Molecular Mechanisms of TULP1-Mediated Photoreceptor Degeneration

抽象的。 TULP1 基因突变是严重的早发性常染色体隐性视网膜炎的基础 色素变性（RP）。我们的长期目标是了解 TULP1 的细胞功能和 视网膜变性的致病机制。 TULP1 是一种光感受器特异性蛋白 参与两个光感受器区室中的囊泡运输。在 tulp1-/- 小鼠中，几种光转导 蛋白质定位错误，表明 TULP1 对于蛋白质转运至外段 (OS) 是必需的。 这些小鼠还存在突触畸形，几乎没有完整的带，表明神经递质存在缺陷 囊泡循环。我们的初步数据表明 TULP1 具有两个不同的相互作用组。我们假设 TULP1 充当连接 OS 结合蛋白转运囊泡与轴丝的衔接蛋白 过渡区和充满神经递质的囊泡到带状复合体。体外初步研究 表明突变的 TULP1 蛋白引起 ER 应激并导致诱导未折叠蛋白反应 （UPR）途径，提示细胞死亡的可能机制。我们的总体假设是 TULP1 是 蛋白质囊泡运输所需的磷脂感应接头蛋白及其突变体 TULP1 的形式会导致蛋白质错误运输，从而诱导慢性 ER 应激，从而导致光感受器 通过蛋白质降解途径超载而导致细胞死亡。目标 1 中的实验将识别 TULP1 区室特异性蛋白质-蛋白质相互作用以及这些相互作用网络是否被 RP-破坏 相关的 TULP1 突变。这将通过进行免疫沉淀、定位来完成 实验、高分辨率成像、邻近蛋白质组学和定量交联质谱 方法。目标 2 中的实验将确定分子特征、时间贡献和 Tulp1 突变模型中疾病进展过程中应激反应通路激活的机制 通过分析每个途径特有的分子标记来分析 RP。对于这些实验，我们生成了两个 新型敲入小鼠品系均表达 RP 相关 TULP1 错义突变。 WT，郁金香1-/-， tulp1D94Y 和 tulp1F491L 小鼠将与三种不同的小鼠品系杂交，每种小鼠品系表达体内报告基因 与 UPR、蛋白酶体活性或自噬相关的基因。综合表型分析将 在小鼠体内进行并比较，以确定突变型 Tulp1 蛋白是否会诱导慢性 ER 压力导致压力反应途径的激活，通过压倒细胞而导致细胞死亡 光感受器蛋白质稳态网络能力。通过澄清参与囊泡运输的蛋白质，这 该项目将显着影响与人类视网膜功能相关的光感受器生物学的一个重要方面 和疾病。重要的是，确定 TULP1 相关 RP 背后的致病机制并识别 激活的途径将有助于发现旨在减缓这种致盲病症的治疗靶点。

Abstract. Mutations in the TULP1 gene underlie a severe, early-onset form of autosomal recessive retinitis pigmentosa (RP). Our long-term objectives are to understand the cellular function of TULP1 and the pathogenic mechanism responsible for retinal degeneration. TULP1 is a photoreceptor-specific protein involved in vesicular trafficking in two photoreceptor compartments. In tulp1-/- mice, several phototransduction proteins are mislocalized, indicating that TULP1 is necessary for protein transport to the outer segment (OS). These mice also have a synaptic malformation with few intact ribbons, indicating a defect in neurotransmitter vesicular cycling. Our preliminary data indicates that TULP1 has two distinct interactomes. We postulate that TULP1 functions as an adapter protein linking OS-bound protein transport vesicles to the axoneme of the transition zone and neurotransmitter-filled vesicles to the ribbon complex. Preliminary studies done in vitro suggest that mutant TULP1 proteins cause ER stress and leads to induction of the unfolded protein response (UPR) pathway, suggesting a possible mechanism for cell death. Our overall hypothesis is that TULP1 is a phospholipid-sensing adapter protein required for the vesicular transport of proteins and that mutant forms of TULP1 cause protein mistrafficking which induces chronic ER stress leading to photoreceptor cell death through overload of protein degradation pathways. Experiments in Aim 1 will identify TULP1 compartment-specific protein-protein interactions and whether these interacting networks are disrupted by RP- associated TULP1 mutations. This will be accomplished by performing immunoprecipitations, localization experiments, high-resolution imaging, proximity proteomics and quantitative cross-linking mass spectroscopy methods. Experiments in Aim 2 will determine the molecular signatures, temporal contributions and mechanism of stress response pathway(s) activation throughout disease progression in Tulp1 mutant models of RP by analyzing molecular markers unique to each pathway. For these experiments, we generated two novel knock-in mouse lines each expressing an RP-associated TULP1 missense mutation. WT, tulp1-/-, tulp1D94Y and tulp1F491L mice will be crossed with three different mouse lines each expressing an in vivo reporter gene relevant to UPR, proteasome activity, or autophagy. Comprehensive phenotypic analyses will be performed and compared across mice to determine in vivo whether mutant Tulp1 proteins induce chronic ER stress leading to activation of stress response pathways causing cell death by overwhelming the photoreceptors proteostasis network capacity. By clarifying the proteins involved in vesicular trafficking, this project will significantly impact an important aspect of photoreceptor biology relevant to human retinal function and disease. Importantly, determining the pathogenic mechanism behind TULP1-associated RP and identifying the activated pathways will aid in discovering therapeutic targets aimed at slowing this blinding condition.