Ufd1 enhances the E3 activity of gp78 and accelerates the degradation of HMGCR (17)

Ufd1 enhances the E3 activity of gp78 and accelerates the degradation of HMGCR (17). mevalonate pathway, promote ubiquitination and degradation of the HMGCR protein (10,C12). Oxysterols can inhibit transcription and stimulate HMGCR degradation (13, 14). Besides sterols, geranylgeraniol, a non-steroid product downstream of mevalonate, acts on the post-ubiquitination step to accelerate sterol-induced HMGCR degradation (7). The sterol-induced degradation JNJ-42041935 of HMGCR initiates when the endoplasmic reticulum (ER)-localized Insig-1 and -2 proteins bind to HMGCR (15) and recruit the ubiquitin ligase (E3) gp78 to catalyze ubiquitination (16). The HMGCR protein is eventually degraded in the proteasome. Ufd1 enhances the E3 activity of gp78 and JNJ-42041935 accelerates the degradation of HMGCR (17). Ablation of in mouse liver increases the stability of HMGCR, Insig-1, and Insig-2 (18, 19). Elevated levels of Insigs inhibit the SREBP pathway and decrease cholesterol synthesis (18). These data suggest that gp78 is a major E3 essential for HMGCR degradation in the hepatocytes. Besides gp78, TRC8 and MARCH6 are two other ER-localized E3s involved in HMGCR degradation (20, 21). TRC8 interacts with Insig-1 and -2 and ubiquitinates HMGCR for proteasomal degradation. JNJ-42041935 In addition to sterol-regulated degradation, the basal turnover of HMGCR is mediated by Hrd1, an ER-anchored E3 homologous to gp78 (22, 23). Interestingly, sterol-induced HMGCR degradation has been found to persist in or alone had partial or little effect on HMGCR degradation in Chinese hamster ovary (CHO) cells. However, knockout of both genes dramatically blunted sterol-induced degradation of HMGCR. The E3 activityCdeficient RNF145 (C537A) failed to promote sterol-induced ubiquitination and degradation of HMGCR. Moreover, we found that Insigs were required for RNF145-catalyzed HMGCR degradation and that RNF145 interacted with Insigs constitutively through its transmembrane domains. We therefore conclude that RNF145 is a new E3 promoting sterol-induced degradation of HMGCR. Results Identification of Rnf145 Mouse monoclonal to S1 Tag. S1 Tag is an epitope Tag composed of a nineresidue peptide, NANNPDWDF, derived from the hepatitis B virus preS1 region. Epitope Tags consisting of short sequences recognized by wellcharacterizated antibodies have been widely used in the study of protein expression in various systems. involved in HMGCR degradation To determine whether gp78 is exclusively responsible for HMGCR degradation, we treated WT CHO and knockout (and and deficiency in plus knockout (double KO) CHO cells using the CRISPR/Cas9 technique (26). Knockout of slightly affected HMGCR degradation relative to WT cells (Fig. 1alone had little influence on HMGCR degradation (Fig. 1indicate nonspecific bands. Results shown are representative of two independent experiments. ubiquitination assay. The recombinant cytosolic domain of gp78 (309C643) was used as a positive control. We found that RNF145 (511C663) could efficiently catalyze the formation of polyubiquitin chains in the presence of E1, E2, FLAG-ubiquitin, and ATP (Fig. 2= 10 m. ubiquitination assay showing that RNF145 (511C663) possesses E3 activity. Recombinant proteins, including E1, E2 (Ubc7), FLAG-ubiquitin (ubiquitination assay comparing RNF145 (511C663) and RNF145 (511C663) (C537A). Experiments were carried out as described in and and (4KO) (Fig. 4and and is knocked out. shows that RNF145 co-immunoprecipitated with both Insig-1 and Insig-2 regardless of sterol levels. Specifically, it was the transmembrane domain (aa 1C510) but not the cytosolic domain (aa 511C663) of RNF145 that bound to Insig-1 (Fig. 5and partially delayed the turnover of HMGCR in response to low concentrations of sterols, and ablation of alone also had little effect (Fig. JNJ-42041935 1). Notably, knockout of both genes largely abolished sterol-induced degradation of HMGCR (Fig. 1as a Liver X receptor (LXR) target gene (32, 33). We hypothesize that activation of LXR might elevate the RNF145 level and subsequently down-regulate cholesterol biosynthesis through degrading HMGCR. Another possibility is that the existence of multiple E3s for HMGCR degradation prevents saturation of specific E3(s) and ensures that ER-associated degradation functions properly when HMGCR is degraded. The protein machineries involved.

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