Biology subjects

Gil, M.

Publications and source records attributed to Gil, M..

At least 19 recordsLinked to original sources

bioRxiv2026

A progressive reduction in basal phosphorylation of glutamate receptors along postweaning development accompanies maturation of hippocampal theta-burst-induced LTP.

Tackling developmental changes in long-term potentiation (LTP) expression during adolescence is crucial to understand disease susceptibility in neurodevelopmental disorders, epileptogenesis or drug abuse. This paper investigated the alterations in the resting phosphorylation state of synaptic proteins essential for synaptic transmission and hippocampal LTP expression from weaning (3 weeks) to adulthood (12 weeks). Synaptic AMPA GluA1 and GluA2 subunit levels increased, yet to different extents, as the GluA1/GluA2 ratio also increased. Conversely, GluA1 phosphorylation in both Ser831 and Ser845 progressively decreased from weaning to adulthood, suggesting an enhanced availability of these sites for activity-dependent phosphorylation. In accordance, LTP induced by different theta-burst stimulation (TBS) intensities in the CA1 area of rat hippocampal slices enhanced gradually in this developmental period. In contrast, basal phosphorylation of GluN1, GluN2B and CaMKII increased during adolescence. Altogether, these findings suggest that alterations in AMPA receptor subunit composition and basal phosphorylation are crucial for the maturation of hippocampal LTP during adolescence, while a mild enhancement in synaptic CaMKII levels may further provide the necessary structural support to LTP expression and stability. Given the reported involvement of GluA1 phosphorylation changes in epileptogenesis and neurodevelopmental disorders, these findings provide important insights into hippocampal synaptic plasticity in normal and altered brain development and epileptogenesis.

neuroscience↗

bioRxiv2026

Serotypic heterogeneity in the response to pneumococcal vaccine

The assessment of pneumococcal vaccine response currently relies on a single IgG concentration threshold, identical for all serotypes, as recommended by the WHO and AAAAI. However, the recommended thresholds do not take the wide inter-serotype variability into account. The purpose of this study was to determine if the antibody threshold linked to a functional immune response varies according to serotype. We performed a retrospective analysis on 729 samples from adults at risk of invasive pneumococcal disease (IPD), sent between 2018 and 2024 to assess vaccine response. Specific IgG concentrations for seven vaccine serotypes were measured by ELISA and compared to opsonophagocytic assay (OPA) results, a functional test considered as the gold standard. For each serotype, we determined the most predictive IgG concentration (PIC) for a positive OPA result using ROC curves, Youdens index, and bootstrap analysis with 1,000 resamples. The resulting PIC were then compared using non-parametric tests (Kruskal-Wallis test followed by Dunns post-hoc test with Holms correction). The PIC varied considerably among serotypes, ranging from 0.84 to 4.74 {micro}g/mL. This variability was found to be statistically significant (p<0.0001). Areas Under the Curve (AUC), ranging from 0.73 to 0.87, demonstrate good diagnostic performance. Overall, the application of serotype-specific thresholds in patients significantly change the classification of vaccination status compared to a single threshold (Cochran, McNemar). These results indicate that the protective antibody threshold is not universal. A serotype-specific approach would allow a more precise and relevant assessment of the pneumococcal vaccine response. Author summaryThe introduction of the 7-valent pneumococcal conjugate vaccine (PCV-7) in the early 2000s significantly changed the epidemiology of invasive pneumococcal disease by modifying serotype distribution. However, vaccine-induced immunity varies across serotypes, and this heterogeneity remains incompletely understood. In this study, we first assessed differences in vaccine responses according to serotype. We then determined predictive serotype-specific immunoglobulin concentration for the seven routinely serotypes tested in our laboratory, defined as the minimal levels required to induce a positive opsonophagocytic activity (OPA) response. The results enable a more accurate assessment of serotype-specific vaccine immunity, supporting improved patient stratification and guiding booster vaccination in individuals with insufficient responses.

immunology↗

bioRxiv2026

AmpliPhy improves gene trees by adding homologs without affecting alignments

In phylogenomics, gene tree reconstruction depends on multiple sequence alignment (MSA) and tree inference, and ongoing work continues to improve inference quality. Denser taxon sampling has been associated with improved gene tree inference, suggesting that adding homologs could be a practical route to higher accuracy as sequence databases continue to expand. However, adding sequences can influence multiple steps of typical inference pipelines, and little is known on its specific effect on the multiple sequence alignment, tree reconstruction, and rooting steps. We performed a large-scale empirical and simulated benchmarks to quantify how homolog enrichment affects alignment and phylogenetic inference. Using an enrichment-impoverishment design and a measure of tree accuracy based on taxonomic congruence, we found that enrichment consistently improves tree inference quality, while effects on alignment quality are marginal. We show that this improvement is associated with, but not restricted to accurate root placement on enriched trees when sensitive homolog search is accompanied. Notably, much of the benefit can be retained with relatively compact alignments produced by sequence addition. Building on these observations, we provide a tool, AmpliPhy, which efficiently improves phylogenetic reconstruction of protein families through homolog enrichment. The AmpliPhy open-source pipeline software is available at https://github.com/DessimozLab/ampliphy.

evolutionary biology↗

bioRxiv2025

Pharmacological inhibition of MCL-1 disrupts mitochondrial cristae and depletes the human neural progenitor cell pool

MCL-1 is a canonical anti-apoptotic protein crucial for early neurodevelopment, and its loss causes embryonic-lethal defects that other BCL-2 family proteins cannot rescue. Here, we pharmacologically inhibit MCL-1 in human neural progenitor cells and uncover non-apoptotic roles in sustaining mitochondrial cristae integrity, fatty acid oxidation, and progenitor identity. MCL-1 inhibition disrupts mitochondrial ultrastructure, destabilizing the OPA1-MICOS machinery. These structural defects are accompanied by ACSL1 displacement from the mitochondria, impaired fatty acid oxidation, lipid droplet accumulation, and reduced oxygen consumption, revealing a tight link between cristae architecture and metabolic competence. Mechanistically, MCL-1 acts through two coordinated functions: maintaining cristae integrity at the inner membrane and retaining ACSL1 at the outer membrane, both independently of caspase activation. Functionally, MCL-1 inhibition selectively depletes intermediate progenitor cells without affecting proliferation, indicating a direct role in lineage progression. Together, our findings position MCL-1 upstream of OPA1, MICOS, and ACSL1 as a critical coordinator of cristae organization, lipid metabolism, and neural progenitor fate, establishing mitochondrial inner membrane architecture as an instructive determinant of human neurogenesis and highlighting non-canonical MCL-1 functions as regulators of brain development.

cell biology↗

bioRxiv2025

circVDJ-seq for T cell clonotype detection in single-cell and spatial multi-omics

Monitoring T cell repertoires in human tissues provides important insights into immune response mechanisms in cancer, infectious diseases, and autoimmunity. However, retrieving VDJ information from single-cell and spatial transcriptomics workflows with 3-barcoding of cDNA remains resource-intensive or requires specialized sequencing equipment. Here, we introduce circVDJ-seq for simplified and cost-efficient T cell receptor (TCR) profiling from 3-directed workflows like single-cell or single-nucleus RNA sequencing, ATAC+RNA multi-omics, and spatial transcriptomics. Application of circVDJ-seq to freshly resected neuroblastomas and postmortem lymph nodes affected by pneumonia or COVID-19 reveals distinct immune microenvironments and T cell clonality patterns, highlighting broad utility across diverse clinical contexts.

genomics↗

bioRxiv2025

Engineered Extracellular Vesicles Enriched with miR-214 Enhance the Efficacy of Chemotherapy for Ovarian Cancer

Recurrent ovarian cancer (OC) remains a major cause of mortality due to chemoresistance and metastasis. Epigenetic dysfunction, particularly through altered microRNA (miRNA) expression, contributes to disease progression. Targeting these molecular aberrations is critical to prevent recurrence, limit metastasis and improve patient outcomes. Here, we identify the miR-214-3p/miR-199a-5p cluster as a stage-associated, tumor-suppressive network that is lost in recurrent and chemoresistant OC, but can be restored using engineered small extracellular vesicles enriched with this cluster (m214-sEVs). Using a clinically relevant mouse model that mimics spontaneous OC relapse following first-line platinum-based chemotherapy, we showed that m214-sEVs were internalized by OC cells and the OC niche fibroblasts via clathrin-mediated endocytosis, resulting in the elevation of miR-214-3p/miR-199a-5p and the downregulation of chemoresistance-associated genes, including toll-like receptor 4 (TLR4), {beta}-catenin, and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein YKT6. Moreover, secondary tumor-derived sEVs (t-sEVs) released by OC and niche cells that internalized m214-sEVs reduced pro-metastatic proteins, such as integrin {beta}1 and matrix metalloproteinase 9 (MMP9), in their cargo and limited their capacity to promote invasion and resistance. In vitro, YKT6 overexpression in ovarian cancer stem cells (OCSCs) attenuated the effect of m214-sEVs on sensitizing carboplatin to block OCSC migration. These findings demonstrate that engineered m214-sEVs designed to restore clinically lost tumor-suppressive miRNAs can concurrently reverse chemoresistance and reprogram tumor-derived EV communication by targeting oncogenic networks. Statement of SignificanceEngineered small extracellular vesicles delivering miR-214-3p/miR-199a-5p overcome chemoresistance and inhibit recurrence in ovarian cancer by targeting oncogenic networks and reprogramming tumor-derived extracellular vesicle communication within the tumor microenvironment.

cancer biology↗

bioRxiv2025

AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

GPR183 is a chemotactic G protein-coupled receptor implicated in immune cell migration and various diseases. Here, we report the discovery of novel GPR183 inverse agonists using an artificial intelligence-driven virtual screening approach combined with biophysical sensors including a first-in-class conformational biosensor. From an initial screen of 70 compounds and a hit expansion, a selected candidate - compound 78 - demonstrated potent inhibition of GPR183s constitutive and agonist-stimulated Gi activation as well {beta}-arrestin2 recruitment. The binding of the compound to the receptor core was confirmed by studies using a new conformational sensor based on intramolecular BRET, molecular dynamics, and site-directed mutagenesis experiments. Finally, the compound 78 was able to block agonist-induced migration of peripheral blood mononuclear cells ex vivo. This work provides a novel chemical scaffold for GPR183 modulation. It introduces a new tool for dissecting receptor function, paving the way for therapeutic exploration in inflammatory, autoimmune, and neoplastic disorders.

pharmacology and toxicology↗

bioRxiv2025

SifA-mediated Remodeling of the Salmonella-Containing Vacuole Prevents Bacterial Dormancy by Promoting Nutrient Accessibility

Salmonella exploits a range of intracellular niches within host cells, adapting to different microenvironments that support its survival and replication. This niche diversity is mediated by bacterial effectors injected via the two type 3 secretion systems (T3SS). Salmonella resides either within membrane-bound Salmonella containing vacuoles (SCV) or they grow rapidly within the cytosol upon SCV rupture. Recently, we identified a third intracellular subpopulation, dormant Salmonella within modified vacuoles in epithelial cells, which can survive for up to 7 days in vitro. To explore how bacterial effectors influence the balance of these three subpopulations, we constructed a panel of mutants lacking genes encoding key effectors and examined their intracellular behaviors at 6 and 24 hours post-infection (hpi). Deletion of the T3SS-2 effector SifA significantly increased the dormant subpopulation at later infection time points, identifying SifA as the first known effector regulating bacterial dormancy in epithelial cells. SifA-induced Salmonella-induced filaments (SIFs) characterize the mature Salmonella-containing vacuole (SCV), and we observed that SIFs were needed for intravacuolar growth of Salmonella. We could show that SIF formation was critical for nutrient acquisition within the SCV; inhibition of SIF formation resulted in a higher proportion of dormant bacteria, akin to the effect of reduced glucose availability during infection. Both bacterial and host glycolysis pathways were required to prevent dormancy, as proper nutrient scavenging through SIF-mediated modification of the SCV is essential for maintaining a replicative, non-dormant state. These results underscore the importance of nutrient access, facilitated by reprogramming host endomembrane trafficking, for Salmonella to avoid dormancy and sustain active replication within its intracellular niche. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/631959v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d4e9cdorg.highwire.dtl.DTLVardef@419d70org.highwire.dtl.DTLVardef@1c75fb0org.highwire.dtl.DTLVardef@5b5d59_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

MCL-1 regulates cellular transitions during oligodendrocyte development

Oligodendrocytes are the myelinating cells of the central nervous system. Regulation of the early stages of oligodendrocyte development is critical to the function of the cell. Specifically, myelin sheath formation is an energetically demanding event that requires precision, as alterations may lead to dysmyelination. Recent work has established that fatty acid {beta}-oxidation is required for the function of oligodendrocytes. We have shown that MCL-1, a well-characterized anti-apoptotic protein, is required for the development of oligodendrocytes in vivo. Further, it was recently uncovered that MCL-1 regulates long- chain fatty acid {beta}-oxidation through its interaction with acyl-CoA synthetase long-chain family member 1 (ACSL1), an enzyme responsible for the conversion of long-chain fatty acids into acyl-CoA. Here, we introduce an in vitro system to isolate human stem cell- derived oligodendrocyte progenitor cells and investigate the involvement of MCL-1 during human oligodendrocyte development. Using this system, we pharmacologically inhibited MCL-1 in oligodendrocyte progenitor cells (OPCs) to elucidate the non-apoptotic function of the protein at this developmental stage. Additionally, we used a motor neuron co-culture system to investigate the downstream effects that MCL-1 inhibition has at later developmental stages when oligodendrocytes begin to contact axons and generate myelin basic protein. We demonstrate that the mitochondrial network changes in human oligodendrocyte development resemble those reported in vivo. Our findings point to MCL-1 as a critical factor essential at the OPC stage for proper oligodendrocyte morphogenesis.

cell biology↗

bioRxiv2024

FhaA plays a key role in mycobacterial polar elongation and 1 asymmetric growth

Mycobacteria, including pathogens like Mycobacterium tuberculosis, exhibit unique growth patterns and cell envelope structures that challenge our understanding of bacterial physiology. This study sheds light on FhaA, a conserved protein in Mycobacteriales, revealing its pivotal role in coordinating cell envelope biogenesis and asymmetric growth. The elucidation of the FhaA interactome in living mycobacterial cells reveals its participation in the protein network orchestrating cell envelope biogenesis and cell elongation/division. By manipulating FhaA levels, we uncovered its influence on cell morphology, cell envelope organization, and the localization of peptidoglycan biosynthesis machinery. Notably, fhaA deletion disrupted the characteristic asymmetric growth of mycobacteria, highlighting its importance in maintaining this distinctive feature. Our findings position FhaA as a key regulator in a complex protein network, orchestrating the asymmetric distribution and activity of cell envelope biosynthetic machinery. This work not only advances our understanding of mycobacterial growth mechanisms but also identifies FhaA as a potential target for future studies on cell envelope biogenesis and bacterial growth regulation. These insights into the fundamental biology of mycobacteria may pave the way for novel approaches to combat mycobacterial infections addressing the ongoing challenge of diseases like tuberculosis in global health.

microbiology↗

bioRxiv2024

Adipose microenvironment promotes hypersialylation of ovarian cancer cells

Sialylation, the addition of negatively charged sialic acid sugars to terminal ends of glycans, is upregulated in most cancers. Hypersialylation supports multiple pro-tumor mechanisms such as enhanced migration and invasion, resistance to apoptosis and immune evasion. A current gap in knowledge is the lack of understanding on how the tumor microenvironment regulates cancer cell sialylation. The adipose niche is a main component of most peritoneal cancers microenvironment. This includes ovarian cancer (OC), which causes most deaths from all gynecologic cancers. In this report, we demonstrate that the adipose microenvironment is a critical regulator of OC cell sialylation. In vitro adipose conditioning led to an increase in both 2,3- and 2,6-linked cell surface sialic acids in both human and mouse models of OC. Adipose-induced sialylation reprogramming was also observed in vivo from intra-peritoneal OC tumors seeded in the adipose-rich omentum. Mechanistically, we observed upregulation of at least three sialyltransferases, ST3GAL1, ST6GAL1 and ST3GALNAC3. Hypersialylated OC cells consistently formed intra-peritoneal tumors in both immune-competent mice and immune-compromised athymic nude mice. In contrast, hyposiaylated OC cells persistently formed tumors only in athymic nude mice demonstrating that sialylation impacts OC tumor formation in an immune dependent manner. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancers.

cancer biology↗

bioRxiv2024

Please Mind the Gap: Indel-Aware Parsimony for Fast and Accurate Ancestral Sequence Reconstruction and Multiple Sequence Alignment including Long Indels

Despite having important biological implications, insertion and deletion (indel) events are often disregarded or mishandled during phylogenetic inference. In multiple sequence alignment, indels are represented as gaps and are estimated without considering the distinct evolutionary history of insertions and deletions. Consequently, indels are usually excluded from subsequent inference steps, such as ancestral sequence reconstruction and phylogenetic tree search. Here, we introduce indel-aware parsimony (indelMaP), a novel way to treat gaps under the parsimony criterion by considering insertions and deletions as separate evolutionary events and accounting for long indels. By identifying the precise location of an evolutionary event on the tree, we can separate overlapping indel events and use affine gap penalties for long indel modelling. Our indel-aware approach harnesses the phylogenetic signal from indels, including them into all inference stages. Validation and comparison to state-of-the-art inference tools on simulated data show that indelMaP is most suitable for densely sampled datasets with closely to moderately related sequences, where it can reach alignment quality comparable to probabilistic methods and accurately infer ancestral sequences, including indel patterns. Due to its remarkable speed, our method is well-suited for epidemiological datasets, eliminating the need for downsampling and enabling the exploitation of the additional information provided by dense taxonomic sampling. Moreover, indelMaP offers new insights into the indel patterns of biologically significant sequences and advances our understanding of genetic variability by considering gaps as crucial evolutionary signals rather than mere artefacts.

genomics↗

bioRxiv2024

Single-character insertion-deletion model preserves long indels in ancestral sequence reconstruction

Insertions and deletions (indels) play a significant role in genome evolution across species. Realistic modelling of indel evolution is challenging and is still an open research question. Several attempts have been made to explicitly model multi-character (long) indels, such as TKF92, by relaxing the site independence assumption and introducing fragments. However, these methods are computationally expensive On the other hand, the Poisson Indel Process (PIP) assumes site independence but allows one to infer single-character indels on the phylogenetic tree, distinguishing insertions from deletions. PIPs marginal likelihood computation has linear time complexity, enabling ancestral sequence reconstruction (ASR) with indels in linear time. Recently, we developed ARPIP, an ASR method using PIP, capable of inferring indel events with explicit evolutionary interpretations Here, we investigate the effect of the single-character indel assumption on reconstructed ancestral sequences on mammalian protein orthologs and on simulated data. We show that ARPIPs ancestral estimates preserve the gap length distribution observed in the input alignment. In mammalian proteins the lengths of inserted segments appear to be substantially longer compared to deleted segments. Further, we confirm the well-established deletion bias observed in real data To date, ARPIP is the only ancestral reconstruction method that explicitly models insertion and deletion events over time. Given a good quality input alignment, it can capture ancestral long indel events on the phylogeny

evolutionary biology↗

bioRxiv2024

Postweaning development influences endogenous VPAC1 modulation of LTP induced by theta-burst stimulation: a link to maturation of the hippocampal GABAergic system?

Long-term potentiation (LTP) induced by theta-burst stimulation (TBS) undergoes postweaning developmental changes partially linked to GABAergic circuit maturation. Endogenous VIP acting on VPAC1 receptors strongly influences LTP induced by theta-burst stimulation (TBS), an effect dependent on GABAergic transmission. Although VPAC1 receptor levels are developmentally regulated during embryogenesis, its variation along postweaning development is unknow, as is VPAC1 modulation of LTP or its relation to hippocampal GABAergic circuit maturation. As such, we investigated how VPAC1 modulation of LTP adjusts from weaning to adulthood along with GABAergic circuit maturation. As described, LTP induced by TBS (5x4) (5 bursts, 4 pulses delivered at 100Hz) was increasingly greater from weaning to adulthood. The influence of the VPAC1 receptor antagonist PG 97-269 (100nM) on TBS-induced LTP was much larger in juvenile (3-week-old) than in young-adult (6-7-week-old) or adult (12-week-old) rats. This effect was not associated to a developmental decrease in synaptic VPAC1 receptor levels. However, an increase in pre and post synaptic GABAergic synaptic markers, suggests an increase in the number of GABAergic synaptic contacts that is more prominent than the one observed in glutamatergic connections during this period. Conversely, endogenous VPAC2 receptor activation did not significantly influence of TBS-induced LTP. VPAC2 receptor levels enhance pronouncedly during postweaning development, but not at synaptic sites. Given the involvement of VIP interneurons in several aspects of hippocampal-dependent learning, neurodevelopmental disorders, and epilepsy, this could provide important insights into the role of VIP modulation of hippocampal synaptic plasticity during normal and altered brain development potentially contributing to epileptogenesis.

neuroscience↗

bioRxiv2022

Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear respectively branched actin filaments. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet. Here, we show that the two family members Evl and VASP are expressed in murine DCs and that their loss negatively affects DC macropinocytosis, spreading, and migration. Our interactome analysis reveals Ena/VASP proteins to be ideally positioned for orchestrating the different actin nucleation pathways by binding to the formin mDia1 as well as to the WAVE regulatory complex, a stimulator of Arp2/3. In fact, Evl/VASP deficient murine DCs are more vulnerable to inhibition of Arp2/3 demonstrating that Ena/VASP proteins contribute to the robustness and efficiency of DC migration.

cell biology↗

bioRxiv2022

Ontology-Aware Biomedical Relation Extraction

MotivationAutomatically extracting relationships from biomedical texts among multiple sorts of entities is an essential task in biomedical natural language processing with numerous applications, such as drug development or repurposing, precision medicine, and other biomedical tasks requiring knowledge discovery. Current Relation Extraction (RE) systems mostly use one set of features, either as text, or more recently, as graph structures. The state-of-the-art systems often use resource-intensive hence slow algorithms and largely work for a particular type of relationship. However, a simple yet agile system that learns from different sets of features has the advantage of adaptability over different relationship types without an extra burden required for system re-design. ResultsWe model RE as a classification task and propose a new multi-channel deep neural network designed to process textual and graph structures in separate input channels. We extend a Recurrent Neural Network (RNN) with a Convolutional Neural Network (CNN) to process three sets of features, namely, tokens, types, and graphs. We demonstrate that entity type and ontology graph structure provide better representations than simple token-based representations for RE. We also experiment with various sources of knowledge, including data resources in the Unified Medical Language System (UMLS) to test our hypothesis. Extensive experiments on four well-studied biomedical benchmarks with different relationship types show that our system outperforms earlier ones. Thus, our system has state-of-the-art performance and allows processing millions of full-text scientific articles in a few days on one typical machine.

bioinformatics↗

bioRxiv2021

Joint Alignment and Tree Inference

Multiple sequence alignment and phylogenetic tree inference are connected problems that are often solved as independent steps in the inference process. Several attempts at doing simultaneous inference have been made, however currently the available methods are greatly limited by their computational complexity and can only handle small datasets. In this manuscript we introduce a combinatorial optimisation approach that will allow us to resolve the circularity of the problem and efficiently infer both alignments and trees under maximum likelihood.

bioinformatics↗

bioRxiv2021

A TPR scaffold couples signal detection to OdhI phosphorylation in metabolic control by the protein kinase PknG

Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of post-translational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here we present an investigation through in vivo experiments as well as biochemical and structural methods of the molecular bases of the regulation of PknG from C. glutamicum (CgPknG), in the light of previous knowledge available for the kinase from M. tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPR) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain and a flexible N-terminal extension of the kinase that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCEBacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. In this way, the metabolic characteristics of a microorganism determine its ecological role and its usefulness in industrial processes. Here, we use genetic, biochemical, and structural approaches to study a key component in a system that regulates glutamate production in C. glutamicum, a species that is used for the industrial production of amino acids. We elucidated molecular mechanisms involved in metabolic control in C. glutamicum, which are conserved in related pathogenic bacteria. The findings have broader significance for diverse actinobacteria, including microorganisms that cause disease as well as environmental species used to produce billions of dollars of amino acids and antibiotics every year.

biochemistry↗