c-IAP1 binds and processes PCSK9 protein: linking the c-IAP1 in a TNF-α pathway to PCSK9-mediated LDLR degradation pathway

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.

Figure 1

Immunoprecipitation (IP)/western blot analysis of novel PCSK9 binding proteins in the PCSK9-FLAG pull-down assay. Equal amount of cellular extracts from a T-Rex 293 cell line stably overexpressing FLAG-tagged wild-type PCSK9 or PCSK9 carrying ADH mutations (p.S127R, D374Y, F216 L) and a negative control cell line, T-Rex-293 cells transfected with the empty vector pcDNA3.1(control) were subjected to anti-FLAG IP and Western blots probed with the antibodies indicated. 5% of the cell lysates prior to IP were used for the input, probed with anti-α-tubulin antibody. The western blots shown are representative of three separate experiments.

Figure 2

c-IAP1 interacts with PCSK9. (A) Co-IP of PCSK9-wt and c-IAP1. Myc-tagged c-IAP1 or c-IAP1-BIR3 was co-transfected with either empty vector (pCMV6-entry vector) or FLAG-tagged wild-type PCSK9 into T-Rex-293 cells. Cell lysates were immunoprecipitated with an anti-myc tag antibody followed by immunoblotting with either anti-PCSK9 or anti-c-IAP1 antibodies; (B) Co-IP of PCSK9-wt, TRAF2 and LDLR. Myc-tagged TRAF2 or LDLR was co-transfected with FLAG-tagged wild-type PCSK9 into T-Rex-293 cells. Cell lysates were immunoprecipitated with an anti-myc tag antibody followed by immunoblotting with either anti-PCSK9 or anti-TRAF or LDLR antibodies. The Western blots shown are representative of three separate experiments.

Figure 3

c-IAP1 Knock-down. (A) A wild-type PCSK9 overexpressed T-Rex-293 stable cell line was transfected with pGB c-IAP1 siRNA and a Western blot was probed with anti-c-IAP1 or anti-PCSK9 antibodies. Scanning densitometry analysis of three western blots is shown below. Data are presented as the percentage conversion to mature PCSK9 (p63), calculated as the p63 value divided by the sum of p63 + p75 (Pro-PCSK9), divided by the tubulin, multiplied by 100. ** indicates a significant difference (p = 0.009) from c-IAP siRNA treated cells from control siRNA cells. Lower panel: Coomassie-Blue-stainedsecreted PCSK9 isolated from the 100mL of cultured medium over 2 days from c-IAP1 siRNA treated or control siRNA treated samples; (B) Western blot analysis of the LDLR and PCSK9 protein in c-IAP1-deficient (c-IAP1^−/−, reference 11) MEFs and the matched wild-type (Wt) MEFs. The percentage conversion to mature PCSK9 level was calculated as described above. LDLR amounts were quantified and normalised to the amount of α-tubulin in three experiments. The ratio of LDLR/tubulin in c-IAP1 deficient cells was assigned a value of 1.00. ** Indicates a significant difference (p < 0.01) between c-IAP1 deficient MEF cells and matched wild-type MEF cells.

Figure 4

Identification of PCSK9 as a substrate of c-IAP1 ubiquitin ligase in vivo and in vitro. (A) pcDNA.3-Empty vector or HA-tagged wild-type ubiquitin plasmid were co-transfected with FlAG-PCSK9wt or FLAG-PCSK9-D374Y expression vector with or without the pCMV–c-IAP1 plasmids(Image clone 3908352)into T-REX 293 cells. 24h later, the transfected cells were treated with MG132 (10 μM) for 1h, the lysates were immunoprecipitated using the FLAG-immunoprecipitation kit, the elution samples were then analyzed by Western blotting with anti- HA-Tag polyclonal antibody to detect the conjugated HA-ubiquitinated PCSK9.Western blots shown are representative of three separate experiments; (B) pcDNA.3-Empty vector or pRK5-HA-Ubiquitin-K27 plasmid were co-transfected with FlAG-PCSK9wt or FLAG-PCSK9-D374Y expression vector with or without the pCMV–c-IAP1 plasmids into T-REX 293 cells. The immunoprecipitation and the western blots analysis were carried out as in A: again, Western blots shown are representative of three separate experiments; (C) FLAG-tagged wild-type-PCSK9 and PCSK9-D374Y were subjected to ubiquitination assays in the presence of recombinant c-IAP1. The ubiquitinated PCSK9 was detected by immunoblotting with anti-PCSK9 antibody; (D) Proposed model of c-IAP1 and PCSK9 interaction. c-IAP1 can bind to PCSK9 and promote its maturation. c-IAP1 is also targeting the PCSK9 by K27-linked ubiquitination, which lead to the PCSK9/LDLR complex into lysosome, eventually lead to the complex degradation.