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Microbiota and feeding modes influence the susceptibility of premature newborns to necrotizing enterocolitis (NEC) through mechanisms that remain unknown. Here, we show that microbiota colonization facilitated by breastmilk feeding promotes NOD-like receptor family CARD domain containing 5 (Nlrc5) gene expression in mouse intestinal epithelial cells (IECs). Notably, inducible knockout of the Nlrc5 gene in IECs predisposes neonatal mice to NEC-like injury in the small intestine upon viral inflammation in an NK1.1 + cell-dependent manner. By contrast, formula feeding enhances neonatal gut colonization with environment-derived tilivalline-producing Klebsiella spp. Remarkably, tilivalline disrupts microbiota-activated STAT1 signaling that controls Nlrc5 gene expression in IECs through a PPAR-γ-mediated mechanism. Consequently, this dysregulation hinders the resistance of neonatal intestinal epithelium to self-NK1.1 + cell cytotoxicity upon virus infection/colonization, promoting NEC development. Together, we discover the underappreciated role of intestinal microbiota colonization in shaping a disease tolerance program to viral inflammation and elucidate the mechanisms impacting NEC development in neonates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities., (© 2024. The Author(s).)

Background: Around 10% of fractures lead to complications. With increasing fracture incidences in recent years, this poses a serious burden on the healthcare system, with increasing costs for treatment. In the present study, we aimed to identify potential 'new' blood markers to predict the development of post-surgical complications in trauma patients following a fracture., Methods: A total of 292 trauma patients with a complete three-month follow-up were included in this cohort study. Blood samples were obtained from 244 of these patients. Two complication groups were distinguished based on the Clavien-Dindo (CD) classification: CD grade I and CD grade III groups were compared to the controls (CD 0). The Mann-Whitney U test was used to compare the complication groups to the control group., Results: Analysis of the patients' data revealed that risk factors are dependent on sex. Both, males and females who developed a CD III complication showed elevated blood levels of B7-1 (p = 0.015 and p = 0.018, respectively) and PlGF-1 (p = 0.009 and p = 0.031, respectively), with B7-1 demonstrating greater sensitivity (B7-1: 0.706 (male) and 0.692 (female), PlGF-1: 0.647 (male) and 0.615 (female)). Further analysis of the questionnaires and medical data revealed the importance of additional risk factors. For males (CD 0: 133; CD I: 12; CD III: 18 patients) alcohol consumption was significantly increased for CD I and CD III compared to control with p = 0.009 and p = 0.007, respectively. For females (CD 0: 107; CD I: 10; CD III: 12 patients) a significantly increased average BMI [kg/m 2 ] from 25.5 to 29.7 with CD III was observed, as well as an elevation from one to three comorbidities (p = 0.003)., Conclusions: These two potential new blood markers hold promise for predicting complication development in trauma patients. Nevertheless, further studies are necessary to evaluate the diagnostic utility of B7-1 and PlGF-1 in predicting complications in trauma patients and consider sex differences before their possible use as routine clinical screening tools., (© 2024. The Author(s).)

Smoking is the most significant and modifiable risk factor for a range of conditions, including cancer, cardiovascular and respiratory diseases. Furthermore, it significantly reduces bone mass and increases the risk of fragility fractures due to its detrimental effects on bone metabolism and regeneration. Moreover, smoking is a known cause of chronic systemic inflammation, leading to an imbalance of cytokines. Comprehending the pathological mechanisms that underlie cytokine production and its impact on post-surgical healing is essential to prevent post-surgical complications. The present study recruited a total of 1144 patients, including 897 patients, among them non-smokers ( N = 413 ), current smokers ( N = 201 ) and ex-smokers ( N = 283 ). Human proteome profiler arrays were used to screen for smoking-dependent differences in the serum cytokine and protein profiles, after matching samples for age, gender, body mass index (BMI), alcohol use, and diabetes risk. Cytokines and immune checkpoint proteins such as CD28, B7-1, MIG, TGFβ2 and IL-1α/β were quantified by ELISA. Our study demonstrates a comprehensive understanding of the relationship between smoking, the development of complications, the systemic immune inflammation index (SII) and cytokine/protein levels. We found that a comparison of non-smokers, former smokers, and active smokers in our study cohort did not exhibit significantly altered cytokine and protein serum levels although other studies reported differences between smokers and non-smokers. We were unable to identify single blood circulating markers that could predict complications in smokers after trauma. However, we found the ratio of women to men to be inverted between non-smokers and active smokers resulting in a ratio of 0.62 in smokers. Furthermore, we demonstrate a higher complication rate, longer hospitalizations and elevated SII values among smokers, indicating an involvement of the immune system. See also the graphical abstract(Fig. 1)., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2024 Tümen et al.)

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions., (© 2024. The Author(s).)

Flavin mononucleotide (FMN)-dependent ene-reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon-carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene-reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH-preferring 12-oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene-reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β-hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex with either NADPH 4 or NADH 4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β-hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β-hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β-hairpin flap favors NADH binding., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Early and reliable detection of infection is vital for successful treatment. Serum markers such as C-reactive protein (CRP) and procalcitonin (PCT) are known to increase with a time lag. Azurocidin 1 (AZU1) has emerged as a promising marker for septic patients, but its diagnostic value in orthopedic and trauma patients remains unexplored. Between July 2020 and August 2023, all patients necessitating inpatient treatment for periprosthetic joint infection (PJI), peri-implant infection (II), soft tissue infection, chronic osteomyelitis, septic arthrodesis, bone non-union with and without infection were enrolled. Patients undergoing elective total joint arthroplasty (TJA) served as the control group. Blood samples were collected and analyzed for CRP, white blood cell count (WBC), PCT, and AZU1. Based on the inclusion and exclusion criteria 222 patients were included in the study (trauma = 38, soft tissue infection = 75, TJA = 33, PJI/II = 39, others = 37). While sensitivity and specificity were comparably high for AZU1 (0.734/0.833), CRP and PCT had higher specificity (0.542/1 and 0.431/1, respectively), and WBC a slightly higher sensitivity (0.814/0.455) for septic conditions. Taken together, the area under the curve (AUC) showed the highest accuracy for AZU1 (0.790), followed by CRP (0.776), WBC (0.641), and PCT (0.656). The Youden-Index was 0.57 for AZU1, 0.54 for CRP, 0.27 for WBC, and 0.43 for PCT. Elevated AZU1 levels effectively distinguished patients with a healthy condition from those suffering from infection. However, there is evidence suggesting that trauma may influence the release of AZU1. Additional research is needed to validate the diagnostic value of this new biomarker and further explore its potential clinical applications., Competing Interests: All authors declare that they have no conflict of interest., (Copyright © 2024 Hemmann et al.)

The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis (MS). Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity. To date, however, the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood. Here, we report that stearoyl-CoA desaturase-1 (SCD1), an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors, acts as an endogenous brake on regulatory T-cell (Treg) differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner. Guided by RNA sequencing and lipidomics analysis, we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase (ATGL). ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma. Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity, with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS., (© 2023. The Author(s).)

The DNA-alkylating metabolite tilimycin is a microbial genotoxin. Intestinal accumulation of tilimycin in individuals carrying til+ Klebsiella spp. causes apoptotic erosion of the epithelium and colitis. Renewal of the intestinal lining and response to injury requires the activities of stem cells located at the base of intestinal crypts. This study interrogates the consequences of tilimycin-induced DNA damage to cycling stem cells. We charted the spatial distribution and luminal quantities of til metabolites in Klebsiella-colonized mice in the context of a complex microbial community. Loss of marker gene G6pd function indicates genetic aberrations in colorectal stem cells that became stabilized in monoclonal mutant crypts. Mice colonized with tilimycin-producing Klebsiella displayed both higher frequencies of somatic mutation and more mutations per affected individual than animals carrying a non-producing mutant. Our findings imply that genotoxic til+ Klebsiella may drive somatic genetic change in the colon and increase disease susceptibility in human hosts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cigarette smoking-induced oxidative stress has harmful effects on bone metabolism. Maqui berry extract (MBE) and ginseng extract (GE) are two naturally occurring antioxidants that have been shown to reduce oxidative stress. By using an osteoblast and osteoclast three-dimensional co-culture system, we investigated the effects of MBE and GE on bone cells exposed to cigarette smoke extract (CSE). The cell viability and function of the co-culture system were measured on day 14. Markers of bone cell differentiation and oxidative stress were evaluated at gene and protein levels on day 7. The results showed that exposure to CSE induced osteoporotic-like alterations in the co-culture system, while 1.5 µg/mL MBE and 50 µg/mL GE improved CSE-impaired osteoblast function and decreased CSE-induced osteoclast function. The molecular mechanism of MBE and GE in preventing CSE-induced bone cell damage is linked with the inhibition of the NF-κB signaling pathway and the activation of the Nrf2 signaling pathway. Therefore, MBE and GE can reduce CSE-induced detrimental effects on bone cells and, thus, prevent smoking-induced alterations in bone cell homeostasis. These two antioxidants are thus suitable supplements to support bone regeneration in smokers.

Aims: Heart failure (HF) is characterized by an overactivation of β-adrenergic signalling that directly contributes to impairment of myocardial function. Moreover, β-adrenergic overactivation induces adipose tissue lipolysis, which may further worsen the development of HF. Recently, we demonstrated that adipose tissue-specific deletion of adipose triglyceride lipase (ATGL) prevents pressure-mediated HF in mice. In this study, we investigated the cardioprotective effects of a new pharmacological inhibitor of ATGL, Atglistatin, predominantly targeting ATGL in adipose tissue, on catecholamine-induced cardiac damage., Methods and Results: Male 129/Sv mice received repeated injections of isoproterenol (ISO, 25 mg/kg BW) to induce cardiac damage. Five days prior to ISO application, oral Atglistatin (2 mmol/kg diet) or control treatment was started. Two and twelve days after the last ISO injection cardiac function was analysed by echocardiography. The myocardial deformation was evaluated using speckle-tracking-technique. Twelve days after the last ISO injection, echocardiographic analysis revealed a markedly impaired global longitudinal strain, which was significantly improved by the application of Atglistatin. No changes in ejection fraction were observed. Further studies included histological-, WB-, and RT-qPCR-based analysis of cardiac tissue, followed by cell culture experiments and mass spectrometry-based lipidome analysis. ISO application induced subendocardial fibrosis and a profound pro-apoptotic cardiac response, as demonstrated using an apoptosis-specific gene expression-array. Atglistatin treatment led to a dramatic reduction of these pro-fibrotic and pro-apoptotic processes. We then identified a specific set of fatty acids (FAs) liberated from adipocytes under ISO stimulation (palmitic acid, palmitoleic acid, and oleic acid), which induced pro-apoptotic effects in cardiomyocytes. Atglistatin significantly blocked this adipocytic FA secretion., Conclusion: This study demonstrates cardioprotective effects of Atglistatin in a mouse model of catecholamine-induced cardiac damage/dysfunction, involving anti-apoptotic and anti-fibrotic actions. Notably, beneficial cardioprotective effects of Atglistatin are likely mediated by non-cardiac actions, supporting the concept that pharmacological targeting of adipose tissue may provide an effective way to treat cardiac dysfunction., Competing Interests: Conflict of interest: M.R. is an employee of Lipidomix GmbH, Berlin, Germany. R.B., A.K.M., R. Z., and G.F.G. have filed for a patent of inhibitors of human ATGL., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Tuberculosis continues to be a major threat to the human population. Global efforts to eradicate the disease are ongoing but are hampered by the increasing occurrence of multidrug-resistant strains of Mycobacterium tuberculosis . Therefore, the development of new treatment, and the exploration of new druggable targets and treatment strategies, are of high importance. Rv0183/mtbMGL, is a monoacylglycerol lipase of M. tuberculosis and it is involved in providing fatty acids and glycerol as building blocks and as an energy source. Since the lipase is expressed during the dormant and active phase of an infection, Rv0183/mtbMGL is an interesting target for inhibition. In this work, we determined the crystal structures of a surface-entropy reduced variant K74A Rv0183/mtbMGL in its free form and in complex with a substrate mimicking inhibitor. The two structures reveal conformational changes in the cap region that forms a major part of the substrate/inhibitor binding region. We present a completely closed conformation in the free form and semi-closed conformation in the ligand-bound form. These conformations differ from the previously published, completely open conformation of Rv0183/mtbMGL. Thus, this work demonstrates the high conformational plasticity of the cap from open to closed conformations and provides useful insights into changes in the substrate-binding pocket, the target of potential small-molecule inhibitors.

Following cardiac injury, increased adrenergic drive plays an important role in compensating for reduced cardiac function. However, chronic excess adrenergic stimulation can be detrimental to cardiac pathophysiology and can also affect other organs including adipose tissue, leading to increased lipolysis. Interestingly, inhibition of adipose triglyceride lipase (ATGL), a rate-limiting enzyme in lipolysis, in adipocytes ameliorates cardiac dysfunction in a heart failure model. Thus, we investigated whether inhibition of adipocyte ATGL can mitigate the adverse cardiac effects of chronic adrenergic stimulation and explored the underlying mechanisms. To do this, isoproterenol (ISO) was continuously administered to C57Bl/6N mice for 2 wk with or without an ATGL inhibitor (Atglistatin). We found that Atglistatin alleviated ISO-induced cardiac remodeling and reduced ISO-induced upregulation of galectin-3, a marker of activated macrophages and a potent inducer of fibrosis, in white adipose tissue (WAT), heart, and the circulation. To test whether the beneficial effects of Atglistatin occur via inhibition of adipocyte ATGL, adipocyte-specific ATGL knockout (atATGL-KO) mice were utilized for similar experiments. Subsequently, the same cardioprotective effects of atATGL-KO following ISO administration were observed. Furthermore, Atglistatin and atATGL-KO abolished ISO-induced galectin-3 secretion from excised WAT. We further demonstrated that activation of cardiac fibroblasts by the conditioned media of ISO-stimulated WAT is galectin-3-dependent. In conclusion, the inhibition of adipocyte ATGL ameliorated ISO-induced cardiac remodeling possibly by reducing galectin-3 secretion from adipose tissue. Thus, inhibition of adipocyte ATGL might be a potential target to prevent some of the adverse effects of chronic excess adrenergic drive. NEW & NOTEWORTHY The reduction of lipolysis by adipocyte ATGL inhibition ameliorates cardiac remodeling induced by chronic β-adrenergic stimulation likely via reducing galectin-3 secretion from adipose tissue. Our findings highlight that suppressing lipolysis in adipocytes may be a potential therapeutic target for patients with heart failure whose sympathetic nervous system is activated. Furthermore, galectin-3 might be involved in the mechanisms by which excessive lipolysis in adipose tissues influences remote cardiac pathologies and thus warrants further investigation.

Bis(monoacylglycerol)phosphate (BMP) is a phospholipid that is crucial for lipid degradation and sorting in acidic organelles. Genetic and drug-induced lysosomal storage disorders (LSDs) are associated with increased BMP concentrations in tissues and in the circulation. Data on BMP in disorders other than LSDs, however, are scarce, and key enzymes regulating BMP metabolism remain elusive. Here, we demonstrate that common metabolic disorders and the intracellular BMP hydrolase α/β-hydrolase domain-containing 6 (ABHD6) affect BMP metabolism in mice and humans. In mice, dietary lipid overload strongly affects BMP concentration and FA composition in the liver and plasma, similar to what has been observed in LSDs. Notably, distinct changes in the BMP FA profile enable a clear distinction between lipid overload and drug-induced LSDs. Global deletion of ABHD6 increases circulating BMP concentrations but does not cause LSDs. In humans, nonalcoholic fatty liver disease and liver cirrhosis affect the serum BMP FA composition and concentration. Furthermore, we identified a patient with a loss-of-function mutation in the ABHD6 gene, leading to an altered circulating BMP profile. In conclusion, our results suggest that common metabolic diseases and ABHD6 affect BMP metabolism in mice and humans., (Copyright © 2019 Grabner et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Despite advancements in therapies for cardiovascular disease and heart failure (HF), the incidence and prevalence of HF are increasing. Previous work has suggested that inhibiting adipose triglyceride lipase (ATGL) in adipose tissue during HF development may assist in the treatment of HF. The ability to specifically target the adipocyte as a potential treatment for HF is a novel approach that could significantly influence the management of HF in the future. Our objectives were to assess the cardiac structural and functional effects of pharmacological inhibition of ATGL in mice with HF, to assess whether ATGL inhibition works in an adipocyte-autonomous manner, and to determine the role that adiposity and glucose homeostasis play in this HF treatment approach. Using a known ATGL inhibitor, atglistatin, as well as mice with germline deletion of adipocyte-specific ATGL, we tested the effectiveness of ATGL inhibition in mice with pressure overload-induced HF. Here, we show that atglistatin can prevent the functional decline in HF and provide evidence that specifically targeting ATGL in the adipocyte is sufficient to prevent worsening of HF. We further demonstrate that the benefit resulting from atglistatin in HF is not dependent on previously suggested improvements in glucose homeostasis, nor are the benefits derived from increased adiposity. Overall, the results of this study suggest that adipocyte-specific pharmacological inhibition of ATGL may represent a novel therapeutic option for HF. NEW & NOTEWORTHY This work shows for the first time that the adipose triglyceride lipase (ATGL)-specific inhibitor atglistatin can prevent worsening heart failure. Furthermore, using mice with adipocyte-specific ATGL ablation, this study demonstrates that ATGL inhibition works in an adipocyte-autonomous manner to ameliorate a functional decline in heart failure. Overall, this work demonstrates that specifically targeting the adipocyte to inhibit ATGL is a potential treatment for heart failure.

Monoacylglycerol lipases (MGLs) are enzymes that hydrolyze monoacylglycerol into a free fatty acid and glycerol. Fatty acids can be used for triacylglycerol synthesis, as energy source, as building blocks for energy storage, and as precursor for membrane phospholipids. In Mycobacterium tuberculosis, fatty acids also serve as precursor for polyketide lipids like mycolic acids, major components of the cellular envelope associated to resistance for drug. We present the crystal structure of the MGL Rv0183 from Mycobacterium tuberculosis (mtbMGL) in open conformation. The structure reveals remarkable similarities with MGL from humans (hMGL) in both, the cap region and the α/β core. Nevertheless, mtbMGL could not be inhibited with JZL-184, a known inhibitor of hMGL. Docking studies provide an explanation why the activity of mtbMGL was not affected by the inhibitor. Our findings suggest that specific inhibition of mtbMGL from Mycobacterium tuberculosis, one of the oldest recognized pathogens, is possible without influencing hMGL.

Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development.

Elevated circulating fatty acids (FAs) contribute to the development of obesity-associated metabolic complications such as insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD). Hence, reducing adipose tissue lipolysis to diminish the mobilization of FAs and lower their respective plasma concentrations represents a potential treatment strategy to counteract obesity-associated disorders. Here we show that specific inhibition of adipose triglyceride lipase (Atgl) with the chemical inhibitor, Atglistatin, effectively reduces adipose tissue lipolysis, weight gain, IR and NAFLD in mice fed a high-fat diet. Importantly, even long-term treatment does not lead to lipid accumulation in ectopic tissues such as the skeletal muscle or heart. Thus, the severe cardiac steatosis and cardiomyopathy that is observed in genetic models of Atgl deficiency does not occur in Atglistatin-treated mice. Our data validate the pharmacological inhibition of Atgl as a potentially powerful therapeutic strategy to treat obesity and associated metabolic disorders.

The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling., (Copyright © 2016 Elsevier B.V. All rights reserved.)

α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. However, the mechanistic link between gene function and disease is incompletely understood. Here we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid, is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little has been known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ∼ 90% of the BMP hydrolase activity detected in the liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live-cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity, implying that it degrades BMP exported from acidic organelles or de novo-formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid-sorting machinery., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Posttranslational modifications (PTMs) control protein function, but established peptide-based proteomic methods often fail to provide a comprehensive view of PTMs. In this issue of Chemistry & Biology, Gersch et al. describe an efficient combination of chromatographic separation and top-down mass spectrometry that together with an intuitive visualization tool allowed them to screen the proteasome for PTMs and covalently binding inhibitors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.