POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation

Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.

Reporter cell line-based screening for anti-varicella-zoster virus compounds / 中华微生物学和免疫学杂志

Objective To establish a novel method to screen for anti-varicella-zoster virus (VZV) compounds with our previously generated reporter cell line for VZV, MV9G. MethodsMV9G cells were directly infected with cell-free virus of Oka vaccine strain (vOka) for 2 hours( CFV direct-infection) or cocultured with vOka-infected MeWo cells containing cell-associated virus for 48 hours (CAV co-culture) to promote expression of the reporter gene firefly luciferase. Antiviral compounds including heparin, mannose-6-phosphate( M-6-P), acyclovir( ACV ), resveratrol and roscovitine were added in the medium before or after the virus infection. Inhibitory effects( IC50 ) of the antiviral compounds were analyzed by comparing firefly luciferase activities of MV9G cells in the presence of antiviral compounds with those in the absence. Results Antiviral compounds inhibited luciferase activities of MV9G cells activated by CFV direct-infection and/or CAV co-culture in different levels. The reductions of luciferase activities statistically correlated with those of viral foci shown by immunostaining with a monoclonal antibody against VZV immediate early 62 antigens (IE62) in controls. Among these compounds, heparin, M-6-P, and 2.5 μmol/L of roscovitine inhibited CFV-activated more strongly than CAV-activated luciferase activities, whereas ACV and resveratrol inhibited CAV-activated more strongly than CFV-activated luciferase activities. Cell-associated ACV-resistant strains,Kanno and rOka YSR, activated luciferase activities of MV9G cells, too. However, the inhibitory concentrations (IC50) of ACV to the ACV-resistant strains were much higher than those to the ACV-sensitive strains,pOka and CaGu. ConclusionThe CFV direct-infection and CAV co-culture assays were useful to screen for antiviral compounds targeting the early and late phases of VZV infection, respectively. The VZV reporter cell-based assays may provide a simple, rapid, sensitive, and high-throughput method to screen for anti-VZV compounds.

The effect of mannose-6-phosphate on adipocyte differentiation and its underlying molecular mechanism / 中华内科杂志

Objective Cathepsin K (CTSK) played an important role in adipocyte differentiation.The activation of CTSK needs to convey by mannose-6-phosphate receptors (M6PR) in osteoclasts. The aim of the present study was to identify the effects of mannose-6-phosphate (M6P) in adipocyte differentiation and its underlying molecular mechanism. Methods Oil red O staining, accumulation of cytoplasmic triglycerides and glycerine release were used to assess its effects on adipocyte differentiation in the 3T3-L1cell line. The enzyme activity of CTSK was observed by laser confocal microscopy. The proliferation of 3T3-L1 preadipocytes was detected by MTT methods. mRNA expression of M6PR was determined by RTPCR. Results M6P could prevent adipocyte differentiation in a dose-dependent manner as evidenced by absence of triglyceride accumulation and glycerol content. Statistical significance was showed when the concentrations of M6P were 5.0 mmol/L and 8. 0 mmol/L respectively(P ＜0. 05). The mRNA expression of M6PR was detected during the whole process of adipocyte differentiation. With the increase of M6Pconcentration, enzyme activity of CTSK was inhibited in a concentration-dependent manner. MTT method showed that the absorbance at 570 nm of 3T3-L1 preadipocytes was 0. 057 ±0. 091, increased about 62. 9%at 10. 0 mmoL/L compared with the control group (P ＜ 0. 05 ). Conclusion M6P inhibits the terminal differentiation of adipocyte, which may be associated with its effect of blocking CTSK activity by competitive binding with M6PR.

The Effect of Transforming Growth Factor-beta and Mannose-6-Phosphate on the Proliferation of Subconjunctival Fibroblast of Rabbit

The main cause of failure in glaucoma filtering surgery is obstruction of aqueous outflow due to subconjunctival fibrosis. Transforming growth factor-beta(TGF-beta) is known to be a growth factor for subconjunctival fibroblast. Recently, mannose-6-phosphate(M-6-P) is reported to be an inhibitor of TGF-beta activity. In this study, we evaluated the effects of TGF-betas and M-6-P on the proliferation of cultured subconjunctival fibroblast of white rabbit in vitro. Cell proliferation was determined by 3H-thymidine DNA incorporation method. TGF-beta1, 2, 3 all promoted proliferation of subconjunctival fibroblast in a concentration dependent fashion and the effect of TGF-beta1 was most prominent among 3 types. Low concentration (0.01mM) of M-6-P paradoxically increased cell proliferation, but with the concentration of 1.0mM, the inhibitory effects were varied in the range of 45% to 7%.

Carbohydrate mediated recognition of lysosomal enzymes by cell surface receptors.

The recognition of lysosomal enzymes by various carbohydrate specific cell surface receptors is reviewed. In particular the biosynthesis of mannose 6-phosphate residues in lysosomal enzymes and their role for targeting of lysosomal enzymes to lysosomes are discussed.