Abstract 1181: Human PCSK9 Mutations And Post-translational Modifications Provide A Novel View On Its Regulation Of Ldl-cholesterol
INTRODUCTION: PCSK9 enhances the degradation of the low density lipoprotein receptor (LDLR). Point mutations in PCSK9 are associated with either familial hypercholesterolemia or hypocholesterolemia. Some PCSK9 single point mutations result in a gain of function of PCSK9 on the degradation of LDLR in acidic compartments, likely endosomes, while others would cause a loss of function, and these would be associated with the development of hypercholesterolemia or hypocholesterolemia, respectively. The current model is that PCSK9 is secreted as a tight complex with its prosegment and as such can drag its partner, the LDLR, towards the endosomal/lysosomal degradation pathway. This involves the co-trafficking of both proteins in clathrin coated vesicles either following cell surface internalization or directly from the Golgi.
HYPOTHESIS: Some natural mutations leading to loss or gain of function may be related to the regulation of the posttranslational modifications of PCSK9.
METHODS: Site directed mutagenesis of human PCSK9 and the analysis of its biosynthetic fate and that of LDLR in cell lines provided the basis of this study.
RESULTS: Using specific PCSK9 membrane-bound chimeras we first showed that both direct and endocytosis degradation pathways are functional in a number of cell lines. Since the acidic segment comprising amino acid 31– 60 is absent from the crystal structures proposed for PCSK9, we next examined the role of the whole prosegment in regulating the function of PCSK9. Accordingly, we used a sliding mutagenesis approach whereby an optimized furin-like cleavage site RRRREL was introduced at strategic positions along the prosegment sequence. Thus, secreted PCSK9 complexed to various truncated forms of its prosegment were tested for their cellular activity on LDLR. Furthermore, specific point mutations within the prosegment such as R46L (hypocholesterolemia natural mutation), Y38F (Tyr-sulfation mutant) suggested that loss of function may be associated with faster degradation of mutant PCSK9 by endogenous proteases, including metalloproteases.
CONCLUSIONS: The prosegment of PCSK9 plays a critical role in regulating not only its biosynthesis and secretion, but also its ability to interact with the LDLR and enhance its degradation.