Abstract
Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the Low density lipoprotein receptor (LDLr). Its autocatalytic cleavage is necessary to its processing within the cell and the targeting of the LDLr. Therefore identifying molecules that would modulate this activity is an important challenge. The possibility of measuring this activity has been debated. Using whole hepatocytes lysates and optimized conditions, we showed that the cleavage of a fluorogenic peptide corresponding to proPCSK9 cleavage site is specific to this proprotein convertase. Indeed, the fluorogenic activity was virtually absent in primary hepatocytes from PCSK9 knockout mice compared with wild type hepatocytes. In human immortalized hepatocytes (IHH), a reduction of PCSK9 expression by 40 to 60% using SiRNA technology reproducibly decreased PCSK9 activity in a range of similar magnitude. Maldi-Toff analysis of the cell lysate following the the experiment showed that the fluorogenic peptide is cleaved at the right site LVFAQ152. In IHH cells exposed to the Liver X agonist T0901317 1 microM or fenofibric acid 250microM, or to both drugs concomitantly for 48h, PCSK9 protein content was respectively increased by 60% (p<0.5), decreased by 70% (p<0.01) or decreased by 30% (p<0.01). We observed similar variations of the fluorogenic activity of these cell lysates. We also observed that PCSK9 autocatalytic activity was increased in cells accumulating PCSK9 upon exposure to pravastatin 10microM for 48h, and that the repressive effect of fenofibric acid 250microM overcame the induction by pravastatin, in agreement with our previous works on PCSK9 transcriptional regulation. We also analysed the catalytic activity of PCSK9 gain of function variants. Altogether, these results show that PCSK9 endogenous autocatalytic activity can be measured in cellulo. Futhermore, the catalytic activity reflects PCSK9 protein abundance. We suggest that this test could also be used to identify postraductional modulators of PCSK9 activity.
Published Version
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