ABSTRACT
Proprotein Convertase Subtilisin Kexin9 (PCSK9), originally called Neural Apoptosis-Regulated Convertase1 (NARC1), is the latest member of mammalian subtilase super-family. Since its discovery in 2003, it has drawn significant attention because of its function in the degradation of Low Density Lipoprotein Receptor (LDL-R). LDL-R removes circulating LDL-cholesterol (LDL-C) in the blood. Increased level of PCSK9 functional activity will lead to an accumulation of cholesterol in the blood - a high risk factor for cardiovascular disease. This is confirmed by PCSK9 knock out and transgenic animals, various biochemical and clinical studies involving "gain and loss of function" genetic mutations of PCSK9 found in various subset of populations. Owing to this finding, development of strategies for inhibition of PCSK9 function has drawn significant research interest for therapeutic intervention of hypercholesterolemia. Thus PCSK9 is a target for the development of new cholesterol lowering drugs.
Subject(s)
Anticholesteremic Agents/pharmacology , Hypercholesterolemia/drug therapy , Hypercholesterolemia/metabolism , Proprotein Convertases/antagonists & inhibitors , Proprotein Convertases/metabolism , Serine Endopeptidases/metabolism , Animals , Humans , Mice , Molecular Targeted Therapy , Proprotein Convertase 9 , Proprotein Convertases/genetics , Serine Endopeptidases/geneticsABSTRACT
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis, mediating degradation of the liver low-density lipoprotein receptor (LDLR). In fact, gain- and loss-of-function PCSK9 variations in human populations associate with hyper- or hypo- cholesterolemia, respectively. Exactly how PCSK9 promotes degradation of the LDLR, the identity of the other biomolecules involved in this process, and the global effect of PCSK9 on other proteins has not been thoroughly studied. Here we employ stable isotope labeling with amino acids in cell culture (SILAC) to present the first quantitative, subcellular proteomic study of proteins affected by the stable overexpression of a gain-of-function PCSK9 membrane-bound chimera (PCSK9-V5-ACE2) in comparison to control, empty vector transfections in a human hepatocyte (HuH7) cell line. The expression level of 327 of 5790 peptides was modified by PCSK9-V5-ACE2 overexpression. Immunoblotting was carried out for the control transferrin receptor, shown to be unaffected in cells overexpressing PCSK9-V5-ACE2, thus validating our SILAC results. We also used immunoblotting to confirm the novel SILAC results of up- and down-regulation of several proteins in cells overexpressing PCSK9-V5-ACE2. Moreover, we documented the novel down-regulation of the EH domain binding protein-1 (EHBP1) in a transgenic PCSK9 mouse model and its up-regulation in a PCSK9 knockout mouse model.
