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

摘要 慢性肾脏疾病（CKD），最常由全身性疾病或基础原发性 肾脏疾病是世界范围内患者发病率和死亡率的重要原因。肾脏学领域是 严重缺乏基于机制的靶向治疗方法来治疗CKD患者。原发性研究，罕见 遗传性肾病会导致常见的、基本的肾脏过程紊乱， 为基础肾脏生物学提供信息，并确定肾脏疾病的潜在治疗靶点， 共同MUC 1肾病（MKD）或常染色体显性肾小管间质性肾病-MUC 1（ADTKD- MUC 1），是一种罕见的遗传性肾脏疾病，由MUC 1基因的移码突变引起。MKD导致 进行性肾小管间质损伤和最终的肾衰竭。最近的研究表明，MKD是 一种突变移码蛋白MUC 1-fs在肾细胞内蓄积的毒性蛋白质病 肾小管上皮细胞，并与细胞毒性水平增加有关。MUC 1-fs进一步显示 在MKD患者肾小管上皮细胞（P细胞）的早期分泌途径中特异性地积累， 在含有TMED 9蛋白的囊泡中。重要的是，已经发现TMED 9的敲除改善了 P细胞中MUC 1-fs积累。TMED 9和MUC 1-fs已被证明在两种细胞中额外共定位， 体内MKD小鼠模型和MKD患者衍生的iPSC类器官。最近的研究表明，TMED 9共同- 与P细胞中的MUC 1-fs免疫沉淀。基于这些观察，本拟议工作旨在 调查以下假设：在MKD的背景下，TMED 9及其相互作用者阻止了 错误折叠的MUC 1-fs并将其保留在早期分泌途径中，导致MUC 1-fs积累和增加 细胞毒性在目的1中，TMED 9对MUC 1-fs通过早期分泌途径运输的作用将被证实。 通过敲除P细胞中的TMED 9和通过抑制分泌性巨噬细胞的MUC 1-fs运输来评估。 途径分支和MUC 1-fs再积累的评估。TMED 9作为细胞凋亡调节因子的作用 在用ER应激诱导剂处理后，通过比较细胞毒性来评估毒性 在P细胞和TMED 9敲除的P细胞中，毒胡萝卜素。在目标2中，介导MUC 1 - 3的TMED 9蛋白复合物被抑制。 将识别FS累积。首先，将使用免疫共沉淀法发现P细胞中TMED 9的相互作用物 和质谱分析。然后，将使用以下方法识别MUC 1-fs累积所需的TMED 9相互作用物： CRISPR/Cas9阵列敲除屏幕。最后，这些必要的相互作用物与MUC 1-fs的结合将是 评估。在目的3中，将在体内探索TMED 9在MUC 1-fs积累中的作用。TMED 9基因敲除 小鼠将与已建立的MKD敲入小鼠杂交，并且TMED 9敲除对MKD敲入小鼠的体内影响将被确定。 将评估MUC 1-fs蓄积和其他已知的MKD敲入小鼠表型。

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.