Regulation of Retinal cGMP phosphodiesterases

描述(申请人提供)：该研究计划的长期目标是阐明杆状和锥状cGMP-磷酸二酯酶(PDE6)的功能和调节的分子机制。PDE6是脊椎动物视觉转导通路中的关键效应酶。转导蛋白通过解除两个β-亚基(P3)对PDE6催化二聚体的抑制来激活PDE6。Rod PDE6是一种由PDE6A和PDE6B催化亚基组成的催化杂二聚体，在环核苷酸PDEs中是独一无二的。我们推测，Rod PDE6的异构化在光传导中起着重要的作用。目标1将考察PDE6A和PDE6B各自的性质以及杆状PDE6异二聚化的功能意义。目的是为了对转导蛋白激活杆状PDE6的机制有一个新的认识，并提出杆状光导级联的现有模型。根据现有证据和我们的初步研究，我们假设P？有了PDE6催化亚基对于全酶在感光细胞中的正确运输是至关重要的。目的2是检验这一假说，并探讨P？在PDE6的表达和运输中。目标1和目标2将通过广泛使用转基因非洲爪哇作为一种表达突变体PDE6的新方法来实现。人类PDE6A和PDE6B基因突变各占隐性视网膜色素变性(RP)病例的3%-4%。作为目标3，我们将研究小鼠PDE6B亚单位中的隐性突变N605S导致小鼠非典型视网膜变性(Atrd3)的机制。根据初步证据，我们推测atrd3是由PDE6的功能变化触发的，可能反映了人类RP的某些形式和机制。将使用突变的小鼠模型与锥体PDE6C突变对应的转基因非洲爪哇模型进行比较，以检验atrd3的机制。在AIM 4中，将通过蛋白质结晶学的方法阐明PDE6-亚基抑制PDE6的分子细节。P？-63-87与西地那非(伟哥)嵌合的PDE5/6催化结构域的结构将首次为PDE6与P？并应该揭示伟哥对视力副作用的结构基础。嵌合的atrd3突变体的结构将被研究，以揭示与PDE6突变相关的视网膜疾病的分子机制。总之，这些研究将促进我们对PDE6调控的理解，并有助于阐明PDE6基因突变引起的RP的机制。公共卫生相关性：杆状和锥状cGMP磷酸二酯酶(PDE6家族)是视觉中的关键效应酶。PDE6基因突变导致人类视网膜疾病，如视网膜色素变性、先天性静止性夜盲和色盲。这项研究将对PDE6的结构、功能和调控发展一个新的水平，这是揭示PDE6在病变视网膜中突变的机制所必需的。

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.