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

摘要 视网膜光感受器细胞可以在人的一生中对光做出反应，这是由于持续的重置 感光分子(视觉色素)及其相关的信号元件。几乎所有蛋白质中的缺陷 参与这些过程会导致光感受器退化，这可能不仅是由于 酶或其他功能活性，但也来自维持复合体的结构元素的损失 杆件外段(ROS)的拓扑。我们的长期目标是阐明其分子机制。 光转导和视网膜变性促进遗传性失明治疗方法的发现 由光传导基因突变引起的人类疾病。编码基因的突变 磷酸二酯酶6(PDE6)是引起这些疾病的主要原因之一。 因此，我们提出了两个主题上和实验上联系在一起的具体目标。目标1：确定高- 单个圆盘的棒外节段(ROS)和轮缘区的分辨率结构由三个磁盘派生而来 三维(3D)低温电子断层扫描。这一目标被细分为三个子目标，将重点放在 关于确定保持盘精确排列的间隔物的分子同一性，确定结构 PDE6水平降低的区域组织及其对区域组织和支柱数量的影响；以及 确定ROS盘环的结构和ABCA4对ROS结构完整性的作用。这项研究将 加深我们对PDE6不仅作为一种酶的功能，而且作为一种结构蛋白的功能的理解。目标 2：确定N-末端马尾螺旋区的作用，并描述PDE6的变构调节。 这一目标被细分为三个子目标，这三个子目标将侧重于铂基序的膜锚定作用， 阐明了PdE6αβγ2络合物的变构活化机理，并确定了其结构 GTα-GTP-PDE6αβγ2复合体。总体而言，这项提案的发现将有助于开发一个理性的 缓解与PDE6基因突变相关的视网膜营养不良的方法。

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.