title: The proprotein convertase subtilisin/kexin type 9 (PCSK9) active site and cleavage sequence differentially regulate protein secretion from proteolysis.
abstract: Biologic-based strategies to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) show promise as anti-hypercholesterolemic and, therefore, anti-atherosclerotic therapies. Despite substantial effort, no small molecule strategy to inhibit PCSK9 has demonstrated feasibility. In this study we interrogated the chemistry of the PCSK9 active site and its adjacent residues to identify a foothold with which to drug the PCSK9 processing pathway and ultimately disrupt the interaction with the LDL receptor. Here, we develop a system in which we amplify the readout of PCSK9 proteolysis with a highly specific substrate in cells, showing that the PCSK9 catalytic domain is capable of proteolysis in trans. We use this system to show that the substrate specificity for PCSK9 proteolysis is distinct from the specificity for PCSK9 secretion, demonstrating that PCSK9 processing occurs in two separate sequential steps: that of proteolysis followed by secretion. We show that specific residues in the protease recognition sequence can differentially modulate the effects on proteolysis and secretion. Additionally, we demonstrate that the clinically described, dominant negative Q152H mutation restricts proteolysis and secretion independently. Our results suggest that the PCSK9 active site and its adjacent residues serve as an allosteric modulator of protein secretion independent of its role in proteolysis, revealing a new strategy for intracellular PCSK9 inhibition.
authors.affiliation: From the Division of Cardiology, San Francisco General Hospital, Department of Medicine, University of California, San Francisco, California 94110, Cardiovascular Research Institute, University of California, San Francisco, California 94158, and john.chorba@ucsf.edu.
authors.forename: John S
authors.initials: JS
authors.lastname: Chorba
authors.affiliation: Cardiovascular Research Institute, University of California, San Francisco, California 94158, and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158 and Department of Chemistry, University of California, Berkeley, California 94720.
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000495:Allosteric Site; D050197:Atherosclerosis / Q000378:metabolism; D020134:Catalytic Domain; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006331:Heart Diseases / Q000378:metabolism; D006801:Humans; D006937:Hypercholesterolemia / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: Active Site; Atherosclerosis; Low Density Lipoprotein (LDL); Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9); Protein Secretion; Substrate Specificity