The Role of TULP1 in Photoreceptor Cells

描述（由申请人提供）：色素性视网膜炎（RP）包括大量以光感受器变性为特征的遗传性视网膜疾病。RP具有遗传和表型异质性，影响全球超过100万人。一种名为TULP1的基因突变是早发性常染色体隐性RP的基础。Tulp1是一种光感受器细胞所独有的蛋白，尽管Tulp1的功能尚不明确，但从缺乏Tulp1的小鼠模型中得到的证据表明，它在光感受器的多个隔室的细胞内蛋白运动中发挥作用。在tulp1-/-小鼠中，在光感受器变性之前，视杆蛋白和视锥蛋白定位错误，携带视紫红质的细胞外囊泡在内节段（IS）周围积聚，表明tulp1是将蛋白质运输到外节段（OS）所必需的。这些小鼠在光感受器退化之前也有突触畸形，很可能会干扰后感受器神经元的正常发育。Tulp1的缺失导致影响多个视网膜部位结构和功能的异常，以及在单独的光感受器区室引起明显的异常。这表明它要么在整个光感受器中发挥一般作用，要么参与多种不同的途径。基于我们已发表的工作和初步数据，本提案的中心假设是，Tulp1是参与两个感光细胞区室中蛋白质囊泡运动的分子机制的一个组成部分。我们的长期目标是了解TULP1的生理功能以及与TULP1突变相关的视网膜变性的致病机制。Aim 1设计的实验将通过蛋白质组学分析确定Tulp1的相互作用伙伴，比较is特异性和突触特异性相互作用组。在Aim 2中，将对缺乏Tulp1和表达导致RP的Tulp1突变形式的IS和突触进行分子解剖，以探测结构或空间干扰。注意力将集中在结合动力蛋白-1的蛋白质上，动力蛋白-1是一种神经元特异性蛋白质，对囊泡运输至关重要。我们认为Tulp1/Dynamin-1/Actin的相互作用构成了Tulp1功能复合物的核心，阐明这一相互作用将解开Tulp1的作用机制。目的3将分析tulp1-/-光感受器突触中的囊泡循环。这将完成使用苯乙烯染料光转换，然后是电子显微镜。在这项工作成功完成后，我们将在功能环境中定位Tulp1并定义其作用机制。我们的研究结果将深入了解光感受器蛋白运输途径的功能组织，以及与TULP1突变相关的视网膜功能扰动。最后，这个项目有可能显著影响与人类视网膜疾病相关的光感受器生物学的一个重要方面。

DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) incorporates a large number of inherited retinal disorders characterized by photoreceptor degeneration. RP is genetically and phenotypically heterogeneous, affecting over 1 million individuals worldwide. Mutations in a gene called TULP1 underlie an early-onset and severe form of autosomal recessive RP. Tulp1 is a protein exclusive to photoreceptor cells, and although the function of Tulp1 remains elusive, there is evidence from the murine model lacking Tulp1 that it plays a role in intracellular protein movement in multiple compartments of the photoreceptor. In tulp1-/- mice, prior to photoreceptor degeneration, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment (IS), indicating that Tulp1 is necessary for the transport of proteins to the outer segment (OS). These mice also have a synaptic malformation that precedes photoreceptor degeneration and most likely interferes with the proper development of post-receptoral neurons. The absence of Tulp1 results in abnormalities that affect structure and function in multiple retinal sites, as well as causing distinct abnormalities in separate photoreceptor compartments. This suggests that it either performs a general role throughout the photoreceptor or participates in multiple distinct pathways. Based on our published work and preliminary data, the central hypothesis of this proposal is that Tulp1 is a component of the molecular machinery involved in the vesicular movement of proteins in two photoreceptor cell compartments. Our long-term objectives are to understand the physiological function of TULP1 and the pathogenic mechanism responsible for retinal degeneration associated with TULP1 mutations. Experiments designed in Aim 1 will identify Tulp1 interacting partners by proteomic analysis, comparing IS-specific to synaptic-specific interactomes. In Aim 2, molecular dissections of the IS and synapse lacking Tulp1 and expressing mutant forms of TULP1 that cause RP will be conducted to probe for structural or spatial disturbances. Attention will focus on proteins that bind Dynamin-1, a neuronal-specific protein that is essential for vesicular trafficking. Elaborating on the Tulp1/Dynamin-1/Actin interaction, which we believe composes the core of Tulp1 functional complexes, will unlock the mechanism of action of Tulp1. Aim 3 will analyze vesicle cycling in the tulp1-/- photoreceptor synapse. This will be accomplished using styryl dye photoconversion followed by electron microscopy. At the successful completion of this work, we will position Tulp1 in a functional context and define its mechanism of action. Our results will provide insight into the functional organization of photoreceptor protein transport pathways, as well as insight into the perturbation of retinal function associated with TULP1 mutations. Finally, this project has potential for significantly impacting an important aspect of photoreceptor biology relevant to human retinal disease.