Extracellular protease modulation of the cilium transition zone in kidney development and disease

肾脏发育和疾病中纤毛过渡区的细胞外蛋白酶调节

• 批准号: 10360154
• 负责人: Sumeda Nandadasa
• 金额: $ 36.85万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10360154
• 关键词: ADAMTS1 gene; ADAMTS9 gene; Affect; Antibodies; Appearance; Brain; C-terminal; CRISPR/Cas technology; Cell Culture Techniques; Cells; Child; Cilia; Coiled-Coil Domain; Corpus Callosum; Cysteine-Rich Domain; Cystic Kidney Diseases; Cystic kidney; Cytoplasmic Tail; Data; Development; Diagnosis; Disease; Disintegrins; Embryo; Endocytic Vesicle; Engineering; Epithelial; Extracellular Domain; Extracellular Matrix; Family; Homeostasis; Human; Integral Membrane Protein; Joubert syndrome; Kidney; Kidney Diseases; Knockout Mice; Mammalian Cell; Mammals; Meckel-Gruber syndrome; Mediating; Metalloproteases; Modeling; Mus; Mutation; N-terminal; Nephronophthisis; Notoencephalocele; Null Lymphocytes; Organelles; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Play; Proteins; Proteolysis; Proteolytic Processing; Proteome; Proteomics; Recycling; Renal tubule structure; Reporting; Research; Role; Signal Transduction; Site; Structural Protein; Structure; Testing; Thrombospondins; Transgenic Mice; Tubular formation; Variant; Vesicle; WNT Signaling Pathway; Western Blotting; base; ciliopathy; cilium biogenesis; conditional knockout; experimental study; extracellular; fluid flow; in vivo Model; insight; kidney cell; loss of function; mechanotransduction; mutant; nephrogenesis; novel; overexpression; receptor

SUMMARY: ADAMTS9 (A Disintegrin and Metalloproteinase with thrombospondin 1 motifs, 9), a secreted metalloproteinase known to regulate extracellular matrix (ECM) dynamics, is essential for primary cilium biogenesis in mice and humans. ADAMTS9 mutations result in nephronophthisis (NPHP), a severe medullary cystic kidney disease caused by loss pf primary cilia. The mechanism of ADAMTS9 function in kidney development and renal disease is not known. Utilizing N-terminomics, we have now identified a novel ADAMTS9 substrate, TMEM67, a key component of the ciliary transition zone. Similar to ADAMTS9, TMEM67 mutations also cause NPHP. The extracellular domain of TMEM67 is known to play crucial roles in canonical and non-canonical Wnt signaling whilst the coil-coiled domain in the cytoplasmic tail is required for cilium transition zone assembly. We hypothesize that ADAMTS9-mediated TMEM67 cleavage is essential for ciliogenesis and hence for the normal development of the mammalian kidney. To investigate ADAMTS9-mediated TMEM67 cleavage in kidney development we will conditionally delete Adamts9 in the murine kidney. To investigate the cellular mechanism downstream of TMEM67 cleavage in ciliogenesis and cell signaling, we will utilize rescue experiments in mammalian cell culture models. Pilot data show that Adamts9 conditional deletion (cKO) in the murine kidney is viable and cKO mice manifest renal pathologies. In wildtype RPE-1 cells, the N-terminal extracellular domain fragment of TMEM67 cleaved-off by ADAMTS9 is not cilium localized while the intracellular C-terminus is. The identified novel cleavage site is 100% conserved in mammals and mutations of the cleavage residues result in cystic kidney disease in humans. We hypothesize that TMEM67 may be a bi-functional and bi-motif molecule and ADAMTS9 cleavage segregates these functions by proteolytic processing. The Specific Aims of this proposal are: 1) To investigate ADAMTS9-mediated TMEM67 cleavage in the murine kidney and 2) To investigate how TMEM67 cleavage affect ciliogenesis by uncovering the downstream mechanism of action. Impact: TMEM67 mutations are the leading cause of MKS (Meckel syndrome) worldwide and understanding its functionality is highly significant to ciliopathy research. Many key ciliary structural proteins that result in devastating renal ciliopathies are also transmembrane molecules (polycystin1, polycystin2, fibrocystin, TMEM67, TCTN 1/2, TMEM216.. etc.). They have distinct extracellular domains, and some are known to be shed by unknown proteases. Here we have identified both the protease and its substrate, giving us a unique opportunity to perform fundamental experiments and gain deep mechanistic insight into the intricate relationship of extracellular proteases and ectodomain shedding of ciliary transmembrane proteins.

摘要：ADAMTS9（一种脱节蛋白和金属蛋白酶，具有血小板蛋白1个图案，9），一个分泌的 已知可以调节细胞外基质（ECM）动力学的金属蛋白酶酶，对于原代纤毛至关重要 小鼠和人类的生物发生。 ADAMTS9突变导致肾植物（NPHP），这是一种严重的髓质 由PF原发性纤毛损失引起的囊性肾脏疾病。 Adamts9功能在肾脏中的机制 发育和肾脏疾病尚不清楚。利用N末端学，我们现在已经确定了一本小说 ADAMTS9底物TMEM67，纤毛过渡区的关键组成部分。类似于adamts9， TMEM67突变也引起NPHP。已知TMEM67的细胞外域在 规范和非典型的Wnt信号传导，而细胞质尾部的线圈式结构域则需要 纤毛过渡区组件。我们假设ADAMTS9介导的TMEM67裂解是必不可少的 用于纤毛发生，因此是哺乳动物肾脏的正常发育。 为了研究ADAMTS9介导的TMEM67裂解在肾脏发育中，我们将有条件地 在鼠肾中删除Adamts9。研究TMEM67下游的细胞机制 在纤毛生成和细胞信号传导中裂解，我们将利用哺乳动物细胞培养的救援实验 型号。试点数据显示，鼠肾中的ADAMTS9条件缺失（CKO）可行，CKO小鼠 明显的肾脏病理。在WildType RPE-1细胞中，TMEM67的N末端细胞外域碎片 在细胞内C末端时，ADAMTS9裂解不是纤毛的。确定的小说 切割位点在哺乳动物中100％保守，裂解残基突变导致囊性肾脏 人类疾病。我们假设TMEM67可能是双官能和双摩托合分子，并且 ADAMTS9裂解通过蛋白水解处理将这些功能隔离。这个特定的目的 提案是：1）调查鼠肾中的ADAMTS9介导的TMEM67裂解和2） 研究TMEM67裂解如何通过发现下游作用机理来影响纤毛发生。 影响：TMEM67突变是全球MK（Meckel综合征）的主要原因，并且 了解其功能对于纤毛病研究至关重要。许多关键睫状结构 导致肾纤毛病的蛋白质也是跨膜分子（polycystin1，polycystin1 polycystin2，纤维细胞蛋白，TMEM67，TCTN 1/2，TMEM216 ..等）。它们具有独特的细胞外域，并且 已知有些是由未知蛋白酶脱落的。在这里，我们确定了蛋白酶及其 底物，为我们提供了执行基本实验并获得深度机械的独特机会 洞悉细胞外蛋白酶和纤毛脱落的异位域的复杂关系 跨膜蛋白。