Mechanisms Of Adapter Protein DAB2 In LDLR-mediated Cholesterol Uptake

Objective: The aim of this study is to find out the related genes and new mutation sites regulating low density lipoprotein receptor(LDLR)at gene-protein-environment levels.We also studied how these new genes and mutation sites are involved in the regulation of cholesterol metabolism.Through the study of the molecular mechanism and function of these genes regulating cholesterol,new targets of lipid-lowering drugs were found,which could provide theoretical basis and experimental basis for reducing the incidence of dyslipidemia and coronary heart disease: 1)Selecting healthy population of Han,Uygur and Kazakh nationalities in Xinjiang,using full exome gene second generation sequencing technology to detect candidate cholesterol metabolic regulatory genes,and to find new mutation sites that can affect blood lipid metabolism;2)To investigate the molecular mechanism of DAB2 involved in the regulation of cholesterol absorption and metabolism by screening new mutation sites of DAB2 gene;3)The labeled SNPs of DAB2 gene were selected to analyze the distribution frequencies of genotypes and alleles of DAB2 gene in Han,Uygur and healthy population in Xinjiang,and to study the interaction between these SNPs and risk factors of coronary heart disease.Methods: 1)According to the reference range of low density lipoprotein cholesterol(LDL-C),the ethnic,gender and age-matched general population in Xinjiang were divided into high-value group and low-value group of low density lipoprotein cholesterol(LDL-C).By consulting the literature,32 candidate genes(635 fragments)that may be involved in the regulation of cholesterol metabolism were sequenced for the second generation of exome genes.The correlation between these gene mutation sites and blood lipids was analyzed;2)ARH-KO cell lines were constructed by CRISPR/Cas9 technique.Eukaryotic overexpression plasmids were constructed from 8 newly discovered mutation sites of DAB2 gene.The effects of these new mutations on LDLR function of target protein were studied by using molecular biology techniques such as protein immunoblotting,real-time fluorescence quantitative PCR and immunoprecipitation.The possible grouping mechanism and functional characteristics were discussed;3)1016 patients with coronary heart disease confirmed by coronary angiography and 976 healthy controls were selected.Age,gender and ethnicity matching were observed in the two groups.SNP genotyping was performed by improved multiplex ligase detection reaction method(i MLDR).The correlation between genotype,allele frequency and incidence of coronary heart disease was analyzed.Results: 1)This study selected 500 people of Han,Uygur and Kazakh nationalities who lived in Xinjiang for a long time.According to the reference range of LDL-C,it can be divided into low-value group and high-value group.Thirty-two candidate genes(635 fragments)known to be involved in cholesterol metabolism,absorption,endogenous synthesis,secretion and Post-regulation were sequenced.The results suggested that NPC1L1,DAB2,LDLR,SREBP1,SREBP2,SCAP,Numb,MBTPS1,FLOT2 and other gene variants appeared simultaneously in three ethnic groups.Compared with Han population,NPC1L1,SCAP,DENND4 C and other gene variants are more frequent in Uygur and Kazakh population.Mutations in AMFR,Insig1,MYLIP9 and TBL2 were mainly found in Uygur and Kazakh populations.Some genes were detected only in a certain ethnic group,such as FBXW7,GRINA,Insig2,etc.Eight new mutation sites of DAB2,a key regulator of cholesterol absorption,were detected,including N226 S,R296C,T505 I,D211Y,F702 C,H425R,Q212 E and V544I;2)The results of mechanism study at cell level in ARH-KO SV589 cell line showed that valine at544 of DAB2 gene mutated into isoleucine.After mutation,the protein level of V544 I mutant was uns Table,half-life was shortened,degradation was accelerated and protein expression was reduced,which resulted in the endocytosis clearance of LDL-C mediated by target protein LDLR decreased,and the level of LDL-C in plasma increased.This is consistent with our screening of this mutant from the high-value group of LDL-C;3)The genotype frequency,dominant model and allele frequency of rs2855512 at SNP locus were significantly different from those of the control group(P=0.015,P=0.010 and P=0.006,respectively).The distribution of genotype frequency,dominant model,recessive model and allele frequency of rs2255280 at SNP locus was significantly different from that of control group(P=0.026,P=0.022,P=0.047 and P=0.011,respectively).The dominant models of rs2855512 and rs2255280 were correlated with the occurrence of coronary heart disease(P=0.008,OR=1.439,95% CI: 1.101-1.88,P=0.011,OR=1.416,95% CI: 1.083).The risk of coronary heart disease in AA genotype carriers may increase.For the Uygur population,there was no significant difference between the four SNPs gene models in CHD group and control group.Conclusion: 1)In this study,32 candidate genes that may be involved in the regulation of metabolism of cholesterol absorption offspring were sequenced by whole-gene exome second-generation sequencing technology.We found that the distribution of these genes and their new mutation sites in three populations of Han,Uygur and Kazakh was different.New mutation sites of DAB2,NPC1L1 and LDLR genes involved in the regulation of cholesterol uptake also appeared in three ethnic groups;2)By studying the mechanism of the new mutation site V544 I of DAB2 gene at the cellular level,we found that the protein expression level decreased significantly after the mutation of V544 I of DAB2 gene,and the level of the gene did not change at that time.Therefore,this new mutation site regulates the endocytosis clearance process of LDL-C mediated by LDLR through post-transcriptional mechanism,which affects the level of LDL-C in plasma and blood.The increase of LDL-C level in plasma and the result of immunoprecipitation also confirmed that the V544 I mutation of DAB2 resulted in less binding of DAB2 protein to LDLR and impaired endocrine function of LDLR,which was consistent with the mutant found in the high-value group of LDL-C;3)The polymorphisms of rs2855512 and rs2255280 loci of DAB2 gene were correlated with the occurrence of coronary heart disease in Xinjiang Han population.People with AA genotype may have an increased risk of coronary heart disease.Allele A may be a risk factor for coronary heart disease in Xinjiang Han population.