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Molecular Mechanisms of TULP1-Mediated Photoreceptor Degeneration

TULP1介导的光感受器变性的分子机制

基本信息

批准号：
10615831
负责人：
STEPHANIE A HAGSTROM
金额：
$ 40.25万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10615831
关键词：
Adaptor Signaling Protein Affect Autophagocytosis Binding Binding Proteins Biological Assay Biology Blindness C-terminal CRISPR/Cas technology Carrier Proteins Cell Death Cell Line Cell physiology Cells Chronic Co-Immunoprecipitations Complement Complex Data Defect Degradation Pathway Disease Disease Progression Fluorescence Resonance Energy Transfer Gene Dosage Genes Goals Golgi Apparatus Histology Human Immunohistochemistry Immunoprecipitation In Situ Nick-End Labeling In Vitro Inherited KRP protein Knock-in Knock-in Mouse Leber&apos s amaurosis Lipid Binding Location Maps Mass Spectrum Analysis Mediating Membrane Methods Missense Mutation Modeling Molecular Molecular Profiling Movement Mus Mutation N-terminal Neurotransmitters Pathogenicity Pathologic Pathway interactions Patients Phenotype Phospholipids Photoreceptors Phototransduction Process Protein Binding Domain Protein Biosynthesis Proteins Proteomics Quantitative Reverse Transcriptase PCR Reporter Reporter Genes Research Project Grants Retina Retinal Degeneration Retinitis Pigmentosa Site Synapses Testing Therapeutic Intervention Time Transgenic Mice Ubiquitin Vesicle Vesicular Transport Proteins Work autosome biological adaptation to stress crosslink early onset endoplasmic reticulum stress experimental study high resolution imaging in vivo link protein malformation microtubule-associated protein 1B molecular marker mouse model multicatalytic endopeptidase complex mutant novel photoreceptor degeneration protein degradation protein protein interaction protein transport proteostasis recruit response ribbon synapse scaffold spatiotemporal superresolution imaging targeted treatment therapeutic target trafficking vesicle transport

项目摘要

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.

抽象的。 TULP 1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沉着（RP）。我们的长期目标是了解TULP 1的细胞功能，视网膜变性的致病机制。TULP 1是一种光受体特异性蛋白参与两个光感受器隔室中的囊泡运输。在tulp 1-/-小鼠中，蛋白质被错误定位，表明TULP 1是蛋白质转运到外节（OS）所必需的。这些老鼠也有一个突触畸形，几乎没有完整的丝带，这表明神经递质的缺陷囊泡循环我们的初步数据表明，TULP 1有两个不同的相互作用组。我们推测 TULP 1作为连接OS结合的蛋白转运囊泡与神经元轴丝的衔接蛋白发挥功能。过渡区和充满神经递质的囊泡到带状复合物。体外初步研究提示突变TULP 1蛋白引起ER应激并导致未折叠蛋白应答诱导 (UPR)途径，表明细胞死亡的可能机制。我们的总体假设是，TULP 1是一个磷脂传感衔接蛋白所需的蛋白质和突变体的囊泡运输 TULP 1的形式引起蛋白质误激活，其诱导慢性ER应激，导致光感受器通过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TULP 1 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TULP 1突变。这将通过进行免疫沉淀、定位实验、高分辨率成像、邻近蛋白质组学和定量交联质谱方法.目标2中的实验将确定分子特征、时间贡献和 Tulp 1突变模型中疾病进展过程中应激反应途径激活的机制通过分析每种途径特有的分子标记物来检测RP。对于这些实验，我们生成了两个新的敲入小鼠系，每个都表达RP相关的TULP 1错义突变。WT，tulp1-/-， tulp 1D 94 Y和tulp 1F 491 L小鼠将与各自表达体内报告基因的三种不同小鼠系杂交与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中进行比较，以确定突变Tulp 1蛋白是否在体内诱导慢性ER 应激导致应激反应途径的激活，通过压倒性地抑制细胞凋亡而导致细胞死亡。光感受器蛋白质稳态网络能力。通过阐明参与囊泡运输的蛋白质，该项目将显著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TULP 1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状况的治疗靶点。