The 3C-like protease of SARS coronavirus (SARS-CoV 3CL(pro)) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CL(pro). S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structural analysis. Our work is expected to provide insight on the dimerization-function relationship of SARS-CoV 3CL(pro).

Background Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. Methods We performed pharmacoproteomic profiling of the mice liver tissues from control, carbon tetrachloride (CCl 4 )-induced fibrosis and NPLC0393 administrated groups. The target gene MAT2A was overexpressed or knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional activity on MAT2A promoter via luciferase assay. Intracellular SAM contents were analyzed by LC-MS method. Findings We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl 4 -induced fibrosis mice, and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65, i.e. , activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl 4 - and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. Interpretation This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. Fund This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873, 81774196 and 31800693), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502).

Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF), characterized by an acute deterioration of liver function in the patients with chronic hepatitis B (CHB), is lack of predicting biomarkers for prognosis. Plasma is an ideal sample for biomarker discovery due to inexpensive and minimally invasive sampling and good reproducibility. In this study, immuno-depletion of high-abundance plasma proteins followed by iTRAQ-based quantitative proteomic approach was employed to analyze plasma samples from 20 healthy control people, 20 CHB patients and 20 HBV-ACLF patients, respectively. As a result, a total of 427 proteins were identified from these samples, and 42 proteins were differentially expressed in HBV-ACLF patients as compared to both CHB patients and healthy controls. According to bioinformatics analysis results, 6 proteins related to immune response (MMR), inflammatory response (OPN, HPX), blood coagulation (ATIII) and lipid metabolism (APO-CII, GP73) were selected as biomarker candidates. Further ELISA analysis confirmed the significant up-regulation of GP73, MMR, OPN and down-regulation of ATIII, HPX, APO-CII in HBV-ACLF plasma samples (p < 0.01). Moreover, receiver operating characteristic (ROC) curve analysis revealed high diagnostic value of these candidates in assessing HBV-ACLF. In conclusion, present quantitative proteomic study identified 6 novel HBV-ACLF biomarker candidates and might provide fundamental information for development of HBV-ACLF biomarker.

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K m values of the two substrates dUMP and CH 2 H 4 folate were determined to be 15.3 6 1.25 lM and 0.35 6 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å ) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.