The mAb approach has also targeted the CHRD domain of PCSK9 and revealed that the best mAb reduces by only 50% the PCSK9dependent inhibitory effects on LDL uptake, without affecting the PCSK9;LDLR interaction. Our previous studies revealed that the R1 domain of AnxA2 can specifically bind the CHRD of PCSK9 and inhibit the function of this protein on LDLR, representing the first example of a natural inhibitor of PCSK9 activity. However, since the expression of AnxA2 is not abundant in liver, the major source of PCSK9, the physiological significance of this observation remained obscure. In this context, it was also observed that injection of PCSK9 in the bloodstream of mice spared the LDLR in a number of tissues, but was very active in selectively reducing the hepatic levels of this receptor. Furthermore, transgenic mice overexpressing PCSK9 in hepatocytes or kidney also had little effect on LDLR levels in a number of MK-0683 extrahepatic tissues. This suggested that the mechanism for PCSK9 induced LDLR degradation might either lack a specific regulator in these tissues, or that PCSK9 function may be inhibited therein. Because AnxA2 is the only known natural inhibitor of PCSK9, we decided to test its possible regulation of PCSK9 function by analyzing the consequences of its genetic deletion. We therefore used AnxA22/2 mice to test the implication of AnxA2 in PCSK9 biology in extrahepatic tissues. In mouse adrenals, cholesterol is mostly obtained from circulating HDL via the SR-BI receptor. Since the adrenal steroid hormone production is normal in mice lacking LDLR, the role of the latter and its lack of regulation by PCSK9 in presence of AnxA2 are yet to be better elucidated. However, in humans, LDLR is also important for cholesterol uptake by adrenals in the acute phase of steroidogenesis and seems to be a major receptor that provides the cholesterol needed for steroid hormone production. How does AnxA2 regulate the functional activity of the PCSK9;LDLR complex? It is possible that by binding to the CHRD, AnxA2 induces a conformational change in PCSK9 such that its EX 527 HDAC inhibitor interaction with the LDLR, and/or its cellular internalization is compromised. This allosteric model is supported by the observation that extracellular mAbs to the CHRD can also inhibit, albeit up to 50%, PCSK9-induced LDLR degradation without affecting the PCSK9;LDLR interaction. This emphasizes the importance of the CHRD in regulating the PCSK9 function on the LDLR internalization and degradation. However, it seems that these mAbs do not compete with AnxA2 in inhibiting PCSK9 function. In our study, the peptides mimicking AnxA2 R1 domain directly inhibit the PCSK9;LDLR interaction. Thus, modulating the CHRD function at multiple sites can reduce the PCSK9;LDLR interaction and/or LDLR degradation. It was recently proposed that the prosegment of PCSK9 could interact with the CHRD within the ER and favors its secretion and may thus affect the R1 AnxA2-CHRD interaction. However, the recent crystal structure of the soluble extracellular ectodomain of LDLR in complex with PCSK9 did not show any interaction between the prosegment in mature PCSK9 and the CHRD at neutral pH. Therefore the role of the prosegment in regulating the extracellular PCSK9-AnxA2 interaction is not yet clear. None of the published therapeutic anti-PCSK9 approaches used a small molecule inhibitor, possibly due to the relative flatness of the surface of interaction of the PCSK9;LDLR complex. Since we had already shown that the R1 domain of AnxA2 is the critical segment interacting with the CHRD, we further investigated the functional structural determinants within this domain using a far Western approach.