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

摘要 慢性肾脏病(CKD)，通常由系统性疾病或潜在的原发疾病的表现引起 肾脏疾病是世界范围内患者发病率和死亡率的重要原因。肾脏病的领域是 严重缺乏基于机制的、有针对性的治疗CKD患者的药物。对原生的、稀有的研究 遗传性肾脏疾病会导致常见的、基本的肾脏过程紊乱，可以加以治理。 向基础肾脏生物学提供信息，并确定肾脏疾病的潜在治疗目标，包括罕见和 很普通。MUC1肾病(MKD)或常染色体显性遗传性肾小管间质肾病(ADTKD- MUC1)，是一种罕见的遗传性肾病，由MUC1基因移码突变引起。MKD导致 进行性肾小管间质损害和最终肾功能衰竭。最近的研究表明，MKD是 一种毒性蛋白病，突变的移码蛋白MUC1-fs在肾脏细胞内积聚 肾小管上皮细胞，并与细胞毒性水平增加有关。进一步显示了MUC1-FS 为了在MKD患者肾小管上皮细胞(P细胞)的早期分泌途径中特异性积聚， 在含有TMED9蛋白的囊泡中。关键的是，已经发现TMED9的敲除可以改善 P细胞内MUC1-fs的积聚。TMED9和MUC1-FS已被证明在两个 活体MKD小鼠模型和MKD患者来源的IPSC有机化合物。最近的研究表明，TMED9联合- 免疫共沉淀物与P细胞中的MUC1-f共沉着。基于这些观察，这项拟议的工作旨在 调查以下假设：在MKD的背景下，TMED9及其相互作用者停止贩运 错误折叠MUC1-fs并将其保留在早期分泌途径中，导致MUc1-fs积累和增加 细胞毒性。在目标1中，TMED9对通过早期分泌途径运输MUC1-fs的影响将 通过敲除P细胞中的TMED9和通过抑制分泌来定位MUC1-fs的运输来评估 MUc1-fs再积聚的通路分支和评估。TMED9作为细胞内信号调节因子的作用 在使用内质网应激诱导剂治疗后，将通过比较细胞毒性来评估毒性 Thapsigargin，在P细胞和TMED9基因敲除的P细胞中。在目标2中，介导MUC1-1的TMED9蛋白复合体 将确定FS累积情况。首先，将通过免疫共沉淀发现P细胞中TMED9的相互作用 和质谱学。然后，将使用以下方法确定MUC1-fs积累所需的TMED9交互作用子 CRISPR/CAS9阵列淘汰屏。最后，这些必要的相互作用因子与MUC1-f的结合将是 评估过了。在目标3中，将探讨TMED9在体内MUC1-fs蓄积中的作用。TMED9基因敲除 将小鼠与已建立的MKD基因敲除小鼠杂交，观察TMED9基因敲除对小鼠体内的影响 将评估MUC1-fs的累积和其他已知的MKD敲入小鼠的表型。

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.