Molecular Mechanisms of TULP1-Mediated Photoreceptor Degeneration

抽象的。 TULP1基因突变是一种严重的、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和TULP1的细胞功能 视网膜变性的致病机制。TULP1是一种光感受器特异性蛋白 参与了两个感光器隔间的囊泡运输。在Tulp1-/-小鼠中，几个光转导 蛋白质定位错误，表明TULP1是蛋白质运输到外段(OS)所必需的。 这些小鼠也有突触畸形，几乎没有完整的条带，表明神经递质存在缺陷。 水泡循环。我们的初步数据表明，TULP1有两个明显的相互作用。我们假定 TULP1是一种连接OS结合蛋白转运泡和轴丝的适配蛋白。 过渡区和充满神经递质的囊泡到达带状复合体。体外进行的初步研究 提示突变的TULP1蛋白引起内质网应激，并导致未折叠蛋白反应的诱导 (UPR)途径，提示了细胞死亡的可能机制。我们的总体假设是TULP1是一个 蛋白质囊泡运输所需的磷脂传感适配蛋白及其突变体 TULP1的不同形式导致蛋白质错配，从而诱导慢性内质网应激导致光感受器 蛋白质降解途径过载导致细胞死亡。目标1中的实验将识别TULP1 隔室特异的蛋白质-蛋白质相互作用以及这些相互作用网络是否被RP- 相关的TULP1突变。这将通过执行免疫沉淀、定位 实验、高分辨率成像、邻近蛋白质组学和定量交联质谱学 方法：研究方法。目标2中的实验将确定分子签名、时间贡献和 Tulp1突变模型中应激反应通路(S)激活在疾病进展中的作用机制 通过分析每条途径所特有的分子标记来分析RP。对于这些实验，我们生成了两个 新的敲入小鼠品系，每个品系都表达一个与Rp相关的TULP1错义突变。Wt，tulp1-/-， Tulp1D94Y和Tulp1F491L小鼠将与三个不同的小鼠品系杂交，每个品系都表达体内报告 与UPR、蛋白酶体活性或自噬相关的基因。全面的表型分析将是 在小鼠之间进行实验和比较，以确定突变的Tulp1蛋白是否在体内诱导慢性ER 应激导致应激反应通路的激活，通过压倒 光感受器蛋白平衡网络能力。通过澄清参与囊泡运输的蛋白质，这 该项目将对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产生重大影响 和疾病。重要的是，确定TULP1相关RP的致病机制并鉴定 激活的通路将有助于发现旨在减缓这种失明情况的治疗靶点。

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.