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

Geschwindner, Stefan ; Andersson, Gunilla M K ; Beisel, Hans-Georg ; Breuer, Sebastian ; Hallberg, Carina ; Kihlberg, Britt-Marie ; Lindqvist, Ann-Margret ; O'Mahony, Gavin ; Plowright, Alleyn T and Raubacher, Florian , et al. (2015) In Protein Engineering Design & Selection
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... (More)
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. (Less)
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Contribution to journal
publication status
published
subject
in
Protein Engineering Design & Selection
publisher
Oxford University Press
external identifiers
  • pmid:25744035
  • scopus:84954309441
  • pmid:25744035
ISSN
1741-0126
DOI
10.1093/protein/gzv008
language
English
LU publication?
yes
id
f643f544-aec3-493a-81b2-de0de4aca42b (old id 5265163)
date added to LUP
2016-04-01 14:19:53
date last changed
2023-03-15 10:17:07
@article{f643f544-aec3-493a-81b2-de0de4aca42b,
  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.}},
  author       = {{Geschwindner, Stefan and Andersson, Gunilla M K and Beisel, Hans-Georg and Breuer, Sebastian and Hallberg, Carina and Kihlberg, Britt-Marie and Lindqvist, Ann-Margret and O'Mahony, Gavin and Plowright, Alleyn T and Raubacher, Florian and Knecht, Wolfgang}},
  issn         = {{1741-0126}},
  language     = {{eng}},
  month        = {{03}},
  publisher    = {{Oxford University Press}},
  series       = {{Protein Engineering Design & Selection}},
  title        = {{Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9.}},
  url          = {{http://dx.doi.org/10.1093/protein/gzv008}},
  doi          = {{10.1093/protein/gzv008}},
  year         = {{2015}},
}