Abstract
Recent genetic studies have shown that PCSK9, one of the key genes in cholesterol metabolism, plays a critical role by controlling the level of low-density lipoprotein receptor. However, how PCSK9 mediates LDLR degradation is still unknown. By combining a shotgun proteomic method and differential analysis of natural occurring mutations of the PCSK9 gene, we found that an E3 ubiquitin ligase c-IAP1 binds and processes PCSK9 protein. One of the ‘gain-of-function’ mutations, S127R, is defective with respect to binding to c-IAP1, and thus has defective autocatalytic activity. Knockdown of c-IAP1 impairs PCSK9 processing and autocatalytic cleavage. In c-IAP1 null mouse embryonic fibroblasts (MEFs), there is a dramatic decrease in secreted mature PCSK9 protein accompanied by a significant increase in LDLR protein levels compared with matched wild-type MEF cells. c-IAP1 also acts as an E3 ligase for ubiquitination of PCSK9. Ubiquitin containing only lysine-27 mediated PCSK9 ubiquitination by c-IAP1. Given K27-linked polyubiquitination promotes lysosomal localization, the finding indicates the c-IAP1 acts on both secretion of PCSK9 and its lysosomal localization. The novel pathway described here will open new avenues for exploring novel disease treatments.
Highlights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is encoded by one of three genes implicated in autosomal dominant hypercholesterolaemia (ADH), in addition to LDLR and APOB
Recent genetic studies have shown that PCSK9 plays a critical role in cholesterol metabolism, by controlling the level of low-density lipoprotein receptor [1,2]
We found that only the region containing the baculoviral IAP repeat 3 (BIR3) domain of c-IAP1 could be used to pull down wild-type PCSK9 (Figure 2A, lane 3), indicating that c-IAP1-BIR3 is the binding site for
Summary
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is encoded by one of three genes implicated in autosomal dominant hypercholesterolaemia (ADH), in addition to LDLR (low-density lipoprotein receptor) and APOB (apolipoprotein B). PCSK9 belongs to a family of secretory serine proteinases known as proprotein convertases (PCs) [5]. It is synthesized as a 73 kDa zymogen and undergoes autocatalytic cleavage in the endoplasmic reticulum and it is secreted as a 63 kDa mature protein, which forms a complex with the N-terminal predomain [5]. Most studies of PCSK9 suggest it acts as a chaperone for LDLR and its related receptors, by directing them to the lysosomes for degradation. PCSK9 expressing cell line and have used anti-FLAG antibody affinity purification and shotgun proteomic analysis to identify potential binding partners of PCSK9 and to study their roles in PCSK9-mediated LDLR degradation
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