Degradation of LDLR protein mediated by ‘gain of function’ PCSK9 mutants in normal and ARH cells

摘要: Dominant gain-of-function mutations in proprotein convertase subtilisin kexin type 9 (PCSK9) cause familial hypercholesterolaemia (FH) and result accelerated atherosclerosis premature coronary heart disease. It is believed that PCSK9 binds to LDL-receptor (LDLR) protein prevents its recycling the cell surface; mutants enhance LDLR degradation. Several new variants of have been identified, but their effect on activity has not determined. We describe a procedure for assessing four putative identified FH patients (D129N, D374H, N425S, R496W). All mutant proteins were secreted normally from transfected HEK293T cells. Immortalized lymphocytes normolipaemic controls incubated with conditioned medium cells cell-surface was determined by FACS. D374H as potent D374Y reducing LDLR, while other three more than wild type, less so D374 mutants; this correlated total serum cholesterol patients. Substitution different amino acids at 374 showed aspartate position critical; even glutamate residue increased When assay carried out ARH-negative are unable internalise D374Y-PCSK9 able reduce 35%, compared ∼70% normal lymphocytes. Thus, PCSK9-mediated degradation entirely dependent ARH function. propose novel ARH-independent pathway LDLR.