Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain.

Recognition of histone covalent modifications by chromatin-binding protein modules ("readers") constitutes a major mechanism for epigenetic regulation, typified by bromodomains that bind acetyllysine. Non-acetyl histone lysine acylations (e.g., crotonylation, butyrylation, propionylation) have been recently identified, but readers that prefer these acylations have not been characterized. Here we report that the AF9 YEATS domain displays selectively higher binding affinity for crotonyllysine over acetyllysine. Structural studies revealed an extended aromatic sandwiching cage with crotonyl specificity arising from π-aromatic and hydrophobic interactions between crotonyl and aromatic rings. These features are conserved among the YEATS, but not the bromodomains. Using a cell-based model, we showed that AF9 co-localizes with crotonylated histone H3 and positively regulates gene expression in a YEATS domain-dependent manner. Our studies define the evolutionarily conserved YEATS domain as a family of crotonyllysine readers and specifically demonstrate that the YEATS domain of AF9 directly links histone crotonylation to active transcription.

Deep-Kcr: accurate detection of lysine crotonylation sites using deep learning method.

As a newly discovered protein posttranslational modification, histone lysine crotonylation (Kcr) involved in cellular regulation and human diseases. Various proteomics technologies have been developed to detect Kcr sites. However, experimental approaches for identifying Kcr sites are often time-consuming and labor-intensive, which is difficult to widely popularize in large-scale species. Computational approaches are cost-effective and can be used in a high-throughput manner to generate relatively precise identification. In this study, we develop a deep learning-based method termed as Deep-Kcr for Kcr sites prediction by combining sequence-based features, physicochemical property-based features and numerical space-derived information with information gain feature selection. We investigate the performances of convolutional neural network (CNN) and five commonly used classifiers (long short-term memory network, random forest, LogitBoost, naive Bayes and logistic regression) using 10-fold cross-validation and independent set test. Results show that CNN could always display the best performance with high computational efficiency on large dataset. We also compare the Deep-Kcr with other existing tools to demonstrate the excellent predictive power and robustness of our method. Based on the proposed model, a webserver called Deep-Kcr was established and is freely accessible at http://lin-group.cn/server/Deep-Kcr.

Engineering the biological conversion of formate into crotonate in Cupriavidus necator.

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse ß-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

Comprehensive analysis of lysine crotonylation modification in patients with chronic renal failure.

BACKGROUND: Post-translational modifications (PTMs) are at the heart of many cellular signaling events, which changes the function of protein. Crotonylation, one of the most important and common PTMs, plays a crucial role in the regulation of various biological processes. However, no study has evaluated the role of lysine crotonylation modification in chronic renal failure (CRF) patients. METHODS: Here, we comparatively evaluated the crotonylation proteome of normal controls and chronic renal failure patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with highly sensitive immune-affinity purification. RESULTS: A total of 1109 lysine modification sites were identified, of which 772 sites were up-regulated and 69 sites were down-regulated. This suggested that crotonylation modification maintains high levels in the patients with chronic renal failure. Gene ontology(GO) enrichment analysis showed that the crotonylated proteins were significantly enriched in the platelet alpha granule lumen, platelet degradulation, and cell adhesion molecule binding. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG)-based functional enrichment analysis in the Kyoto encyclopedia showed that crotonylated protein was enriched in CD36, which is closely linked to renal failure. CONCLUSIONS: This is the first report of the global crotonylation proteome in chronic renal failure patients. Crotonylation of histone and non-histone may play important roles in delaying the continuous deterioration of renal function in patients with chronic renal failure.

Crotonylation at serine 46 impairs p53 activity.

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.

Chemical genetic inhibition of DEAD-box proteins using covalent complementarity.

DEAD-box proteins are an essential class of enzymes involved in all stages of RNA metabolism. The study of DEAD-box proteins is challenging in a native setting since they are structurally similar, often essential and display dosage sensitivity. Pharmacological inhibition would be an ideal tool to probe the function of these enzymes. In this work, we describe a chemical genetic strategy for the specific inactivation of individual DEAD-box proteins with small molecule inhibitors using covalent complementarity. We identify a residue of low conservation within the P-loop of the nucleotide-binding site of DEAD-box proteins and show that it can be mutated to cysteine without a substantial loss of enzyme function to generate electrophile-sensitive mutants. We then present a series of small molecules that rapidly and specifically bind and inhibit electrophile-sensitive DEAD-box proteins with high selectivity over the wild-type enzyme. Thus, this approach can be used to systematically generate small molecule-sensitive alleles of DEAD-box proteins, allowing for pharmacological inhibition and functional characterization of members of this enzyme family.

Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

iKcr-PseEns: Identify lysine crotonylation sites in histone proteins with pseudo components and ensemble classifier.

Lysine crotonylation (Kcr) is an evolution-conserved histone posttranslational modification (PTM), occurring in both human somatic and mouse male germ cell genomes. It is important for male germ cell differentiation. Information of Kcr sites in proteins is very useful for both basic research and drug development. But it is time-consuming and expensive to determine them by experiments alone. Here, we report a novel predictor called iKcr-PseEns that is established by incorporating five tiers of amino acid pairwise couplings into the general pseudo amino acid composition. It has been observed via rigorous cross-validations that the new predictor's sensitivity (Sn), specificity (Sp), accuracy (Acc), and stability (MCC) are 90.53%, 95.27%, 94.49%, and 0.826, respectively. For the convenience of most experimental scientists, a user-friendly web-server for iKcr-PseEns has been established at http://www.jci-bioinfo.cn/iKcr-PseEns, by which users can easily obtain their desired results without the need to go through the complicated mathematical equations involved.

Bioconversion of methane to C-4 carboxylic acids using carbon flux through acetyl-CoA in engineered Methylomicrobium buryatense 5GB1C.

Methane, the primary component of natural gas, is the second most abundant greenhouse gas (GHG) and contributes significantly to climate change. The conversion of methane to industrial platform chemicals provides an attractive opportunity to decrease GHG emissions and utilize this inexpensive and abundantly available gas as a carbon feedstock. While technologies exist for chemical conversion of methane to liquid fuels, the technical complexity of these processes mandate high capital expenditure, large-scale commercial facilities to leverage economies of scale that cannot be efficiently scaled down. Alternatively, bioconversion technologies capable of efficient small-scale operation with high carbon and energy efficiency can enable deployment at remote methane resources inaccessible to current chemical technologies. Aerobic obligate methanotrophs, specifically Methylomicrobium buryatense 5GB1, have recently garnered increased research interest for development of such bio-technologies. In this study, we demonstrate production of C-4 carboxylic acids non-native to the host, specifically crotonic and butyric acids, from methane in an engineered M. buryatense 5GB1C by diversion of carbon flux through the acetyl-CoA node of central 'sugar' linked metabolic pathways using reverse ß-oxidation pathway genes. The synthesis of short chain carboxylic acids through the acetyl-CoA node demonstrates the potential for engineering M. buryatense 5GB1 as a platform for bioconversion of methane to a number of value added industrial chemicals, and presents new opportunities for further diversifying the products obtainable from methane as the feedstock.

Effect of teriflunomide on QuantiFERON-TB Gold results.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by damage to myelin and axons, over time leading to progressive neuronal degeneration and microglial activation. There is still no curative treatment, but during the last 20 years eight different therapies have become available including interferon beta, glatiramer acetate, teriflunomide, dimethyl fumarate, natalizumab, fingolimod, alemtuzumab, mitoxantrone and teriflunomide. Teriflunomide is an immunomodulatory drug that exerts an inhibitory effect on T cell activation in central nervous system of the patients with multiple sclerosis. We determined whether teriflunomide affect the production of interferon-gamma, interleukin-2 and tumor-necrosis-factor-α in the QuantiFERON-TB in-Tube-assay. Blood from 24 adults with latent tuberculosis infection was added to one standard set of QuantiFERON tubes and one further set containing teriflunomide. Teriflunomide resulted in a change in QuantiFERON results from positive to negative in four patients with a marked reduction in interferon-γ. Our data indicated that results from QuantiFERON in patients on teriflunomide therapy should be interpreted with caution.

Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity.

AIM: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. METHODS: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed. RESULTS: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 µmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 µmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. CONCLUSION: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.

Physiological and biochemical characteristics of the ethyl tiglate production pathway in the yeast Saprochaete suaveolens.

A yeast identified as Saprochaete suaveolens was investigated for its capacity to produce a large panel of flavouring molecules. With a production of 32 compounds including 28 esters, S. suaveolens seems to be a good producer of fruity flavours and fragrances and especially of unsaturated esters, such as ethyl tiglate. Physiological and biochemical analyses were performed in this study in an attempt to comprehend the metabolic route to the formation of this compound. We show that the accumulation of ethyl tiglate by S. suaveolens is specifically induced by isoleucine. However, and contrary to S. cerevisiae, which harbours a classical Ehrlich pathway leading to the production of 2-methylbutanol from isoleucine, our results provide phenotypic and enzymological evidence of ethyl tiglate biosynthesis in S. suaveolens through the catabolism of this amino acid by the ß-oxidation pathway, which generates tiglyl-CoA as a probable intermediate. A kinetic analysis of this flavour molecule during growth of S. suaveolens on glucose and isoleucine showed a phase of production of ethyl tiglate that culminated concurrently with isoleucine exhaustion, followed by a disappearance of this compound, likely due to reassimilation by the yeast.

Microwave-assisted synthesis, molecular docking, and cholinesterase inhibitory activities of new ethanediamide and 2-butenediamide analogues.

A novel series of meta-substituted ethanediamide and 2-butenediamide derivatives were synthesized and tested for their ability to inhibit electric eel acetylcholinesterase (AChE) and equine serum butyrylcholinesterase (BuChE). The synthesized compounds were evaluated against ChE enzymes using the colorimetric method described by Ellman et al. (Biochem. Pharmacol., 7, 1961). It was revealed that some synthesized compounds exhibited high anticholinesterase activity, among which compounds 1f and 2f were the most active inhibitors against BuChE (IC50 value=1.47 µM) and AChE (IC50 value=2.09 µM), respectively. Docking simulations revealed that the inhibitors 1f and 2f are capable of simultaneously binding the peripheral anionic site as well as the catalytic anionic site of both ChE enzymes. These derivatives are considered interesting candidates for Alzheimer's disease treatment.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Cyclohexanecarboxyl-coenzyme A (CoA) and cyclohex-1-ene-1-carboxyl-CoA dehydrogenases, two enzymes involved in the fermentation of benzoate and crotonate in Syntrophus aciditrophicus.

The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.

Crotonylation of GAPDH regulates human embryonic stem cell endodermal lineage differentiation and metabolic switch.

BACKGROUND: Post-translational modifications of proteins are crucial to the regulation of their activity and function. As a newly discovered acylation modification, crotonylation of non-histone proteins remains largely unexplored, particularly in human embryonic stem cells (hESCs). METHODS: We investigated the role of crotonylation in hESC differentiation by introduce crotonate into the culture medium of GFP tagged LTR7 primed H9 cell and extended pluripotent stem cell lines. RNA-seq assay was used to determine the hESC transcriptional features. Through morphological changes, qPCR of pluripotent and germ layer-specific gene markers and flow cytometry analysis, we determined that the induced crotonylation resulted in hESC differentiating into the endodermal lineage. We performed targeted metabolomic analysis and seahorse metabolic measurement to investigate the metabolism features after crotonate induction. Then high-resolution tandem mass spectrometry (LC-MS/MS) revealed the target proteins in hESCs. In addition, the role of crotonylated glycolytic enzymes (GAPDH and ENOA) was evaluated by in vitro crotonylation and enzymatic activity assays. Finally, we used knocked-down hESCs by shRNA, wild GAPDH and GAPDH mutants to explore potential role of GAPDH crotonylation in regulating human embryonic stem cell differentiation and metabolic switch. RESULT: We found that induced crotonylation in hESCs resulted in hESCs of different pluripotency states differentiating into the endodermal lineage. Increased protein crotonylation in hESCs was accompanied by transcriptomic shifts and decreased glycolysis. Large-scale crotonylation profiling of non-histone proteins revealed that metabolic enzymes were major targets of inducible crotonylation in hESCs. We further discovered GAPDH as a key glycolytic enzyme regulated by crotonylation during endodermal differentiation from hESCs. CONCLUSIONS: Crotonylation of GAPDH decreased its enzymatic activity thereby leading to reduced glycolysis during endodermal differentiation from hESCs.

Enoyl-Coenzyme A Respiration via Formate Cycling in Syntrophic Bacteria.

Syntrophic bacteria play a key role in the anaerobic conversion of biological matter to methane. They convert short-chain fatty acids or alcohols to H2, formate, and acetate that serve as substrates for methanogenic archaea. Many syntrophic bacteria can also grow with unsaturated fatty acids such as crotonate without a syntrophic partner, and the reducing equivalents derived from the oxidation of one crotonate to two acetate are regenerated by the reduction of a second crotonate. However, it has remained unresolved how the oxidative and reductive catabolic branches are interconnected and how energy may be conserved in the reductive branch. Here, we provide evidence that during axenic growth of the syntrophic model organism Syntrophus aciditrophicus with crotonate, the NAD+-dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA is coupled to the reduction of crotonyl-CoA via formate cycling. In this process, the intracellular formate generated by a NAD+-regenerating CO2 reductase is taken up by a periplasmic, membrane-bound formate dehydrogenase that in concert with a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone oxidoreductase, ETF, and an acyl-CoA dehydrogenase reduces intracellular enoyl-CoA to acyl-CoA. This novel type of energy metabolism, referred to as enoyl-CoA respiration, generates a proton motive force via a methylmenaquinone-dependent redox-loop. As a result, the beneficial syntrophic cooperation of fermenting bacteria and methanogenic archaea during growth with saturated fatty acids appears to turn into a competition for formate and/or H2 during growth with unsaturated fatty acids. IMPORTANCE The syntrophic interaction of fermenting bacteria and methanogenic archaea is important for the global carbon cycle. As an example, it accomplishes the conversion of biomass-derived saturated fatty acid fermentation intermediates into methane. In contrast, unsaturated fatty acid intermediates such as crotonate may serve as growth substrate for the fermenting partner alone. Thereby, the reducing equivalents generated during the oxidation of one crotonate to two acetate are regenerated by reduction of a second crotonate to butyrate. Here, we show that the oxidative and reductive branches of this pathway are connected via formate cycling involving an energy-conserving redox-loop. We refer to this previously unknown type of energy metabolism as to enoyl-CoA respiration with acyl-CoA dehydrogenases serving as cytoplasmic terminal reductases.

Toxoplasma gondii Infection Inhibits Histone Crotonylation to Regulate Immune Response of Porcine Alveolar Macrophages.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that can infect almost all warm-blooded animals, causing serious public health problems. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM), which is first identified on histones and has been proved relevant to procreation regulation, transcription activation, and cell signaling pathway. However, the biological functions of histone crotonylation have not yet been reported in macrophages infected with T. gondii. As a result, a total of 1,286 Kcr sites distributed in 414 proteins were identified and quantified, demonstrating the existence of crotonylation in porcine alveolar macrophages. According to our results, identified histones were overall downregulated. HDAC2, a histone decrotonylase, was found to be significantly increased, which might be the executor of histone Kcr after parasite infection. In addition, T. gondii infection inhibited the crotonylation of H2B on K12, contributing on the suppression of epigenetic regulation and NF-κB activation. Nevertheless, the reduction of histone crotonylation induced by parasite infection could promote macrophage proliferation via activating PI3K/Akt signaling pathway. The present findings point to a comprehensive understanding of the biological functions of histone crotonylation in porcine alveolar macrophages, thereby providing a certain research basis for the mechanism research on the immune response of host cells against T. gondii infection.