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
- 批准号:10360154
- 负责人:
- 金额:$ 36.85万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ADAMTS1 geneADAMTS9 geneAffectAntibodiesAppearanceBrainC-terminalCRISPR/Cas technologyCell Culture TechniquesCellsChildCiliaCoiled-Coil DomainCorpus CallosumCysteine-Rich DomainCystic Kidney DiseasesCystic kidneyCytoplasmic TailDataDevelopmentDiagnosisDiseaseDisintegrinsEmbryoEndocytic VesicleEngineeringEpithelialExtracellular DomainExtracellular MatrixFamilyHomeostasisHumanIntegral Membrane ProteinJoubert syndromeKidneyKidney DiseasesKnockout MiceMammalian CellMammalsMeckel-Gruber syndromeMediatingMetalloproteasesModelingMusMutationN-terminalNephronophthisisNotoencephaloceleNull LymphocytesOrganellesPathologyPathway interactionsPatientsPeptide HydrolasesPlayProteinsProteolysisProteolytic ProcessingProteomeProteomicsRecyclingRenal tubule structureReportingResearchRoleSignal TransductionSiteStructural ProteinStructureTestingThrombospondinsTransgenic MiceTubular formationVariantVesicleWNT Signaling PathwayWestern Blottingbaseciliopathycilium biogenesisconditional knockoutexperimental studyextracellularfluid flowin vivo Modelinsightkidney cellloss of functionmechanotransductionmutantnephrogenesisnoveloverexpressionreceptor
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.
摘要:ADAMTS9(一种带有血栓反应蛋白1基序的去整合素和金属蛋白酶,9),一种分泌型
已知的金属蛋白酶调节细胞外基质(ECM)的动态,对原发纤毛是必不可少的。
老鼠和人类的生物发生。ADAMTS9基因突变导致严重的髓质肾单位病
由于原生纤毛丧失而引起的囊性肾病。ADAMTS9在肾脏中的作用机制
发育和肾脏疾病之间的关系尚不清楚。利用N-末端组学,我们现在已经确定了一种新的
ADAMTS9底物,TMEM67,纤毛过渡区的关键成分。与ADAMTS9类似,
TMEM67突变也会导致NPHP。已知TMEM67的胞外结构域在
规范的和非规范的Wnt信号转导,而细胞质尾部的螺旋结构域需要
纤毛移行区集结。我们假设ADAMTS9介导的TMEM67切割是必不可少的
用于纤毛发生,从而促进哺乳动物肾脏的正常发育。
为了研究ADAMTS9介导的TMEM67切割在肾脏发育中的作用,我们将有条件地
删除小鼠肾脏中的Adamts9。探讨TMEM67下游的细胞机制
纤毛发生和细胞信号的分裂,我们将利用哺乳动物细胞培养中的挽救实验
模特们。先导数据显示Adamts9条件缺失(CKO)在小鼠肾脏中是存活的和CKO小鼠
明显的肾脏病变。在野生型RPE-1细胞中,TMEM67的N端胞外区片段
ADAMTS9的裂解不定位于纤毛,而胞内C末端定位于纤毛。被认同的小说
哺乳动物中的裂解位点是100%保守的,裂解残基的突变导致囊性肾
人类的疾病。我们推测TMEM67可能是一个双功能、双基序的分子,并且
ADAMTS9裂解通过蛋白降解过程分离这些功能。这样做的具体目的是
建议:1)研究ADAMTS9介导的TMEM67在小鼠肾脏中的切割作用;2)研究ADAMTS9在小鼠肾脏中的切割作用
通过揭示下游的作用机制,研究TMEM67裂解如何影响纤毛发生。
影响:TMEM67突变是全球MKS(梅克尔综合征)的主要原因,
了解其功能对纤毛疾病的研究具有重要意义。多关键睫状结构
导致破坏性肾纤毛疾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、TMEM67、TCTN 1/2、TMEM216.等)。它们有不同的胞外结构域,并且
一些已知是由未知的蛋白酶脱落的。在这里我们已经鉴定了这两种酶及其
衬底,给了我们一个独特的机会来进行基础实验并获得更深层次的机制
纤毛胞外蛋白水解酶与胞外区脱落的复杂关系
跨膜蛋白。
项目成果
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Sumeda Nandadasa其他文献
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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: