Pathogenic gain-of-function mutations in the prodomain and C-terminal domain of PCSK9 inhibit LDL binding

Area of Science:

• Biochemistry
• Genetics
• Molecular Biology

Background:

• Proprotein convertase subtilisin/kexin type-9 (PCSK9) regulates low-density lipoprotein receptor (LDLR) levels, impacting cholesterol metabolism.
• Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH) by impairing LDL clearance.
• PCSK9 binds to LDL particles, a process potentially affected by disease-associated mutations.

Purpose of the Study:

• To identify and characterize novel GOF mutations in PCSK9 that affect LDL binding.
• To investigate the role of specific PCSK9 domains, including the prodomain and cysteine-histidine-rich domain module 1 (CM1), in LDL association.
• To elucidate the functional consequences of PCSK9 mutations on LDLR interaction and degradation.

Main Methods:

• Site-directed mutagenesis to introduce specific PCSK9 GOF mutations.
• In vitro assays to assess the binding affinity of PCSK9 mutants to LDL particles.
• Cell-based assays to evaluate the impact of PCSK9 mutants on LDLR cell surface levels and degradation.

Main Results:

• Multiple FH-associated GOF mutations, located in CM1 and the PCSK9 prodomain, were found to inhibit LDL binding.
• A specific prodomain mutation, Ser-127 to Arginine (S127R), significantly reduced PCSK9-LDL association.
• Mutations at position 127 of PCSK9, including alanine and proline substitutions, also impaired LDL binding.
• The S127R mutation abrogated the ability of PCSK9 to inhibit LDLR binding and degradation, even in the presence of a known PCSK9 GOF mutation (D374Y).

Conclusions:

• FH-associated GOF mutations in distinct regions of PCSK9 disrupt LDL binding.
• The Ser-127 residue in the PCSK9 prodomain is critical for effective LDL association.
• Understanding these mutation mechanisms provides insights into PCSK9 function and FH pathogenesis.