atrd3) in mice. Based on the initial evidence, we hypothesize that atrd3 is triggered by functional changes in PDE6 and may reflect certain forms and mechanisms of human RP. The atrd3 mechanism will be examined using the mutant mouse model in comparison to a transgenic Xenopus laevis model of the cone PDE6C mutant counterpart. The molecular details of PDE6 inhibition by its ?-subunit will be elucidated by means of protein crystallography in aim 4. The structure of the chimeric PDE5/6 catalytic domain complexed with P?-63-87 and sildenafil (Viagra) will provide the first structural insights into the critical interaction between PDE6 and P? and should reveal the structural basis for the side effects of Viagra on vision. The structure of the chimeric atrd3 mutant will be investigated to uncover the molecular mechanism for retinal disease associated with this PDE6 mutation. Overall, these studies will advance our understanding of the regulation of PDE6 and help to elucidate the mechanisms of RP caused by mutations of PDE6 genes. PUBLIC HEALTH RELEVANCE: Rod and cone cGMP phosphodiesterases (PDE6 family) are the key effector enzymes in vision. Mutations in PDE6 lead to human retinal diseases such as retinitis pigmentosa, congenital stationary night blindness, and achromatopsia. This research would develop a new level of understanding of structure, function, and regulation of PDE6 necessary to uncover the mechanisms of PDE6 mutations in diseased retina.