56 research outputs found
Microbiota alterations in proline metabolism impact depression
The microbiota-gut-brain axis has emerged as a novel target in depression, a disorder with low treatment efficacy. However, the field is dominated by underpowered studies focusing on major depression not addressing microbiome functionality, compositional nature, or confounding factors. We applied a multi-omics approach combining pre-clinical models with three human cohorts including patients with mild depression. Microbial functions and metabolites converging onto glutamate/GABA metabolism, particularly proline, were linked to depression. High proline consumption was the dietary factor with the strongest impact on depression. Whole-brain dynamics revealed rich club network disruptions associated with depression and circulating proline. Proline supplementation in mice exacerbated depression along with microbial translocation. Human microbiota transplantation induced an emotionally impaired phenotype in mice and alterations in GABA-, proline-, and extracellular matrix-related prefrontal cortex genes. RNAi-mediated knockdown of proline and GABA transporters in Drosophila and mono-association with L. plantarum, a high GABA producer, conferred protection against depression-like states. Targeting the microbiome and dietary proline may open new windows for efficient depression treatment
3D Structure Prediction of TAS2R38 Bitter Receptors Bound to Agonists Phenylthiocarbamide (PTC) and 6-n-Propylthiouracil (PROP)
Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations
Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors
Obesity status and obesity-associated gut dysbiosis effects on hypothalamic structural covariance
[Background]: Functional connectivity alterations in the lateral and medial hypothalamic networks have been associated with the development and maintenance of obesity, but the possible impact on the structural properties of these networks remains largely unexplored. Also, obesity-related gut dysbiosis may delineate specific hypothalamic alterations within obese conditions. We aim to assess the effects of obesity, and obesity and gut-dysbiosis on the structural covariance differences in hypothalamic networks, executive functioning, and depressive symptoms.[Methods]: Medial (MH) and lateral (LH) hypothalamic structural covariance alterations were identified in 57 subjects with obesity compared to 47 subjects without obesity. Gut dysbiosis in the subjects with obesity was defined by the presence of high (n = 28) and low (n = 29) values in a BMI-associated microbial signature, and posthoc comparisons between these groups were used as a proxy to explore the role of obesity-related gut dysbiosis on the hypothalamic measurements, executive function, and depressive symptoms.[Results]: Structural covariance alterations between the MH and the striatum, lateral prefrontal, cingulate, insula, and temporal cortices are congruent with previously functional connectivity disruptions in obesity conditions. MH structural covariance decreases encompassed postcentral parietal cortices in the subjects with obesity and gut-dysbiosis, but increases with subcortical nuclei involved in the coding food-related hedonic information in the subjects with obesity without gut-dysbiosis. Alterations for the structural covariance of the LH in the subjects with obesity and gut-dysbiosis encompassed increases with frontolimbic networks, but decreases with the lateral orbitofrontal cortex in the subjects with obesity without gut-dysbiosis. Subjects with obesity and gut dysbiosis showed higher executive dysfunction and depressive symptoms.[Conclusions]: Obesity-related gut dysbiosis is linked to specific structural covariance alterations in hypothalamic networks relevant to the integration of somatic-visceral information, and emotion regulation.This study has been funded by the Project Grant IRONMET (PI15/01934) from the ISCIII, and the Project ThinkGut (EFA345/19) 65% co-financed by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra program (POCTEFA 2014–2020) (JM Fernández-Real). Partial support was also obtained by the Ministerio de Economia y Competitividad, Spain, reference MTM2015-64465-C2-1-R (ML Calle). O Contreras-Rodriguez is funded by a “PERIS” postdoctoral fellowship (SLT006/17/00236) from the Health Department of the Catalan Government and by a “Miguel Servet” contract (CP20/00165) from the ISCIII. M Arnoriaga-Rodríguez is funded by a predoctoral Rio Hortega contract (CM19/00190) co-funded by European Social Fund “Investigating in your future” from the ISCIII.Peer reviewe
SNi from SN2: a front-face mechanism ‘synthase’ engineered from a retaining hydrolase
SNi or SNi-like mechanisms, in which leaving group departure and nucleophile approach occur on the same ‘front’ face, have been observed previously experimentally and computationally in both the chemical and enzymatic (glycosyltransferase) substitution reactions of α-glycosyl electrophiles. Given the availability of often energetically comparable competing pathways for substitution (SNi vs SN1 vs SN2) the precise modulation of this archetypal reaction type should be feasible. Here, we show that the drastic engineering of a protein that catalyzes substitution, a retaining β-glycosidase (from Sulfolobus solfataricus SSβG), apparently changes the mode of reaction from “SN2” to “SNi”. Destruction of the nucleophilic Glu387 of SSβG-WT through Glu387Tyr mutation (E387Y) created a catalyst (SSβG-E387Y) with lowered but clear transglycosylation substitution activity with activated substrates, altered substrate and reaction preferences and hence useful synthetic (‘synthase’) utility by virtue of its low hydrolytic activity with unactivated substrates. Strikingly, the catalyst still displayed retaining β stereoselectivity, despite lacking a suitable nucleophile; pH-activity profile, mechanism-based inactivators and mutational analyses suggest that SSβG-E387Y operates without either the use of nucleophile or general acid/base residues, consistent with a SNi or SNi-like mechanism. An x-ray structure of SSβG-E387Y and subsequent metadynamics simulation suggest recruitment of substrates aided by a π-sugar interaction with the introduced Tyr387 and reveal a QM/MM free energy landscape for the substitution reaction catalyzed by this unnatural enzyme similar to those of known natural, SNi-like glycosyltransferase (GT) enzymes. Proton flight from the putative hydroxyl nucleophile to the developing p-nitrophenoxide leaving group of the substituted molecule in the reactant complex creates a hydrogen bond that appears to crucially facilitate the mechanism, mimicking the natural mechanism of SNi-GTs. An oxocarbenium ion-pair minimum along the reaction pathway suggests a step-wise SNi-like DN*ANss rather than a concerted SNi DNAN mechanism. This first observation of a front face mechanism in a β-retaining glycosyl transfer enzyme highlights, not only that unusual SNi reaction pathways may be accessed through direct engineering of catalysts with suitable environments, but also suggests that ‘β-SNi’ reactions are also feasible for glycosyl transfer enzymes and the more widespread existence of SNi or SNi-like mechanism in nature
Insights into the Binding of Phenyltiocarbamide (PTC) Agonist to Its Target Human TAS2R38 Bitter Receptor
Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models
IL-1β Is Upregulated in the Diabetic Retina and Retinal Vessels: Cell-Specific Effect of High Glucose and IL-1β Autostimulation
Many molecular and cellular abnormalities detected in the diabetic retina support a role for IL-1β-driven neuroinflammation in the pathogenesis of diabetic retinopathy. IL-1β is well known for its role in the induction and, through autostimulation, amplification of neuroinflammation. Upregulation of IL-1β has been consistently detected in the diabetic retina; however, the mechanisms and cellular source of IL-1β overexpression are poorly understood. The aim of this study was to investigate the effect of high glucose and IL-1β itself on IL-1β expression in microglial, macroglial (astrocytes and Müller cells) and retinal vascular endothelial cells; and to study the effect of diabetes on the expression of IL-1β in isolated retinal vessels and on the temporal pattern of IL-1β upregulation and glial reactivity in the retina of streptozotocin-diabetic rats. IL-1β was quantified by RealTime RT-PCR and ELISA, glial fibrillar acidic protein, α2-macroglobulin, and ceruloplasmin by immunoblotting. We found that high glucose induced a 3-fold increase of IL-1β expression in retinal endothelial cells but not in macroglia and microglia. IL-1β induced its own synthesis in endothelial and macroglial cells but not in microglia. In retinal endothelial cells, the high glucose-induced IL-1β overexpression was prevented by calphostin C, a protein kinase C inhibitor. The retinal vessels of diabetic rats showed increased IL-1β expression as compared to non-diabetic rats. Retinal expression of IL-1β increased early after the induction of diabetes, continued to increase with progression of the disease, and was temporally associated with upregulation of markers of glial activation. These findings point to hyperglycemia as the trigger and to the endothelium as the origin of the initial retinal upregulation of IL-1β in diabetes; and to IL-1β itself, via autostimulation in endothelial and macroglial cells, as the mechanism of sustained IL-1β overexpression. Interrupting the vicious circle triggered by IL-1β autostimulation could limit the progression of diabetic retinopathy
Well-Tempered Metadynamics as a Tool for Characterizing Multi-Component, Crystalline Molecular Machines
Prevalence of Age-Related Macular Degeneration in Europe: The Past and the Future
Purpose Age-related macular degeneration (AMD) is a frequent, complex disorder in elderly of European ancestry. Risk profiles and treatment options have changed considerably over the years, which may have affected disease prevalence and outcome. We determined the prevalence of early and late AMD in Europe from 1990 to 2013 using the European Eye Epidemiology (E3) consortium, and made projections for the future. Design Meta-analysis of prevalence data. Participants A total of 42 080 individuals 40 years of age and older participating in 14 population-based cohorts from 10 countries in Europe. Methods AMD was diagnosed based on fundus photographs using the Rotterdam Classification. Prevalence of early and late AMD was calculated using random-effects meta-analysis stratified for age, birth cohort, gender, geographic region, and time period of the study. Best-corrected visual acuity (BCVA) was compared between late AMD subtypes; geographic atrophy (GA) and choroidal neovascularization (CNV). Main Outcome Measures Prevalence of early and late AMD, BCVA, and number of AMD cases. Results Prevalence of early AMD increased from 3.5% (95% confidence interval [CI] 2.1%–5.0%) in those aged 55–59 years to 17.6% (95%
Expression of acute-phase response proteins in retinal Müller cells in diabetes. Invest Ophthalmol Vis Sci 46: 349–357
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