ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides.

BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120.

The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The Km values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 µM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu2+ and Zn2+. Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations.

MicroRNA-125a suppresses intestinal mucosal inflammation through targeting ETS-1 in patients with inflammatory bowel diseases.

MicroRNA (miR)-125a is highly expressed in T cells and regulates the functions of Treg through the IL-6-STAT3 signaling pathway. However, the role of miR-125a in regulating immune responses in intestinal mucosa of patients with inflammatory bowel diseases (IBD) is still not understood. Here we showed that miR-125a expression was decreased in PBMC and inflamed intestinal mucosa from IBD patients compared with that in healthy controls. Transduction with LV-miR-125a into IBD CD4+ T cells could significantly inhibit proinflammatory cytokine production, including IFN-γ, TNF-α and IL-17A. RNA-seq analysis of miR-125a-/- CD4+ T cells revealed enhanced genes (e.g., Stat1, Stat3, RORγt, Irf4, Klf13) in T cell activation and effector pathways, while ETS-1 as its functional target promoted IBD CD4+ T cell differentiation into Th1 cells. Consistently, miR-125a-/- mice developed more severe colitis induced by TNBS compared with WT mice. Thus, our data suggest that miR-125a protects intestinal mucosa from inflammatory injury and that ETS-1 as its target participates in the pathogenesis of IBD.

Two isocitrate dehydrogenases from a plant pathogen Xanthomonas campestris pv. campestris 8004. Bioinformatic analysis, enzymatic characterization, and implication in virulence.

Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylate (TCA) cycle, which may play an important role in the virulence of pathogenic bacteria. Here, two structurally different IDHs from a plant pathogen Xanthomonas campestris pv. campestris 8004 (XccIDH1 and XccIDH2) were characterized in detail. The recombinant XccIDH1 forms homodimer in solution, while the recombinant XccIDH2 is a typical monomer. Phylogenetic analysis showed that XccIDH1 belongs to the type I IDH subfamily and XccIDH2 groups into the monomeric IDH clade. Kinetic characterization demonstrated that XccIDH1's specificity towards NAD(+) was 110-fold greater than NADP(+) , while XccIDH2's specificity towards NADP(+) was 353-fold greater than NAD(+) . The putative coenzyme discriminating amino acids (Asp268, Ile269 and Ala275 for XccIDH1, and Lys589, His590 and Arg601 for XccIDH2) were studied by site-directed mutagenesis. The coenzyme specificities of the two mutants, mXccIDH1 and mXccIDH2, were completely reversed from NAD(+) to NADP(+) , and NADP(+) to NAD(+) , respectively. Furthermore, Ser80 of XccIDH1, and Lys256 and Tyr421 of XccIDH2, were the determinants for the substrate binding. The detailed biochemical properties, such as optimal pH and temperature, thermostability, and metal ion effects, of XccIDH1 and XccIDH2 were further investigated. The possibility of taking the two IDHs into consideration as the targets for drug development to control the plant diseases caused by Xcc 8004 were described and discussed thoroughly.

Identification and characterization of a novel citrate synthase from Streptomyces diastaticus No. 7 strain M1033.

Citrate synthase (CS) is a key enzyme of the tricarboxylic acid cycle and is widely distributed among prokaryotes and eukaryotes. Here, we report for the first time, the cloning, expression, and characterization of a novel CS from Streptomyces diastaticus No. 7 strain M1033 (SdCS). Gel filtration chromatography and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analyses indicate that SdCS forms homodimers with a molecular mass of approximately 100.0 kDa. The predicted amino acid sequence of SdCS is highly similar to those of bacterial homodimeric type I CSs. The pH and temperature optima for SdCS activity were 8.0 and 35 °C, respectively. The half-life (t1/2 ) of SdCS was 10 Min at 50 °C and was increased to 210 Min in the presence of oxaloacetate. The kinetic parameters of SdCS (kcat = 262.8 and 230.7 s(-1); Km = 58.4 and 11.2 µM for acetyl-CoA and oxaloacetate, respectively) were comparable to those of dimeric CSs isolated from Gram-positive bacteria and eukaryotes. Moreover, SdCS activity was inhibited by ATP and ADP and stimulated by AMP. These findings provide a foundation for further investigations on the three-dimensional structure and mechanism of catalysis of SdCS.

Enzymatic Characterization and Coenzyme Specificity Conversion of a Novel Dimeric Malate Dehydrogenase from Bacillus subtilis.

Malate is an important material to various industrials and clinical applications. Bacillus subtilis is a widely used biocatalyst tool for chemical production. However, the specific enzymatic properties of malate dehydrogenase from Bacillus subtilis (BsMDH) remain largely unknown. In the present study, BsMDH was cloned, recombinantly expressed and purified to test its enzymatic properties. The molecular weight of single unit of BsMDH was 34,869.7 Da. Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight Mass Spectrometry and gel filtration analysis indicated that the recombinant BsMDH could form dimers. The kcat/Km values of oxaloacetate and NADH were higher than those of malate and NAD+, respectively, indicating a better catalysis in the direction of malate synthesis than the reverse. Furthermore, six BsMDH mutants were constructed with the substitution of amino acids at the coenzyme binding site. Among them, BsMDH-T7 showed a greatly higher affinity and catalysis efficiency to NADPH than NADH with the degree of alteration of 2039, suggesting the shift of the coenzyme dependence from NADH to NADPH. In addition, BsMDH-T7 showed a relatively lower Km value, but a higher kcat and kcat/Km than NADPH-dependent MDHs from Thermus flavus and Corynebacterium glutamicum. Overall, these results indicated that BsMDH and BsMDH-T7 mutant might be promising enzymes for malate production.

Simulation of integrated anaerobic digestion-gasification systems using machine learning models.

In this study, the anaerobic digestion model M-ADM1 was integrated with the gasification model T-ANN to form a set of integrated models that can efficiently simulate the biomass AD-GS integration technology. Biogas slurry is used as feedstocks to prepare biogas slurry fertilizer. Solid residue is used feedstocks for gasification reactions. Biogas and syngas from the gasification of solid residue are used for energy. In this process, carbon emission is regarded as an important index for the comprehensive evaluation and optimization of AD-GS integration process. This study found that when the anaerobic digestion duration was 0 to 15 days, the carbon emission reduction increased rapidly. The amount of carbon emission reduction peaks on day 15. The value of carbon emission reduction is 0.1828 gCO2eq. In addition, when FEAG reached the maximum value at 15 days of anaerobic digestion, the decreasing trend of FEAG rate change value started to become significant.

Characteristics prediction of hydrothermal biochar using data enhanced interpretable machine learning.

Hydrothermal biochar is a promising sustainable soil remediation agent for plant growth. Demands for biochar properties differ due to the diversity of soil environment. In order to achieve accurate biochar properties prediction and overcome the interpretability bottleneck of machine learning models, this study established a series of data-enhanced machine learning models and conducted relevant sensitivity analysis. Compared with traditional support vector machine, artificial neural network, and random forest models, the accuracy after data enhancement increased in average from 5.8% to 15.8%, where the optimal random forest model showed the average of accuracy was 94.89%. According to sensitivity analysis results, the essential factors influencing the predicting results of the models were reaction temperature, reaction pressure, and specific element of biomass feedstock. As a result, data-enhanced interpretable machine learning proved promising for the characteristics prediction of hydrothermal biochar.

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680.

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolor A3(2).

We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD(+)-specific and displayed about 400-fold (k(cat)) and 1,050-fold (k(cat)/K(m)) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K(i)=5.8 mM) and excess L-malate (K(i)=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe(2+) and Zn(2+). Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.

Expression and characterization of a thermostable citrate synthase from Microcystis aeruginosa PCC7806.

Citrate synthase (CS) is an important enzyme in energy conversion and material circulation, participating in many important biochemical processes. In the present study, CS from Microcystis aeruginosa PCC7806 (MaCS) was cloned and expressed in Escherichia coli Rosetta (DE3). The recombinant MaCS was purified and its enzymological properties were characterized. The results showed that MaCS formed dimers in native status. The optimum temperature and pH of MaCS was 30°C and 8.2, respectively. MaCS displayed relative high thermal stability. Treatment at 50°C for 20 min only decreased 11.30% activity of MaCS and the half-life of MaCS was approximately 35 min at 55°C. The kcat and Km of acetyl-CoA and oxaloacetic acid were 17.133 s-1 (kcat) and 11.62 µM (Km), 24.502 s-1 and 103.00 µM, respectively. MaCS activity was not drastically inhibited by monovalent ions and NADH but depressed by divalent ions and some small molecular compounds, especially Mg2+, Zn2+, Co2+ and DTT. Overall, these data contributed to further understanding of energy metabolism in cyanobacteria and also provided basic information for industrial application of CS.

Critical Role of CD6highCD4+ T Cells in Driving Th1/Th17 Cell Immune Responses and Mucosal Inflammation in IBD.

BACKGROUND AND AIMS: CD6 is a crucial regulator of T cell activation and is implicated in the pathogenesis of multiple autoimmune diseases. ALCAM is the first identified endogenous ligand of CD6. We sought to investigate potential roles of CD6 in regulating intestinal mucosal inflammation in inflammatory bowel disease [IBD]. METHODS: We analysed the expression of CD6 and ALCAM in the inflamed mucosa of IBD patients using qRT-PCR and immunohistochemistry. Phenotypic properties of CD6low/- and CD6highCD4+ T cells were determined by flow cytometry, qRT-PCR, and ELISA. ALCAM Fc chimeric protein was used to evaluate the role of CD6-ALCAM engagement in regulating IBD CD4+ T cell activation and differentiation. RESULTS: Expression of CD6 and its ligand ALCAM was markedly increased in the inflamed mucosa of IBD patients compared with that in normal controls, and was significantly correlated with disease activity indices of IBD patients. Interestingly, CD6highCD4+ T cells of IBD patients exhibited significantly higher pathogenicity compared with CD6low/-CD4+ T cells, characterized by enhanced T cell activation and preferential Th1 and Th17 cell phenotypes, but a markedly decreased proportion of nTreg [CD25highFoxp3+, CD25highCD127low] cells. Importantly, inclusion of ALCAM Fc chimeric protein significantly facilitated IBD CD4+ T cell, especially CD6highCD4+ T cell, differentiation into Th1/Th17 cells compared with hIgG1 Fc-treated controls. CONCLUSIONS: These data indicate that overexpression of CD6 and ALCAM in the inflamed mucosa of IBD patients accelerates intestinal mucosal immune responses via promoting CD4+ T cell proliferation and differentiation into Th1/Th17 cells. Thus, CD6 may serve as a novel therapeutic target for treatment of IBD.

NADP(+)-specific isocitrate dehydrogenase from oleaginous yeast Yarrowia lipolytica CLIB122: biochemical characterization and coenzyme sites evaluation.

NADP(+)-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg(2+) was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP(+) and no NAD-dependent activity could be detected. The K m values displayed for NADP(+) and isocitrate were 59 and 31 µM (Mg(2+)), 120 µM and 58 µM (Mn(2+)), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K m values for NADP(+) of R322D mutant was 2,410 µM, being about 41-fold higher than that of wild type enzyme. NAD(+)-dependent activity was detected for R322D mutant and the K m and k cat values for NAD(+) were 47,000 µM and 0.38 s(-1), respectively. Although the R322D mutant showed low activity with NAD(+), it revealed the feasibility of engineering an eukaryotic IDP to a NAD(+)-dependent one.