The complex role of phosphodiesterase 6 in rod photoreceptor health and function

磷酸二酯酶 6 在视杆光感受器健康和功能中的复杂作用

• 批准号: 10455528
• 负责人: Krzysztof Palczewski
• 金额: $ 63.34万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455528
• 关键词: 3-Dimensional; Affect; Allosteric Regulation; Architecture; Binding; Biochemical; Biological; Brain; Catalytic Domain; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cyclic GMP; Data; Defect; Development; Dimensions; Disease; Electrons; Elements; Enzymes; Equilibrium; Event; G Protein-Coupled Receptor Signaling; Genes; Goals; Guanosine Triphosphate; Health; Heterozygote; Human; Hydrolase; Individual; Inherited; Isoenzymes; Knowledge; Life; Light; Link; Liposomes; Maintenance; Mediating; Membrane; Molecular; Molecular Conformation; Mus; Mutation; N-terminal; Neurons; Photoreceptors; Phototransduction; Play; Process; Proteins; Regulation; Research; Resolution; Retina; Retinal Degeneration; Retinal Dystrophy; Retinal Photoreceptors; Retinal Pigments; Retinoids; Rod Outer Segments; Role; Signal Transduction; Structural Protein; Structure; Tail; Therapeutic; Tomogram; algorithm training; data acquisition; density; disease-causing mutation; inhibitor; nanometer; neural network; novel therapeutics; peripherin; phosphodiesterase 6; photoreceptor degeneration; reconstruction; retinal rods; rod outer segment disc; virtual; visual phototransduction

ABSTRACT Retinal photoreceptor cells can respond to light throughout an individual’s life due to continuous resetting of the light sensing molecules (visual pigments) and their associated signaling elements. Defects in almost all proteins involved in these processes cause photoreceptor degeneration, which could result not only from a deficiency in enzymatic or other functional activity, but also from a loss of structural elements that maintain the complex topology of the rod outer segment (ROS). Our long-term goal is to elucidate the molecular mechanisms of phototransduction and retinal degeneration to promote the discovery of therapeutics for inherited blinding diseases in humans caused by mutations in phototransduction genes. Mutations in the gene encoding phosphodiesterase 6 (PDE6) are among the main causes of such diseases. Accordingly, we propose two thematically and experimentally linked specific aims. Aim 1: Determine the high- resolution structure of rod outer segments (ROS) and rim region of individual disks derived from three- dimensional (3D) cryo-electron tomograms. This aim has been subdivided into three sub-aims that will focus on determining the molecular identity of the spacers that hold disks precisely arranged, determining the structure of ROS with reduced levels of PDE6 and its impact on ROS organization and the number of pillars, and determining the structure of ROS disk rims and the role of ABCA4 on ROS structural integrity. This research will advance our understanding of the function of PDE6 not only as an enzyme but also as a structural protein. Aim 2: Define the role of the N-terminal pony-tail helical region and delineate the allosteric regulation of PDE6. This aim has been subdivided into three sub-aims that will focus on the membrane anchoring role of the Pt-motif, elucidating the allosteric activation mechanism of the PDE6αβγ2 complex, and determining the structure of the Gtα-GTP-PDE6αβγ2 complex. Overall, the findings from this proposal will facilitate the development of a rational approach to alleviate retinal dystrophies related to mutations in the PDE6 gene.

摘要 视网膜光感受器细胞可以在个体的一生中对光做出响应，这是由于视网膜光感受器细胞的连续重置。 感光分子（视色素）及其相关的信号元件。几乎所有蛋白质的缺陷 参与这些过程导致感光细胞变性，这可能不仅是由于缺乏 酶或其他功能活性，而且还来自维持复合物的结构元件的损失 视杆细胞外段（ROS）的拓扑结构。我们的长期目标是阐明 光传导和视网膜变性，以促进遗传性致盲疗法的发现 由光传导基因突变引起的人类疾病。基因编码突变 磷酸二酯酶6（PDE 6）是这些疾病的主要原因之一。 因此，我们提出了两个主题和实验相关的具体目标。目标1：确定高- 分辨率结构的杆外节（ROS）和边缘区域的个别磁盘来自三个- 三维（3D）冷冻电子断层扫描。这一目标又细分为三个次级目标， 在确定将圆盘精确排列的间隔物的分子身份时， ROS与PDE 6水平降低及其对ROS组织和支柱数量的影响， 确定ROS盘边缘的结构和ABCA 4对ROS结构完整性的作用。这项研究将 推进我们对PDE 6功能的理解，不仅是一种酶，而且是一种结构蛋白。目的 2：定义N-末端马尾螺旋区的作用，并描绘PDE 6的变构调节。 该目标已被细分为三个子目标，其将集中于Pt基序的膜锚定作用， 阐明了PDE 6 αβγ2复合物的变构活化机制，并确定了PDE 6 αβγ2复合物的结构。 Gtα-GTP-PDE 6 αβγ2复合物。总的来说，这项建议的结果将有助于制定一项合理的 缓解与PDE 6基因突变相关的视网膜营养不良的方法。