Asociation of PCSK9 with Low Density Lipoproteins (LDL) in the Regulation of LDL-Cholesterol Levels

• Main Author: Sarkar, Samantha Khadija
• Other Authors: Lagace, Thomas
• Language: en
• Published: Université d'Ottawa / University of Ottawa 2015
• Subjects: PCSK9; LDL; lipoprotein; cholesterol
• Online Access: http://hdl.handle.net/10393/32825; http://dx.doi.org/10.20381/ruor-4155

Proprotein Convertase Subtilisin / Kexin Type-9 (PCSK9) has emerged as a major regulator of plasma cholesterol levels. PCSK9 is secreted mainly from the liver and circulates as a plasma protein. PCSK9 binds cell surface low-density lipoprotein (LDL) receptors and mediates their degradation upon endocytosis in the liver. This decreases the liver’s ability to clear LDL-cholesterol from the blood. PCSK9 is also capable of binding LDL particles themselves; this interaction inhibits the ability of PCSK9 to bind and mediate LDLR degradation in cultured hepatic cells, but its effect on PCSK9 function in vivo remains unknown. A disordered N-terminal region of the PCSK9 prodomain is necessary for binding to isolated LDL particles in vitro. This N-terminal region is also autoinhibitory to PCSK9-LDL receptor binding. We hypothesized that the N-terminal of the PCSK9 prodomain plays a role in an allosteric mechanism that regulates PCSK9 function. Through mutagenesis studies, we found that both a conserved stretch of acidic residues and an adjacent conserved stretch of hydrophobic residues are crucial for the PCSK9-LDL interaction; the hydrophobicity of the residue at position 38 (Tyr) within the conserved acidic stretch was also found to be important for this. Helical wheel modeling of the prodomain N-terminal sequence revealed the potential for a lipid-ordered amphipathic helix to form, which may aid PCSK9 docking onto LDL. Replacing residues A44 and L41 with helix-disrupting proline residues abolished LDL binding. Co-pelleting ultracentrifugation assays also show that wild-type PCSK9 is capable of associating with liposomes, while the A44P mutation disrupts this lipid association. The A44P-PCSK9 mutation, showing an 80-90% decrease in LDL association but with LDL receptor binding and degrading functions intact, may serve as an important tool in future studies investigating the PCSK9-LDL interaction in vivo. Elucidation of the mechanism by which LDL-binding naturally inhibits PCSK9 activity may also help to develop new anti-PCSK9 therapeutics in the future.