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

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.