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

基本信息

项目摘要

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.
ADAMTS 9(A Disintegrin and Metalloproteinase with thrombospondin 1 motifs,9),一种分泌型的 已知调节细胞外基质(ECM)动力学的金属蛋白酶是初级纤毛所必需的 在小鼠和人类中的生物发生。ADAMTS 9突变导致肾单位结核(NPHP),这是一种严重的骨髓 由于初级纤毛丧失而引起的囊性肾病。ADAMTS 9在肾脏中的作用机制 发展和肾脏疾病是未知的。利用N-末端组学,我们现在已经确定了一种新的 ADAMTS 9底物,TMEM 67,纤毛过渡区的关键成分。与ADAMTS 9类似, TMEM 67突变也会导致NPHP。已知TMEM 67的胞外结构域在肿瘤的发生中起关键作用。 典型和非典型Wnt信号传导,而细胞质尾区中的卷曲螺旋结构域是 纤毛过渡区组装。我们假设ADAMTS 9介导的TMEM 67切割是必需的, 对于纤毛发生和哺乳动物肾脏的正常发育。 为了研究肾脏发育中ADAMTS 9介导的TMEM 67切割,我们将有条件地 删除小鼠肾脏中的Adamts 9。研究TMEM 67下游的细胞机制 我们将利用哺乳动物细胞培养中的拯救实验, 模型初步数据显示,小鼠肾脏中的Adamts 9条件性缺失(cKO)是可行的,并且cKO小鼠 明显的肾脏病变。在野生型RPE-1细胞中,TMEM 67的N-末端胞外结构域片段 被ADAMTS 9切断的蛋白质不位于纤毛上,而位于细胞内的C-末端。鉴定的新型 切割位点在哺乳动物中是100%保守的,切割残基的突变导致囊性肾 人类的疾病。我们假设TMEM 67可能是一种双功能和双基序分子, ADAMTS 9裂解通过蛋白水解加工分离这些功能。具体目标是 建议是:1)研究ADAMTS 9介导的小鼠肾脏中的TMEM 67切割和2) 通过揭示下游作用机制来研究TMEM 67切割如何影响纤毛发生。 影响:TMEM 67突变是全球MKS(Meckel综合征)的主要原因, 了解其功能对纤毛疾病的研究具有重要意义。许多关键的纤毛结构 导致破坏性肾纤毛病的蛋白质也是跨膜分子(多囊蛋白1, 多囊蛋白2、纤维囊蛋白、TMEM 67、TCTN 1/2、TMEM 216。等)。它们具有不同的细胞外结构域, 一些已知由未知蛋白酶脱落。在这里,我们已经确定了蛋白酶及其 基板,给我们一个独特的机会,进行基础实验,并获得深入的机制 深入了解细胞外蛋白酶和纤毛细胞胞外域脱落的复杂关系 跨膜蛋白

项目成果

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Sumeda Nandadasa其他文献

Sumeda Nandadasa的其他文献

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{{ truncateString('Sumeda Nandadasa', 18)}}的其他基金

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:
    10539298
  • 财政年份:
    2022
  • 资助金额:
    $ 36.85万
  • 项目类别:
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