Machine learning of plasma metabolome identifies biomarker panels for metabolic syndrome: Findings from the China Suboptimal Health Cohort

Background: Metabolic syndrome (MetS) has been proposed as a clinically identifiable high-risk state for the prediction and prevention of cardiovascular diseases and type 2 diabetes mellitus. As a promising “omics” technology, metabolomics provides an innovative strategy to gain a deeper understanding of the pathophysiology of MetS. The study aimed to systematically investigate the metabolic alterations in MetS and identify biomarker panels for the identification of MetS using machine learning methods. Methods: Nuclear magnetic resonance-based untargeted metabolomics analysis was performed on 1011 plasma samples (205 MetS patients and 806 healthy controls). Univariate and multivariate analyses were applied to identify metabolic biomarkers for MetS. Metabolic pathway enrichment analysis was performed to reveal the disturbed metabolic pathways related to MetS. Four machine learning algorithms, including support vector machine (SVM), random forest (RF), k-nearest neighbor (KNN), and logistic regression were used to build diagnostic models for MetS. Results: Thirteen significantly differential metabolites were identified and pathway enrichment revealed that arginine, proline, and glutathione metabolism are disturbed metabolic pathways related to MetS. The protein-metabolite-disease interaction network identified 38 proteins and 23 diseases are associated with 10 MetS-related metabolites. The areas under the receiver operating characteristic curve of the SVM, RF, KNN, and logistic regression models based on metabolic biomarkers were 0.887, 0.993, 0.914, and 0.755, respectively. Conclusions: The plasma metabolome provides a promising resource of biomarkers for the predictive diagnosis and targeted prevention of MetS. Alterations in amino acid metabolism play significant roles in the pathophysiology of MetS. The biomarker panels and metabolic pathways could be used as preventive targets in dealing with cardiometabolic diseases related to MetS

Structural studies of class A and D plexins

Semaphorin signalling via the plexin receptors, important in the nervous, cardiovascular, immune and skeletal systems, requires cytoplasmic domain dimerisation but its extracellular regulation and activation mechanisms remain unclear. In the thesis I conducted structural characterisation for two particular classes of verterbrate plexins, PlxnAs and PlxnD1. Here I present crystal structures of PlxnA1, PlxnA2 and PlxnA4 full ectodomains. The first nine domains of PlxnAs forma 230 Å long ring-like stalk fromwhich the tenth C-terminal domain points away. In agreement, negative stain electron microscopy of the PlxnA1 ectodomain reveals a predominant ring-like conformation and a minor twisted-open conformation of the stalk. All PlxnA crystal structures also make intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by structure-guided biophysical assays and live cell fluorescence microscopy. Functional assays performed in COS-7 cell and dentate gyrus growth cones revealed an autoinhibitory mechanism for PlxnAs based on this head-to-stalk interface. My results reveal a two-fold role for the PlxnA ectodomains: imposing a presignalling autoinhibitory separation of the PlxnA membrane proximal domains via a head-to-stalk, intermolecular interaction and supporting PlxnA cytoplasmic domain dimerisation post semaphorin-binding. For PlxnD1, I present high-resolution crystal structures of its semaphorinbinding segment (domains 1-2) as well as an initial structural model of its complete ectodomain (domains 1-10). The sema (domain 1) of PlxnD1 has the classic fold of a plexin sema domain as well as distinct insertions potentially important for its ligand-binding specificity. The PlxnD1 ectodomain adopts a closed-ring conformation in its crystal structure, in which the tail (domain 9) interacts with the sema domain head. In-solution experiments suggest structural variability and potential weak oligomerization for the PlxnD1 ectodomain. These new structures shed light on the unique and previously uncharacterised structural basis for PlxnD1 signalling.</p

Structural studies of class A and D plexins

Semaphorin signalling via the plexin receptors, important in the nervous, cardiovascular, immune and skeletal systems, requires cytoplasmic domain dimerisation but its extracellular regulation and activation mechanisms remain unclear. In the thesis I conducted structural characterisation for two particular classes of verterbrate plexins, PlxnAs and PlxnD1. Here I present crystal structures of PlxnA1, PlxnA2 and PlxnA4 full ectodomains. The first nine domains of PlxnAs forma 230 &Aring; long ring-like stalk fromwhich the tenth C-terminal domain points away. In agreement, negative stain electron microscopy of the PlxnA1 ectodomain reveals a predominant ring-like conformation and a minor twisted-open conformation of the stalk. All PlxnA crystal structures also make intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by structure-guided biophysical assays and live cell fluorescence microscopy. Functional assays performed in COS-7 cell and dentate gyrus growth cones revealed an autoinhibitory mechanism for PlxnAs based on this head-to-stalk interface. My results reveal a two-fold role for the PlxnA ectodomains: imposing a presignalling autoinhibitory separation of the PlxnA membrane proximal domains via a head-to-stalk, intermolecular interaction and supporting PlxnA cytoplasmic domain dimerisation post semaphorin-binding. For PlxnD1, I present high-resolution crystal structures of its semaphorinbinding segment (domains 1-2) as well as an initial structural model of its complete ectodomain (domains 1-10). The sema (domain 1) of PlxnD1 has the classic fold of a plexin sema domain as well as distinct insertions potentially important for its ligand-binding specificity. The PlxnD1 ectodomain adopts a closed-ring conformation in its crystal structure, in which the tail (domain 9) interacts with the sema domain head. In-solution experiments suggest structural variability and potential weak oligomerization for the PlxnD1 ectodomain. These new structures shed light on the unique and previously uncharacterised structural basis for PlxnD1 signalling.</p

Advanced In Vivo Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions

International audienceChemical cross-linking (XL) coupled to mass spectrometry (MS) has become a powerful approach to probe the structure of protein assemblies. Although most of the applications concerned purified complexes, latest developments focus on large-scale in vivo studies. Pushing in this direction, we developed an advanced in vivo cross-linking mass spectrometry platform to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 3300 cross-links for the LC-MS/MS analysis of a biological triplicate using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our proteome-wide approach. Our workflow paves new avenues for the large-scale and nonbiased analysis of protein-protein interactions. All raw data, databases, and processing parameters are available on ProteomeXchange via PRIDE repository (data set identifier PXD021553)

Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly

International audienceDespite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections

Blood transcriptome profiling as potential biomarkers of suboptimal health status: Potential utility of novel biomarkers for predictive, preventive, and personalized medicine strategy

The early identification of Suboptimal Health Status (SHS) creates a window opportunity for the predictive, preventive, and personalized medicine (PPPM) in chronic diseases. Previous studies have observed the alterations in several mRNA levels in SHS individuals. As a promising “omics” technology offering comprehension of genome structure and function at RNA level, transcriptome profiling can provide innovative molecular biomarkers for the predictive identification and targeted prevention of SHS. To explore the potential biomarkers, biological functions, and signalling pathways involved in SHS, an RNA sequencing (RNA-Seq)–based transcriptome analysis was firstly conducted on buffy coat samples collected from 30 participants with SHS and 30 age- and sex-matched healthy controls. Transcriptome analysis identified a total of 46 differentially expressed genes (DEGs), in which 22 transcripts were significantly increased and 24 transcripts were decreased in the SHS group. A total of 23 transcripts were selected as candidate predictive biomarkers for SHS. Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that several biological processes were related to SHS, such as ATP-binding cassette (ABC) transporter and neurodegeneration. Protein–protein interaction (PPI) network analysis identified 10 hub genes related to SHS, including GJA1, TWIST2, KRT1, TUBB3, AMHR2, BMP10, MT3, BMPER, NTM, and TMEM98. A transcriptome predictive model can distinguish SHS individuals from the healthy controls with a sensitivity of 83.3% (95% confidence interval (CI): 73.9–92.7%), a specificity of 90.0% (95% CI: 82.4–97.6%), and an area under the receiver operating characteristic curve of 0.938 (95% CI: 0.882–0.994). In the present study, we demonstrated that blood (buffy coat) samples appear to be a very promising and easily accessible biological material for the transcriptomic analyses focused on the objective identification of SHS by using our transcriptome predictive model. The pattern of particularly determined DEGs can be used as predictive transcriptomic biomarkers for the identification of SHS in an individual who may, subjectively, feel healthy, but at the level of subcellular mechanisms, the changes can provide early information about potential health problems in this person. Our findings also indicate the potential therapeutic targets in dealing with chronic diseases related to SHS, such as T2DM and CVD, and an early onset of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, as well as the findings suggest the targets for personalized interventions as promoted in PPPM

PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments

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Structural Basis for Plexin Activation and Regulation

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1–9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular “head-to-stalk” (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors