Biology subjects

Schittenhelm, R. B.

Publications and source records attributed to Schittenhelm, R. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery

Mass spectrometry based immunopeptidomics is limited by peptide identification. HLA class I ligands are non-tryptic, frequently carry low charge states and often fragment incompletely, yet nearly all workflows rely on a single activation method, higher-energy collisional dissociation (HCD). Unlike proteomics, where a protein is inferred from several peptides, in immunopeptidomics the peptide is itself the analyte and the candidate therapeutic target, so a ligand that is not identified is a target that is not found. Here we evaluate whether two alternative activation chemistries, ion trap collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD), recover ligands that HCD does not. To our knowledge this is the first application of either method to HLA class I immunopeptidomics, made practical by the integrated infrared laser of the Orbitrap Tribrid Apex MultiOmics. Four HLA-typed cell lines were analysed in technical triplicate against the canonical human proteome and a Ribo-seq-derived cryptic ORF database. Because fragment-intensity predictors are trained almost exclusively on HCD spectra, AlphaPeptDeep was fine-tuned separately for each method; fine-tuning and Percolator rescoring increased unique peptide identifications in every cell line and method combination (median +17.1% canonical, +54.5% cryptic), and fine-tuned models transferred across cell lines with a median loss of 0.4%, placing the three methods on a common analytical footing. On that footing the three methods yielded immunopeptidomes of similar size and composition, but each recovered peptides the others missed. HCD produced the most complete fragment-ion ladders and recovered more peptides from the basic-anchor alleles A*11:01 and A*33:03, both members of the A3 supertype. CID and IRMPD recovered a larger share of alleles without a basic C-terminal anchor and, for peptides containing no lysine, arginine or histidine, produced higher search scores and more matched b/y ion current in three of four cell lines. Each method therefore contributed exclusive peptides, and at a fixed budget of three LC-MS/MS runs, one run per method yielded on average more unique peptides than triplicate runs of a single method in all four cell lines (+0.4% to +10.9%). Of 239 novel canonical predicted HLA binders in the two cancer cell lines (HCT116 and MDA-MB-231) absent from the Immune Epitope Database, 55% were detected by only one method; nine derive from cancer-associated antigens. CID and IRMPD can therefore provide sequences that may not be identified by HCD alone, or with better spectral quality for a chemically defined subset of ligands. Their value lies alongside HCD rather than in its place, and realising it will require fragment-intensity models and acquisition schemes built for these chemistries rather than adapted from HCD.

immunology↗

bioRxiv2026

Temporal expression of liver-stage malaria antigens shapes vaccine efficacy

Vaccine-induced cytotoxic T cells can prevent malaria by killing parasite-infected hepatocytes during the liver stage. While several antigenic targets have been identified, little consideration has been given to their temporal expression. Here, we identified SERA1 of Plasmodium berghei as a late liver-stage target in rodent malaria and further showed that the classic vaccine antigen thrombospondin-related adhesion protein (TRAP) is only an early target. While vaccination with either antigen alone was modestly protective, combining these antigens enabled killing over the entire liver-stage, greatly improving efficacy. Given the relatively long liver-stage in human malaria, our findings imply TRAP-dependent vaccines likely utilize only a small proportion of the available liver-stage to eradicate parasites. Our findings further indicate that considerations of temporal coverage when selecting vaccine antigens will improve efficacy. One-Sentence SummaryTemporally defining presentation of liver-stage antigens informs rational combinations that maximize malaria vaccine efficacy.

immunology↗

bioRxiv2026

Monocyte Migration Emerges from a Divergent Chemokine Signaling Network

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

systems biology↗

bioRxiv2026

Di-codon organization links tRNA modifications to cancer cell proteome composition

Hormone-signalling modulates levels of tRNA-modifying enzymes, including ELP3, in cancer cells. Here we show that ELP3 is required to sustain proliferation in prostate cancer cells. Intriguingly, although ELP3 modifies tRNA at the U34-position, the ELP3-sensitive proteome was poorly explained by frequencies of codons requiring U34-modified tRNA for decoding. Instead, we identified six codon pairs (herein denoted ELP3 down di-codons "E3dDCs") that, in concert with local sequence context and 5UTR features affecting translation initiation, explain ELP3-sensitive protein expression. Moreover, despite activation of the canonical integrated stress response upon ELP3 suppression, the associated expression program was paradoxically suppressed in a fashion correlating with higher E3dDC frequencies. E3dDCs were also enriched in mitotic regulators, and ELP3-suppression caused mitotic defects that limited proliferation. Therefore, ELP3 regulates proteome composition via E3dDCs that, depending on their local sequence context and translation initiation activity, determine protein expression and downstream cellular phenotypes upon reduced levels of U34-modified tRNA.

molecular biology↗

bioRxiv2026

If the shoe fits? -- technical nuances of plasma proteomic workflows in clinical and preclinical contexts

The push for new clinical biomarkers has seen rapid innovation in biofluid analysis, particularly for plasma. For mass-spectrometry (MS)-based analysis, achieving depth and quantitative accuracy whilst ensuring throughput continues to shape plasma methods development. Numerous workflows have emerged that mitigate high-abundance suppression and expand dynamic range, especially when paired with next-generation MS instrumentation. Yet systematic evaluations that also consider biological variables (e.g., biofluid type, species) and technical parameters (e.g., MS methods) are limited. Here, we benchmarked eight sample-preparation workflows spanning neat approaches (SP3, STrap), depletion (perchloric acid, PerCA), and corona-enrichment strategies (MagNet HILIC/SAX, Enrich-iST, ProteoNano). We compared their performance across human plasma, human serum, and rat plasma, analysing all samples on an Orbitrap Astral (Thermo) using two plasma-optimised data-independent acquisition (DIA) methods: one discovery-maximised and one throughput-maximised. We identified 2,726 human and 3,767 rat proteins across workflows and methods, including [~]1,000 from neat plasma. Increasing throughput incurred a [~]20-30% reduction in depth, depending on workflow and species. EV-enrichment produced the deepest proteomes but with distinct compositions relative to neat, depleted, and secreted-protein-enriched samples, revealing a unique sub-proteome niche. Several workflows also performed markedly better in rat plasma, supporting improved sensitivity for preclinical analyses. Enrichment or depletion dramatically reshaped the balance of tissue-and cell-specific proteins detectable in plasma, suggesting that workflow choice should be guided by the organs, immune targets, or inflammatory signals most relevant to the study. In this vein, statistical analysis of differentially abundant proteins showed that >90% of detected proteins were significantly altered between workflows, with the largest numbers arising from the corona-enrichment strategies, underscoring how strongly workflow choice shapes the downstream proteome. Taken together, these findings emphasise a rapidly expanding plasma methodological landscape, where the most effective workflow is the one most precisely tailored to a cohorts biology.

systems biology↗

bioRxiv2025

Validation of Solvent Proteome Profiling for Antimalarial Drug Target Deconvolution

Malaria remains a global health threat, with rising drug resistance accelerating the urgent need for new therapeutics. Target elucidation is a critical step in antimalarial drug discovery, enabling a deeper understanding of the molecular mechanisms of action of both existing and novel compounds. This study validates solvent-induced proteome profiling (SPP) as a proteomics-based approach for identifying drug-protein interactions in Plasmodium falciparum. SPP detects shifts in protein stability induced by ligand binding, allowing the identification of drug target/s without the need for compound modifications. Here, we successfully generated solvent denaturation curves for the P. falciparum proteome, and demonstrated the utility of SPP with five antimalarial compounds: pyrimethamine, atovaquone, cipargamin, MMV1557817 and OSM-S-106. In addition to measuring each compounds impact across the full denaturation curve, investigating protein levels at individual solvent percentages preserved specific stability changes that would otherwise be masked in pooled analyses performed by integral SPP. This strategy was critical for the identification of the cipargamin target, non-SERCA-type Ca2+-transporting P-ATPase (PfATP4). Notably, we propose live-cell treatment SPP as a novel approach, demonstrating its ability to identify the validated target of pyrimethamine, bifunctional dihydrofolate reductase-thymidylate synthase (PfDHFR), with high specificity. We also introduced the novel one-pot mixed-drug SPP which enables the evaluation of multiple drugs within a single lysate and experimental setup. This alternative method simplifies the experimental workflow and includes positive controls to affirm the performance of the experiment. Overall, this study demonstrates that SPP can be successfully applied in both lysates and live cells to elucidate drug targets in P. falciparum, as well as providing additional information regarding the mechanisms of drug action, offering insights for the optimisation of existing antimalarials and the development of novel therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/670428v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b83c75org.highwire.dtl.DTLVardef@1895f23org.highwire.dtl.DTLVardef@1c51ba0org.highwire.dtl.DTLVardef@198c822_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

H3K27M Mutation Reshapes the Antigenic Landscape in Diffuse Midline Glioma

Simple SummaryDiffuse midline gliomas (DMGs) are aggressive childhood brain tumours with no effective treatments. Over 80% of cases carry the histone H3K27M mutation, which alters chromatin structure and gene regulation and induce tumours growth. Immunotherapy, which uses the bodys immune system to fight cancer, relies on tumour- specific antigen molecules that immune cells can recognise. However, because DMGs have few mutations, finding suitable antigens for therapeutic purposes has been challenging. In this study, we investigated how the H3K27M mutation affects tumour antigen presentation in DMG. Using patient-derived DMG models, we found that H3K27M alters the landscape of antigens displayed on tumour cell surface, creating unique immune targets. We identified six immunogenic peptides, that triggered strong T cell responses. These antigens were absent when H3K27M was removed, confirming their link to the mutation. Our findings provide a blueprint for developing T cell-based immunotherapies for DMG, offering new hope for targeted treatments against this devastating disease. AbstractO_ST_ABSBackgroundC_ST_ABSDiffuse midline gliomas (DMGs) are among the most aggressive paediatric brain tumours, with the pathognomonic H3K27M mutation present in over 80% of cases. This mutation drives epigenetic dysregulation and transcriptional reprogramming, yet its impact on the tumour antigenic landscape remains poorly understood. Given the low mutational burden of DMG, an expanded search beyond neoantigens to include epigenetically dysregulated tumour-associated antigens (TAAs) is critical for advancing antigen-specific immunotherapies. MethodsTo assess how H3K27M influences antigenic landscape of DMG, we performed a comprehensive immunopeptidomic analysis using patient-derived DMG cell line models (SU-DIPG13 and BT245) that harbour the H3K27M mutation and their CRISPR-edited H3K27M-knockout (KO) counterparts. High-resolution mass spectrometry and bioinformatics were employed to define H3K27M-driven changes in the immunopeptidome. Functional T cell assays using HLA-matched healthy donor PBMCs were conducted to evaluate the immunogenicity of H3K27M-associated peptides. ResultsOur findings reveal that the H3K27M mutation reshapes the tumour antigenic landscape in a model-specific manner. While H3K27M knockout increased HLA-I expression in SU-DIPG13 but not BT245, immunopeptidomic profiling uncovered distinct shifts in the presentation of tumour-associated peptides, independent of direct effects on antigen processing machinery. Among these, we identified six immunogenic peptides, derived from SLITRK2, PRAME, XKR5, and CBX2, that elicited CD8 T cell responses in in vitro functional assays. Notably, PRAME, a well-characterised cancer-testis antigen was confirmed as an H3K27M-associated immunogenic target, reinforcing its therapeutic relevance. Peptides identified exclusively in H3K27M+ cells were absent in KO models, demonstrating a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. ConclusionsThis study provides the first systematic assessment of how H3K27M reshapes the antigenic landscape in DMG, uncovering novel, immunogenic tumour- associated peptides that could serve as targets for precision immunotherapy. By demonstrating that H3K27M mutation drives context-dependent antigen presentation, our findings establish a foundation for T cell-based therapies targeting H3K27M-associated antigens. These insights pave the way for next-generation personalised immunotherapies for this otherwise treatment-refractory disease. Key PointsH3K27M mutation induces expression of tumour-associated antigens in DMG H3K27M alters the DMG immunopeptidome without uniformly changing HLA-I levels PRAME- and CBX2-derived peptides are immunogenic and targetable by CD8 T cells Importance of StudyDiffuse midline gliomas (DMGs) are universally fatal paediatric brain tumours with limited treatment options and poor immune visibility. While the H3K27M mutation is a defining hallmark, its impact on tumour immunogenicity remains unclear. This study presents the first comprehensive to explore the effect of H3K27M-mution on the DMG immunopeptidome, revealing six immunogenic peptides derived from epigenetically dysregulated tumour- associated antigens, including SLITRK2, PRAME, XKR5, and CBX2. These antigens elicited CD8 T cell responses, establishing a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. By leveraging these altered antigens, we highlight actionable vulnerabilities for T cell-based immunotherapy.

cancer biology↗

bioRxiv2024

Ribosomal Biogenesis Hyperactivation and ErbB signalling Mediated Network Rewiring Causes Adaptive Resistance to FGFR2 Inhibition

Fibroblast Growth Factor Receptor 2 inhibition presents a promising therapeutic approach for restraining the growth and survival of cancer cells, particularly in breast tumours. However, the emergence of resistance to FGFR2 inhibitors like PD173074 highlights the importance of understanding the molecular mechanisms driving resistance and identifying effective therapeutic strategies. In this study, we employed temporal quantitative proteomics and phosphoproteomics, complemented by computational clustering, PTM-SEA analysis, and kinase activity prediction, to monitor the response of MFM223 triple negative breast cancer cells to FGFR2 inhibition. Strikingly, we observed a marked enrichment of ribosome biogenesis function modules immediately following treatment, a phenomenon not observed with other inhibitors. Additionally, we discovered that CX-5461, an RNA polymerase I inhibitor, synergistically enhanced the growth inhibition induced by PD173074, mechanistically attributed to its significant suppression of rDNA transcription stimulated by PD173074. Moreover, our phosphoproteomics dynamic profiling identified the clustering of kinases within MAPK and ErbB signalling pathways, indicative of their reactivation in response to FGFR2 inhibition. Experimentally validating this finding, we observed a notable rebound in phosphorylation levels of key kinases such as ERK1/2 and ErbB3, and demonstrated a substantial synergistic effect of PD173074 in combination with Trametinib, a MEK inhibitor, in suppressing cancer cell growth. Collectively, our findings provide critical insights into the network rewiring triggered by FGFR2 inhibition and offer a foundation for the rational design of combinatorial therapeutic strategies to overcome resistance mechanisms associated with FGFR2 inhibitors.

cancer biology↗

bioRxiv2024

Valine restriction extends survival in a Drosophila model of short-chain enoyl-CoA hydratase 1 (ECHS1) deficiency

Short-chain enoyl-CoA hydratase 1 deficiency (ECHS1D) is a rare genetic disorder caused by biallelic pathogenic variants in the ECHS1 gene. ECHS1D is characterised by severe neurological and physical impairment that often leads to childhood mortality. Therapies such as protein and single nutrient-restricted diets show poor efficacy, whereas development of new treatments is hindered by the low prevalence of the disorder and a lack of model systems for treatment testing. Here we report on the establishment of a Drosophila model of ECHS1D. Flies carrying mutations in Echs1 (CG6543) were characterised for their physical and metabolic phenotypes, and dietary intervention to improve fly model health was explored. The Echs1 null larvae recapitulated human ECHS1D phenotypes including elevated biomarkers (S-(2-carboxypropyl)cysteamine and 2,3-dihydroxy-2-methylbutyric acid), poor motor behaviour and early mortality, and could be rescued by expression of a human ECHS1 transgene. We observed that both restriction of valine in isolation, or all branched-chain amino acids (BCAAs - leucine, isoleucine, and valine) together, extended larval survival, supporting the idea that reducing BCAA pathway catabolic flux is beneficial in this disorder. Further, metabolic profiling revealed substantial changes to carbohydrate metabolism, suggesting that Echs1 loss causes widespread metabolic dysregulation beyond valine metabolism. The similarities between Drosophila and human ECHS1D suggest that the fly model is a valuable animal system in which to explore mechanisms of pathogenesis and novel treatment options for this disorder.

genetics↗

bioRxiv2024

Haptoglobin and glutamine synthetase may biomark cachexia induced by anti-acute myeloid leukemia chemotherapy

BackgroundAnti-cancer chemotherapy is an underappreciated contributor to cancer cachexia, an often irreversible body-wasting condition that causes 20-30% of cancer-related deaths. An obstacle to predicting, monitoring and understanding the mechanisms underlying chemotherapy cachexia is that each cancer (and sub-type) is assigned different chemotherapeutic compounds, typically in multi-agent regimens. Here, we investigate the chemotherapy induction regimen (CIR) used in the haematological cancer, acute myeloid leukemia (AML). We hypothesized that the AML CIR would induce cachexia, including loss of lean tissue mass and skeletal muscle atrophy. MethodsUsing an unbiased proteomics approach we interrogated the underlying molecular mechanisms. 3-month-old male Balb/c mice were treated with the AML CIR via intraperitoneal injections of daunorubicin (1.7 mg/kg) on days 1-3, and cytarabine (33.2 mg/kg) administered on days 1-7 or vehicle. Mice were assessed 24 hours after the last treatment, on day 8, or allowed to recover for 2 weeks and assessed on day 22. A third cohort was given access to running wheels in cages. We assessed body composition, whole body metabolism and assessed the muscle proteome using quantitative tandem mass tag labelling LC-MS/MS analysis. ResultsThe AML CIR-induced acute cachexia involved a [~]10% loss of body mass, [~]10% loss of lean mass and [~]20% reduction in skeletal muscle fibre size. Whole body metabolism and ambulatory activity declined. This cachexic phenotype did not recover over the 2-week post-CIR period (lean mass loss post-CIR: 1 week [~]7% vs 2 weeks [~]9%). In voluntarily active CIR-treated mice, body wasting was exacerbated due to unchecked loss of fat mass (CIR sedentary: [~]31% vs CIR active: [~]51%). Muscle proteome studies revealed upregulation of haptoglobin (Hp) and glutamine synthetase (Glul), which were positively correlated with body and lean mass loss. Hp was sensitive to the conditional induction, recovery and exacerbation of AML CIR-mediated cachexia, suggestive of biomarker potential. ConclusionsThe AML CIR induces an acute reduction of body, lean and fat mass underpinned by skeletal muscle atrophy, hypermetabolism and catabolism. Our data uncovered a conditionally sensitive muscle biomarker in Hp, which may be useful as a prognostic tool across other scenarios of chemotherapy-induced myopathy and cachexia or as a target for therapeutic discovery in follow-up studies.

physiology↗

bioRxiv2024

Nitration of a ribosomal pentapeptide generates a noncanonical precursor for nonribosomal peptide synthesis

Peptide natural products possess a fascinating array of complex structures and diverse functions. Central to this is a repertoire of modified amino acid building blocks, which stem from fundamentally different biosynthesis pathways for peptides of nonribosomal and ribosomal origins. Given these origins, integration of nonribosomal and ribosomal pathways have previously been thought unlikely. Now, we demonstrate that ribosomal biosynthesis generates a key noncanonical 3-nitrotyrosine building block for the nonribosomal synthesis of rufomycin. In this pathway, a biarylitide-type ribosomal peptide is nitrated by a modified cytochrome P450 crosslinking enzyme, with the nitrated residue liberated by the actions of a dedicated protease found within the rufomycin gene cluster before being incorporated into rufomycin by the rufomycin nonribosomal peptide synthetase. This resolves the enigmatic origins of 3-nitrotyrosine within rufomycin biosynthesis and demonstrates unexpected integration of ribosomal peptide synthesis as a mechanism for the generation of noncanonical building blocks within nonribosomal synthesis pathways.

biochemistry↗

bioRxiv2024

Analysis and visualization of quantitative proteomics data using FragPipe-Analyst

The FragPipe computational proteomics platform is gaining widespread popularity among the proteomics research community because of its fast processing speed and user-friendly graphical interface. Although FragPipe produces well-formatted output tables that are ready for analysis, there is still a need for an easy-to-use and user-friendly downstream statistical analysis and visualization tool. FragPipe-Analyst addresses this need by providing an R shiny web server to assist FragPipe users in conducting downstream analyses of the resulting quantitative proteomics data. It supports major quantification workflows including label-free quantification, tandem mass tags, and data-independent acquisition. FragPipe-Analyst offers a range of useful functionalities, such as various missing value imputation options, data quality control, unsupervised clustering, differential expression (DE) analysis using Limma, and gene ontology and pathway enrichment analysis using Enrichr. To support advanced analysis and customized visualizations, we also developed FragPipeAnalystR, an R package encompassing all FragPipe-Analyst functionalities that is extended to support site-specific analysis of post-translational modifications (PTMs). FragPipe-Analyst and FragPipeAnalystR are both open-source and freely available.

bioinformatics↗

bioRxiv2024

Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates and citrate synthase activity but that these deficits can be offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggests that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

pathology↗

bioRxiv2023

Structure of a Rhs effector clade domain identifies new mechanistic insights into type VI secretion system toxin delivery.

The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins directly into adjacent bacteria, often providing a competitive advantage. Rearrangement hotspot (Rhs) effectors are known T6SS cargo effectors, but the precise delivery and activation of these toxins is poorly defined. We present the structure of a novel T6SS Rhs effector (Tse15) from the multidrug resistant nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered y-cocoon Rhs domain with an N-terminal -helical clade domain and an unfolded C-terminal toxin domain located entirely inside the Rhs cage. We identify that Tse15 is cleaved into three domains, through two independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for the cleavage of the N-terminal clade domain. Proteomic analyses showed that the N-terminal clade and toxin domains, but not the y-strand rich Rhs cage, are delivered outside of the cell, suggesting a novel mechanism for Rhs toxin delivery and activation. Our findings suggest that this delivery mechanism requires an interaction between the N-terminal clade and toxin domains, with the clade domain acting as the internal chaperone to mediate tethering of the toxin to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggest this may be a common mechanism for T6SS toxin delivery.

microbiology↗

bioRxiv2023

GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing

Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Here we investigated this mechanism, finding that flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and that challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, we show that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. We therefore propose a mechanism by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine our understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment.

molecular biology↗

bioRxiv2023

Hypoxic injury triggers maladaptive repair in human kidney organoids

Acute kidney injury (AKI) is a common clinical disorder linked to high rates of illness and death. Ischemia is a leading cause of AKI, which can result in chronic kidney disease (CKD) through maladaptive repair marked by impaired epithelial regeneration, inflammation, and metabolic dysregulation. There are no targeted therapies for AKI or to prevent progression to CKD and insight into human disease mechanisms remains limited. Here we show that human kidney organoids recapitulate key molecular and metabolic signatures of AKI and maladaptive repair in response to hypoxic injury. Transcriptional, proteomic, and metabolomic profiling revealed tubular injury, cell death, cell cycle arrest and metabolic reprogramming in organoids exposed to hypoxia. Following return to normoxic conditions, injured organoids had increased signatures of TNF and NF-{kappa}B signalling pathways and S100A8/9, associated with maladaptive repair. Single cell RNA sequencing localized AKI and maladaptive repair markers including GDF15, MMP7, ICAM1, IL32, SPP1, C3 and CCN1 to injured tubules. Metabolic phenotypes linked to CKD were also evident, including dysregulated gluconeogenesis, altered amino acid metabolism and lipid peroxidation. iPSC-derived macrophages incorporated into organoids displayed a robust activation and inflammatory response to hypoxia. Spatial transcriptomics revealed a shift from a tissue resident-like to inflammatory macrophage states and localized effects on tubular injury and inflammation. This multi-omic analysis defines conserved mechanisms of human ischemic AKI and maladaptive repair, highlighting new opportunities to test therapeutics and model immune-mediated interactions.

systems biology↗

bioRxiv2023

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

microbiology↗

bioRxiv2023

A defined diet for pre-adult Drosophila melanogaster

Drosophila melanogaster is unique among animal models because it has a fully defined synthetic diet available to study nutrient-gene interactions. However, use of this diet is limited to adult studies due to impaired larval development and survival. Here, we provide an adjusted formula that improves larval growth, development rate, and rescues survival. We demonstrate its use for exploring pre-adult diet compositions of therapeutic potential in a model of an inherited metabolic disorder.

genetics↗