PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/ lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N-and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln 152 2, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg 46 2 by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ϳ4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ϳ3-fold higher binding to LDLR in vitro, in >4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ϳ2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg 248 , generating a two-chain PCSK9-⌬N 248 . At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.Complications resulting from cardiovascular disorders are the main cause of death worldwide. High levels of circulating low density lipoprotein-cholesterol represent a major risk factor that leads to coronary heart disease associated with increased death and morbidity worldwide (1). LDL is constantly cleared by internalization into cells by the LDL receptor (LDLR), 4 which binds and internalizes LDL via its unique apolipoprotein B (apoB) protein. Mutations in LDLR or APOB genes are major causes for the frequent autosomal dominant genetic disorder known as familial hypercholesterolemia (2, 3). More recently, the PCSK9 gene (4), which is highly expressed in liver and small intestine (5), was identified as the third locus associated with familial hypercholesterolemia (6). PCSK9 binds the LDLR and triggers its intracellular degradation in acidic endosomes/lysosomes, resulting in increased circulating LDL-cholesterol (7-10).Following its autocatalytic cleavage, PCSK9 is secreted as a stable noncovalent complex with its 122-amino acid (aa 31-152) N-terminal prosegment (5, 7). This cleavage results in a conformational change (11), which favors the binding of PCSK9 to the EGFA domain of the LDLR (12), with much increased affinity at acidic pH values (11).Overexpression studies in liver suggested that PCSK9 targets the LDLR (9, 13, 14) toward degradation in late endosomes/l...

Background: PCSK9 regulates low-density lipoprotein receptor levels in the liver. The importance of the M1, M2, and M3 modules within the C terminus of PCSK9 is unknown. Results: The M2 module is needed for the extracellular, but not intracellular, activity of PCSK9. Conclusion:The integrity of the M2 module is essential for the extracellular function of PCSK9. Significance: Targeting the M2 module should neutralize circulating PCSK9 and reduce LDL-cholesterol.

Background: Annexin A2 (AnxA2) is an extracellular endogenous inhibitor of the PCSK9-LDLR protein-protein interaction. Results: AnxA2 mRNA knockdown in Huh7 cells increases PCSK9 protein levels, and its overexpression in HepG2 cells has the opposite effect. Conclusion: AnxA2 inhibits mRNA translation of PCSK9 and interacts with the M1 ϩ M2 domains of PCSK9. Significance: AnxA2 is a negative regulator of PCSK9 protein levels and activity.

Objective-Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a downregulator of the low density lipoprotein receptor.The aims of this cross-sectional cohort-study were to examine whether the PCSK9 R46L loss of function variant found in a cohort of familial hypercholesterolemia (FH) patients was associated with lower low density lipoprotein cholesterol, lower frequency of xanthomata, and cardiovascular risk. Approach and Results-We studied FH patients attending the IRCM (Institut de Recherches Cliniques de Montréal) LipidClinic and whose DNA genotyping was positive for a low density lipoprotein receptor mutation. The presence of the PCSK9 loss of function R46L missense variant was determined among a cohort of 582 FH patients by genotyping. Frequency of the R46L variant was 3%. Carriers had significantly lower low density lipoprotein cholesterol (11%, P=0.002), total cholesterol (9%, P=0.007), apolipoprotein B (10%, P=0.037), and non-high density lipoprotein (12%, P<0.001) concentrations compared with noncarriers. Furthermore, R46L carriers showed a decreased average number of xanthoma per individual compared with noncarriers (0.33 and 0.76, respectively; P<0.001). Importantly, the R46L genetic variant was associated with a significant 86% lower odd of presenting a cardiovascular event (odds ratio, 0.14; 95% confidence interval, 0.032-0.63; P=0.001). Conclusions-Even though the R46L variant was present in 3% of our FH population, carriers of this polymorphism showed attenuated effect of the low density lipoprotein receptor mutation on parameters, such as low density lipoprotein cholesterol, apolipoprotein B, total cholesterol, and non-high density lipoprotein. More importantly, this mutation is associated with a significant lower risk of cardiovascular disease compared with noncarriers. It is therefore likely that targeting PCSK9 in FH patients with novel anti-PCSK9 therapies will be useful in reducing cardiovascular risk in affected subjects. plasma LDL-C, total cholesterol and, importantly, showed also a decrease in coronary heart disease. 11 Additionally, important observations from meta-analysis have highlighted the association of the R46L carriers to a reduced risk of ischemic heart disease and of early-onset myocardial infarction. 22,23 Presently, statins are the first line medications used to lower LDL-C and the risk of CVD. Unfortunately, statins also upregulate the PCSK9 gene expression, therefore, compromising their LDL lowering effect. 24 Nevertheless, a previous report observed that compound carriers of the PCSK9 R46L variation and of a LDLR mutation associated with FH responded much better to statin therapy compared with controls. 25 A decade of scientific research on PCSK9 resulted in the development of neutralizing molecules, such as monoclonal antibodies. These molecules are currently undergoing phase II/III clinical trials and have been shown to lower LDL-C efficiently by ≈50% to 60%. 26,27,27a,27b Outcome trials are underway to test the ability of these drugs to reduce cardiovascular even...