Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a low-abundance plasma protein that reduces the expression of the LDL receptor (LDLR) and therefore plays a critical role in cholesterol trafficking. Its discovery revolutionized our understanding of cholesterol metabolism, shifting the homeostatic paradigm from a system where all the molecular determinants of cholesterol production and internalization are regulated by cellular responses, to one where the highest level of regulation is based extracellularly. Although the mechanism of action of PCSK9 on LDLR is well established, our understanding of the functional output and kinetic pathway of PCSK9 remains limited. We present preliminary results on three common post-translational modifications that alter PCSK9 and drive kinetic and functional changes: 1. Binding to apoB-containing lipoproteins (we now show that it prolongs plasma half-life of PCSK9 and increases its binding affinity to the LDLR); 2. Cleavage by furin (we now show that this is an extracellular event, and that furin-cleaved PCSK9 has accelerated clearance, impaired apoB-binding, and preserved ability to induce LDLR degradation intracellularly); and 3. N- glycosylation (we now show that non-glycosylated PCSK9 is less stable than its glycosylated form). Thus, the first aim of this competing renewal application focuses on how these post-translational modifications impact PCSK9 kinetics and function and affect cholesterol metabolism. Improved understanding of the global and net effect of plasma PCSK9 will open avenues for innovative diagnostic tools and tailored interventions to block PCSK9 action and lower plasma LDL cholesterol levels. Moreover, we have recently reported that therapeutic monoclonal antibodies inhibiting PCSK9 activity also induce a rapid and drastic (up to 20-fold) increase in plasma PCSK9 levels in patients receiving a few injections. This effect offers an opportunity to investigate the regulation of PCSK9 in plasma and to refine our knowledge of the turnover of this critical protein. We present preliminary data showing that the rise in plasma PCSK9 in humans occurs within a few hours after the first antibody injection, and can be reproduced in the mouse model, an observation that we will exploit for rigorous investigations of PCSK9 synthesis, secretion, processing, and clearance. Thus, the second aim of the program will elucidate the effect of plasma PCSK9 blockade on the regulation of hepatic PCSK9 synthesis or secretion in relation to its expected influence on PCSK9 clearance. Overall, the proposed work will provide an improved understanding of PCSK9 kinetics and activity on LDLR regulation, and may identify novel targets for pharmacologic inhibition of PCSK9.
Lowering of LDL cholesterol is critical to reduce the risk of heart attacks. PCSK9 is a plasma protein that regulates the LDL receptor in the liver, and its inhibition causes extreme LDL cholesterol lowering and reduces cardiovascular risk. In the first cycle of our grant we discovered that PCSK9 binds to some lipoproteins and that PCSK9-inhibiting antibodies raise plasma PCSK9 levels. These observations set the stage for our new plan of work, which aims at understanding common factors affecting residence time and function of plasma PCSK9 and how these can be exploited to develop new diagnostics or therapeutic leads.
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