Related Experiment Video
Updated: Jun 8, 2026
A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
Published on: August 28, 2018
Effects of the prosegment and pH on the activity of PCSK9: evidence for additional processing events
Suzanne Benjannet1, Yascara Grisel Luna Saavedra, Josée Hamelin
1Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada.
Abstract:
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)↓, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg(46)↓ by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ∼4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ∼3-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 ∼2.5-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-ΔN(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.
Insights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) activity is regulated by its prosegment and pH. Cleavage of the PCSK9 prosegment and acidic endosomal pH enhance its LDL receptor degradation activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of LDL cholesterol levels by promoting LDL receptor (LDLR) degradation.
- The precise roles of PCSK9's N- and C-terminal domains and regulatory mechanisms are not fully understood.
- PCSK9 undergoes autocatalytic processing, but further regulatory cleavage events remain to be elucidated.
Purpose of the Study:
- To investigate the functional roles of PCSK9's N-terminal prosegment and the impact of pH on its activity.
- To identify novel cleavage sites and their effect on PCSK9's interaction with LDLR.
- To understand the mechanisms regulating PCSK9 function in the context of dyslipidemia treatment.
Main Methods:
- Site-directed mutagenesis to analyze PCSK9 prosegment cleavage at Arg46 and Arg248.
- In vitro binding assays to assess PCSK9-LDLR interaction.
- Cellular assays using insect and mammalian cells to measure LDLR degradation and PCSK9 activity.
- pH shift experiments to mimic endosomal conditions.
Main Results:
- PCSK9 can be further cleaved at Arg46 by cellular proteases, enhancing its activity up to 4-fold.
- Removal of the prosegment acidic stretch increases PCSK9 binding to LDLR, cellular activity, and internalization rate.
- Exposure to acidic pH (6.5-5.5) increases PCSK9 activity and promotes a second cleavage at Arg248, forming a two-chain PCSK9.
- PCSK9 dissociates from its prosegment and loses activity below pH 5.5.
Conclusions:
- The N-terminal prosegment of PCSK9 contains an acidic stretch that inhibits its function.
- Proteolytic cleavage of the PCSK9 prosegment and acidic endosomal pH are novel mechanisms for PCSK9 activation.
- These findings provide a deeper understanding of PCSK9 regulation and potential therapeutic strategies for dyslipidemia.
Related Concept Videos
01:25cAMP-dependent Protein Kinase Pathways
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:09GPCRs Regulate Adenylyl Cylase Activity
Two...
01:12GPCR Desensitization
01:16Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
01:22Amplifying Signals via Enzymatic Cascade