Molecular Mechanisms And Functions Of APLP2 Variants Regulation Cholesterol Metabolism

Objective: The aim of this study was to investigate the molecular mechanisms and functions of amyloid precursor-like protein 2(APLP2)-related genes and new mutations,and to study the mechanisms and functions of cholesterol metabolism in gene/protein/environment multi-levels.Offering a novel therapeutic approach complementary to statins for treating hyperlipidemia and cardiovascular disease.1)Mutants of the APLP2 gene were investigated in Xinjiang population by using extreme genotype and second generation sequenceing techinque,and APLP2 gene methylation levels were measured by using the Methyltarget high-throughput sequencing method.2)To reveal the molecular mechanisms of the new variants of APLP2 in cholesterol metabolism.3)To investigate the relationships between single nucleotide polymorphisms(SNPs)of APLP2 gene and the prevalence of dyslipidemia in Xinjiang populations.Methods: 1)Using extreme genotype strategy,60 subjects from Xinjiang Kazakh and Uygur ethnic groups were selected,and the correlations between APLP2 gene and lipid parameters were analyzed.Using case-group study method,the methylation levels of APLP2 gene was calculated.2)At the cellular level,using different molecular biology research methods including immunoprecipitation,real-time quantitative PCR to investigate the molecular mechanisms of APLP2 protein expression,stability,degradation and other characteristics and effects.The effects of target proteins ApoE/LRP1 and PCSK9/LDLR function,and explore the mechanisms and functions involved in regulating cholesterol metabolism.3)1738 subjects from Han,Uygur,and Kazakh populations in Xinjiang were selected in ?35 years old adults,and the APLP2 gene tag-SNPs were expanded sample size verification by using Taqman technology.Results: 1)A total of 11 new unreported non-synonymous variants(Q194E,G42E,G295S,M323 I,D329N,S447 N,R468C H534 Q,V535M,D573Y and P601A)were found.Q194 E,G42E,M323 I,S447N,R468C,and P601A were appeared in the very high LDL-C group,and the rest wae appeared in the very low LDL-C group.Methylation levels of APLP2 gene was higher in the case group.2)The results of the mechanism study at the cellular levels showed that compared with wild-type APLP2,the protein expression level of APLP2 G42E variant was significantly reduced,and the protein expression level of APLP2 Q194E variant was significantly increased.However,there were no significant differences in two variants at the m RNA levels.APLP2 G42E has a relatively short half-life and relatively rapid degradation speed,while APLP2 Q194E has a relatively long half-life and relatively slow degradation speed.Wild-type APLP2 could interact with Apo E.Compared with wild-type,APLP2 G42 E and APLP2 Q194E have no significant difference in binding to Apo E.APLP2 wild type interacts with LDLR and is a dose-dependent increase.Compared with the wild type,APLP2 G42E and APLP2 Q194 have no significant differences in PCSK9 mediated LDLR degradations.3)The rs2054247 polymorphism of APLP2 gene was correlated with serum total cholesterol level and low-density lipoprotein cholesterol levels(P<0.05).The rs2054247 polymorphism of APLP2 gene was associated with serum high-density lipoprotein cholesterol(P<0.05).However,the rs2054247 polymorphism was not associated with serum triglyceride levels(P>0.05).There was no significant associations between rs374088 SNP and serum total high cholesterol,low density lipoprotein cholesterol,high density lipoprotein cholesterol and triglyceride levels(P>0.05).The rs747180 SNP was only correlated with serum total cholesterol and low-density lipoprotein cholesterol levels(P<0.05).Conclusion: 1)In the present study,11 non-synonymous mutation sites of APLP2 gene were screened from Xinjiang Uygur and Kazakh populations.The methylation levels of APLP2 gene in patients with coronary heart disease was higher.2)By investigating the mechanisms of new APLP2 gene variants G42E and Q194E at the cellular level,it was found that G42E and Q194E variants did not affect cholesterol levels.Meanwhile,the two variants did not affect the binding of Apo E/LRP1,nor did they affect the degradation of PCSK9-mediated LDLR.3)APLP2 gene rs2054247 and rs747180 polymorphisms are associated with high TC and high LDL-C levels in Xinjiang population.The rs429358 polymorphism of Apo E gene is associated with the incidence of coronary heart disease in Xinjiang Uygur population.