Objective Hepatitis B virus X protein (HBx) is a pivotal factor for HBV-induced hepatitis. Herein, we sought to investigate HBx-mediated NLR pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis under oxidative stress. Methods The effect of HBx on the NLRP3 inflammasome was analyzed by enzyme-linked immunosorbent assays, quantitative reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence in hepatic HL7702 cells. Pyroptosis was evaluated by western blotting, lactate dehydrogenase release, propidium iodide staining, and transmission electron microscopy. NLRP3 expression in the inflammasome from liver tissues was assessed by immunohistochemistry. Results In hydrogen peroxide (H 2 O 2)-stimulated HL7702 cells, HBx triggered the release of pro-inflammatory mediators apoptosis-associated speck-like protein containing a CARD (ASC), interleukin (IL)-1β, IL-18, and high-mobility group box 1 (HMGB1); activated NLRP3; and initiated pro-inflammatory cell death (pyroptosis). HBx localized to the mitochondria, where it induced mitochondrial damage and production of mitochondrial reactive oxygen species (mitoROS). Treatment of HL7702 cells with a mitoROS scavenger attenuated HBx-induced NLRP3 activation and pyroptosis. Expression levels of NLRP3, ASC, and IL-1β in liver tissues from patients were positively correlated with HBV DNA concentration. Conclusions The NLRP3 inflammasome was activated by elevated mitoROS levels and mediated HBx-induced liver inflammation and hepatocellular pyroptosis under H 2 O 2-stress conditions.

Stereocomplementary bioreduction of 33 α-nitro ketones catalyzed by ketoreductases (KREDs) with publicly known sequences furnished both enantiomers of β-nitro alcohols.

We report here the discovery of a novel ketoreductase (KRED), named KmCR2, with a broad substrate spectrum on bioreduction of sterically bulky diaryl‐ and aryl(heteroaryl)methanones. The position of the substituent on aromatic rings (meta versus para or ortho) was revealed to control the stereospecificity of KmCR2. The stereoselective preparation of both enantiomers of diaryl‐ or aryl(heteroaryl)methanols using strategically engineered substrates with a traceless directing group (bromo group) showcased the potential application of this substrate‐controlled bioreduction reaction. The combined use of substrate engineering and protein engineering, was demonstrated to be a useful strategy in efficiently improving stereoselectivity or switching stereopreference of enzymatic processes.magnified image

Metrics & MoreArticle Recommendations * sı Supporting Information ABSTRACT: (S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline [(S)-1-(4-methoxybenzyl)-OHIQ, (S)-1a] is a key synthetic intermediate in the industrial production of dextromethorphan, one of the most widely used over-the-counter antitussives. We report here that a new cyclohexylamine oxidase discovered by genome mining, named CHAO CCH12-C2 , was able to completely deracemize 100 mM 1a under Turner's deracemization conditions to afford (S)-1a in 80% isolated yield and 99% ee at a semipreparative scale (0.4 mmol). When this biocatalytic reaction was scaled up to a gram scale (5.8 mmol), without reaction optimization (S)-1a was still isolated in 67% yield and 96% ee. The relatively higher k cat determined for CHAO CCH12-C2 was rationalized as one major factor rendering this enzyme capable of oxidizing 1a effectively at elevated substrate concentrations. Protein sequence alignment, analysis of our co-crystal structure of CHAO CCH12-C2 complexed with the product 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline [1-(4-methoxybenzyl)-HHIQ, 2a], and the structure-guided mutagenesis study together indicated L295 is one of the critical residues for this efficient enzymatic oxidation process and supported the presence of two cavities as well as a catalytically important "aromatic cage" formed by F342, Y433, and FAD. The synthetic applicability of CHAO CCH12-C2 was further underscored by the stereoselective synthesis of various enantioenriched 1-benzyl-OHIQ derivatives of potential pharmaceutical importance at a semipreparative scale.

Primary cilia are critically involved in the coordination of diverse signaling pathways and ciliary defects are associated with a variety of human diseases. The past decades have witnessed great progress in the core machinery orchestrating ciliary assembly. However, the upstream epigenetic cues that direct ciliogenesis remain elusive. Herein, we demonstrate that mixed-lineage leukemia protein 2 (MLL2), a histone methyltransferase, plays a negative role in ciliogenesis. RNA-sequencing analysis reveals that the expression of five actin-associated proteins is significantly downregulated in MLL2-depleted cells. Overexpression of these proteins partially rescues ciliary abnormality elicited by MLL2 depletion. Our data also show that actin dynamics is remarkably changed in MLL2-depleted cells, resulting in the impairment of cell adhesion, spreading, and motility. In addition, MLL2 depletion promotes ciliary vesicle trafficking to the basal body in an actin-related manner. Together, these results reveal that MLL2 inhibits ciliogenesis by modulating actin dynamics and vesicle transport, and suggest that alteration of MLL2 may contribute to the pathogenesis of cilium-associated diseases.