The Function Of SNX17 In Ciliogenesis

The purpose of this article is to study the function of the SNX17 gene.The Sorting nexin(SNX)protein family is a class of proteins involved in protein transport and endosomal sorting pathways.They contain a common Phox homology(PX)domain that binds to specific phosphatidylinositol phosphates and localize to the vesicles.As a member of the SNX family,in addition to the PX domain,SNX 17 also contains an atypical cargo-recognition 4.1/exrin/radixin/moesin(FERM)domain.Similar to many SNX proteins,SNX 17 is located in early endosomes.Studies have shown that SNX 17 is associated with low-density lipoprotein receptor(LDLR)members,integrins,P-selectin,amyloid precursor proteins,integrins,cytoplasmic factors Kritl,Kif1B and the scavenger receptor FEEL-1/stabilin-1 by regulation of their internalization or circulation from the endosomes to the cell surface.In vivo,SNX17 is also involved in the activation of T cells and plays an important role in receptor recycling.SNX 17 activates the Notch signaling pathway by promoting the cycling of ligand Jagla from the cytoplasm to the cell membrane and maintains different subpopulations of Notch activity in zebrafish pancreatic precursor cells,thereby regulating the differentiation fate of these different precursor cells.The snxl7 mutant mouse showed congenital heart disease phenotypes,such as right ventricle double outlet right ventricle(DORV)and atrioventricular septal defects(AVSD),but the cause of this phenotype was not yet clear.These findings suggest that SNX17 plays an important role in different cellular processes and different developmental stages.Cilia(or flagella)are organelles based on microtubules that extend outward from the cell surface.They are found in almost vertebrate cells and play an important physiological role in a wide range of fields.Cilia can transmit extracellular signal stimulation,regulate fluid flow and determine the left and right asymmetric development of organs.Cilia defects can cause different organ developmental disorders,such as polydactyly,cardiac abnormalities,cystic kidneys,retinal atrophy,hypogonadism.We first suppressed the expression of snx17 protein in the zebrafish resulting in the phenotype of polycystic kidney.Further analysis revealed that the cilia structure was damaged and we preliminary demonstrated the regulation of ciliogenesis through snx17.In order to further study the function mechanism of SNX17 in the process of ciliogenesis,we knocked down the SNX17 gene in the human retinal pigment epithelial RPE1 cell line firstly and found that the proportion of cells that can induce growth of ciliated cells was significantly reduced.We knocked out SNX17 by using CRISPR/Cas9 gene editing system in the RPE1 cell line and the mutant cell line had reduced cilia growth.To verify the specificity of the SNX17,we found that transfecting a mouse-derived SNX17 plasmid in a mutant cell line can rescue defected cilia phenotype.SNX17 moved to the basal body in the ciliogenesis.These results indicate that SNX17 regulates cilia and may be involved in vesicle trafficking.Results from electron microscopy experiment showed that the mutant cell line had an obstacle in vesicles transport and fusion in the early stage of ciliogenesis and the assembly of the basal body was affected,confirming our conclusions.We detected the centriole and satellite centriole proteins associated with cilia in serum-and serum-free culture.We found that PCM1,CEP131,and OFD1 levels were significantly reduced in mutant-free cells cultured in serum-free for 48 hours.Others such as PCNT,TUBG1 were not affected.This demonstrated that SNX17 selectively maintained the stability of satellite centrosome in serum-free culture.The lysosomal inhibitor bafilomycin A1 prevented PCM1 from being degraded,whereas the proteasome inhibitor MG132 did not.This indicated that the loss of SNX17 during cilia formation would resulted in the degradation of PCM1 into the lysosome pathway.Previous studies have shown that the E3 ligase MIB1 can ubiquitinate PCM1 and inhibit the growth of cilia.In mutant cells,we found that ubiquitination of PCM1 reached the highest level in serum-free culture starved for 24 hours and ubiquitination mainly through the K63 site.To verify whether MIB1 plays an important role in the ubiquitination of PCM1,we knocked down MIB1 and found that ubiquitination of PCM1 in the 24-hour mutant of starvation was significantly reduced.Mutant cells knocked down by MIB1 could prevent PCM1 from being degraded.To a certain extent,this could rescue the phenotype of cilia defect.The deubiquitinated enzyme USP9X can stabilize the proteins on satellite centrosome proteins,particularly PCM1,and the mutants lacking SNX17 also degraded USP9X after serum-starvation,indicating that SNX17 stabilized the normal distribution of USP9X.The results of co-immunoprecipitation experiments showed that compared with normal culture,the interaction between SNX17 and PCM1 was significantly enhanced after starvation culture,but there was no interaction with MIB1 and the interaction between SNX17 and USP9X kept stable.It showed that under starvation conditions,SNX17 maintained the stability of PCM1 through USP9X to prevent degradation by MIB1.SNXs were rarely involved in the regulation of cilia.Apart from vesicle trafficking,we first demonstrated that SNX17 has regulated the basal body of cilia by affecting the stability of the satellite centrosome.This belonged to an indirect regulatory pathway that influences ciliogenesis.Our work can provide some theoretical basis for future cilia diseases research.