Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9.

Research output: Contribution to journalArticle

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the hepatic low-density lipoprotein receptor (LDL-R) and is therefore a prominent therapeutic target for reducing LDL-cholesterol. The C-terminal domain of PCSK9 is unlikely to be involved in a direct extracellular interaction with the LDL-R. We probed the importance of the C-terminus for the degradation of the LDL-R by designing seven de novo mutants of PCSK9 that fill potential druggable cavities. The mutants were tested for their ability to diminish LDL uptake in human HepG2 cells and for affinity towards a calcium independent mutant of the EGF(A) domain of the human LDL-R. The later was done by a newly developed surface plasmon resonance-based assay format. We identified three mutant proteins (G517R, V610R and V644R) with decreased ability to block LDL uptake into HepG2 cells. These mutations define areas outside the direct interaction area between PCSK9 and the LDL-R that could be targeted to inhibit the PCSK9 triggered degradation of the LDL-R. We also describe the mechanistic rationalisation of the affinity changes seen with the natural occurring human D374Y (gain of function) mutation causing severe hypercholesterolaemia. The action of this mutant is due to a significantly decreased dissociation rate constant, whereas the mutation does not affect the association rate constant.

Details

Authors
  • Stefan Geschwindner
  • Gunilla M K Andersson
  • Hans-Georg Beisel
  • Sebastian Breuer
  • Carina Hallberg
  • Britt-Marie Kihlberg
  • Ann-Margret Lindqvist
  • Gavin O'Mahony
  • Alleyn T Plowright
  • Florian Raubacher
  • Wolfgang Knecht
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biological Sciences
Original languageEnglish
JournalProtein Engineering, Design and Selection
Early online date2015 Mar 4
Publication statusPublished - 2015
Publication categoryResearch
Peer-reviewedYes