title: Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor.
abstract: Circulating low-density lipoprotein cholesterol (LDLc) is regulated by membrane-bound LDL receptor (LDLr). Upon LDLc and LDLr interaction the complex is internalized by the cell, leading to LDLc degradation and LDLr recycling back to the cell surface. The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein regulates this cycling. PCSK9 is secreted from the cell and binds LDLr. When the complex is internalized, PCSK9 prevents LDLr from shuttling back to the surface and instead targets it for degradation. PCSK9 is a serine protease expressed as a zymogen that undergoes autoproteolysis, though the two resulting protein domains remain stably associated as a heterodimer. This PCSK9 autoprocessing is required for the protein to be secreted from the cell. To date, direct analysis of PCSK9 autoprocessing has proven challenging, as no catalytically active zymogen has been isolated. A PCSK9 loss-of-function point mutation (Q152H) that reduces LDLc levels two-fold was identified in a patient population. LDLc reduction was attributed to a lack of PCSK9(Q152H) autoprocessing preventing secretion of the protein. We have isolated a zymogen form of PCSK9, PCSK9(Q152H), and a related mutation (Q152N), that can undergo slow autoproteolysis. We show that the point mutation prevents the formation of the mature form of PCSK9 by hindering folding, reducing the rate of autoproteolysis, and destabilizing the heterodimeric form of the protein. In addition, we show that the zymogen form of PCSK9 adopts a structure that is distinct from the processed form and is unable to bind a mimetic peptide based on the EGF-A domain of the LDLr.
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142. cgarvie@broadinstitute.org.
authors.forename: Colin W
authors.initials: CW
authors.lastname: Garvie
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142.
authors.forename: Cara V
authors.initials: CV
authors.lastname: Fraley
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142.
authors.forename: Nadine H
authors.initials: NH
authors.lastname: Elowe
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142.
authors.forename: Elizabeth K
authors.initials: EK
authors.lastname: Culyba
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Lemke
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hubbard
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142.
authors.forename: Virendar K
authors.initials: VK
authors.lastname: Kaushik
authors.affiliation: Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142. doug.daniels@udayton.edu.
authors.forename: Douglas S
authors.identifier: 0000-0003-0238-9403
authors.initials: DS
authors.lastname: Daniels
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D000071449:Proprotein Convertase 9 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C573412:LDLR protein, human; D010455:Peptides; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9
keywords: LDLR; PCSK9; autoprocessing; autoproteolysis; low-density lipoprotein receptor; point mutations; protein refolding; serine protease; zymogen