Investigation of the role of TMED9 in the accumulation of a mutant protein in MUC1 kidney disease (MKD)

研究 TMED9 在 MUC1 肾病 (MKD) 突变蛋白积累中的作用

基本信息

批准号：
10252805
负责人：
Alissa Campbell Goss
金额：
$ 3.78万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2024-06-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10252805
关键词：
Address Binding Biology CRISPR/Cas technology Cells Cellular biology Chronic Kidney Failure Co-Immunoprecipitations Development Disease Epithelial Epithelial Cells Familial Hypercholesterolemia Foundations Frameshift Mutation Genetic Health In Vitro Inherited Institutes Investigation Kidney Kidney Diseases Kidney Failure Knock-in Mouse Knock-out Knockout Mice Laboratories Lead Low-Density Lipoproteins MUC1 gene Mass Spectrum Analysis Mediating Mediator of activation protein Morbidity - disease rate Mucin 1 protein Nephrology Organoids Pathology Pathway interactions Patients Pattern Phenotype Population Process Proteins Rare Diseases Research Resources Role Systemic disease Testing Thapsigargin Therapeutic Toxic effect Translating Tubular formation Vesicle Work base endoplasmic reticulum stress graduate student in vivo induced pluripotent stem cell innovation insight interest medical schools mortality mouse model mutant new therapeutic target protein complex proteostasis targeted treatment therapeutic target trafficking

项目摘要

Abstract Chronic kidney disease (CKD), most often caused by manifestations of systemic disease or underlying primary kidney disease, is a significant cause of worldwide patient morbidity and mortality. The field of nephrology is severely lacking in mechanism-based, targeted therapeutics to treat patients with CKD. Studies of primary, rare genetic kidney diseases, which lead to perturbations of common, essential kidney processes, can be harnessed to inform foundational kidney biology and identify potential therapeutic targets for kidney diseases, both rare and common. MUC1 kidney disease (MKD), or autosomal dominant tubulointerstitial kidney disease-MUC1 (ADTKD- MUC1), is a rare genetic kidney disease caused by a frameshift mutation in the MUC1 gene. MKD leads to progressive kidney tubulointerstitial damage and eventual renal failure. Recent studies have shown that MKD is a toxic proteinopathy in which the mutant frameshift protein, MUC1-fs, intracellularly accumulates in kidney tubular epithelial cells and is associated with increased levels of cellular toxicity. MUC1-fs has been further shown to accumulate specifically in the early secretory pathway of MKD patient kidney tubular epithelial cells (P cells), in vesicles that contain the protein TMED9. Critically, it has been found that knockout of TMED9 ameliorates MUC1-fs accumulation in P cells. TMED9 and MUC1-fs have been shown to additionally colocalize in both an in vivo MKD mouse model and MKD patient-derived iPSC organoids. Recent work has shown that TMED9 co- immunoprecipitates with MUC1-fs in P cells. Based on these observations, this proposed work aims to investigate the following hypothesis: In the setting of MKD, TMED9 and its interactors halt the trafficking of misfolded MUC1-fs and retain it in the early secretory pathway, leading to MUC1-fs accumulation and increased cellular toxicity. In Aim 1, the effect of TMED9 on MUC1-fs trafficking through the early secretory pathway will be assessed by knockout of TMED9 in P cells and mapping of MUC1-fs trafficking by inhibition of secretory pathway branches and assessment of MUC1-fs reaccumulation. The role of TMED9 as a mediator of cellular toxicity will be assessed by comparison of cellular toxicity, following treatment with the ER stress-inducer thapsigargin, in P cells and TMED9 knockout P cells. In Aim 2, the TMED9 protein complex that mediates MUC1- fs accumulation will be identified. First, interactors of TMED9 in P cells will be found using co-immunoprecipitation and mass spectrometry. Then, TMED9 interactors necessary for MUC1-fs accumulation will be identified using a CRISPR/Cas9 arrayed knockout screen. Finally, the binding of these necessary interactors to MUC1-fs will be assessed. In Aim 3, the role of TMED9 in MUC1-fs accumulation will be explored in vivo. A TMED9 knockout mouse will be crossed with the established MKD knock-in mouse and the in vivo effect of TMED9 knockout on MUC1-fs accumulation and other known MKD knock-in mouse phenotypes will be assessed.

抽象的慢性肾脏疾病（CKD），通常是由全身性疾病表现或基本原发性引起的肾脏疾病是全球患者发病率和死亡率的重要原因。肾脏科领域是严重缺乏基于机制的，有针对性的治疗药物来治疗CKD患者。主要，罕见的研究可以利用常见的基本肾脏过程扰动的遗传肾脏疾病为基础肾脏生物学提供信息，并确定罕见和肾脏疾病的潜在治疗靶点常见的。 MUC1肾脏疾病（MKD）或常染色体显性tubulointerstitial肾脏疾病 - MUC1（ADTKD- MUC1）是一种罕见的遗传肾脏疾病，是由MUC1基因中的移码突变引起的。 MKD导致渐进的肾小管间隙损害和最终的肾衰竭。最近的研究表明MKD是一种有毒蛋白质病，其中突变的移料器蛋白MUC1-FS细胞内积聚在肾脏中管状上皮细胞，与细胞毒性水平升高有关。 MUC1-FS已进一步显示专门在MKD患者肾小管上皮细胞（P细胞）的早期分泌途径中积累在包含蛋白质TMed9的囊泡中。至关重要的是，已经发现TMED9的敲除可以改善 MUC1-FS在P细胞中积累。 TMED9和MUC1-FS已被证明在两个中都额外进行了共定位 Vivo MKD小鼠模型和MKD患者衍生的IPSC类器官。最近的工作表明，TMed9共同 P细胞中用MUC1-FS免疫沉淀。基于这些观察，这项提出的工作旨在研究以下假设：在MKD的情况下，TMed9及其交互者停止了贩运错误折叠的MUC1-FS并将其保留在早期的分泌途径中，导致MUC1-FS积累并增加细胞毒性。在AIM 1中，TMED9对通过早期分泌途径的MUC1-FS贩运的影响将通过在P细胞中敲除TMED9的敲除和通过抑制分泌的MUC1-FS运输的映射来评估途径分支和MUC1-FS重新汇总的评估。 TMED9作为细胞中介体的作用在与ER应激诱导者治疗后，将通过比较细胞毒性来评估毒性 thapsigargin，在P细胞和TMED9基因敲除P细胞中。在AIM 2中，介导MUC1-的TMED9蛋白复合物 FS积累将被确定。首先，将使用共免疫沉淀找到P细胞中TMED9的相互作用者和质谱。然后，将使用MUC1-FS积累所需的TMED9交流器使用 CRISPR/CAS9阵列淘汰赛屏幕。最后，这些必要的相互作用者与MUC1-FS的结合将是评估。在AIM 3中，将在体内探索TMED9在MUC1-FS积累中的作用。 TMed9淘汰赛鼠标将与已建立的MKD敲入鼠标交叉，并且TMED9敲除对将评估MUC1-FS积累和其他已知的MKD敲入小鼠表型。