Biology subjects

Jones, A. R.

Publications and source records attributed to Jones, A. R..

At least 19 recordsLinked to original sources

bioRxiv2026

A Single-Cell Framework for Classifying Human Th17 Pathogenicity Links Acylcarnitine Metabolism to Non-Pathogenic Inflammation in Type 2 Diabetes

Based on in vitro and animal studies, Th17 cells are classified as pathogenic (pTh17) or non-pathogenic (nTh17), but the inability to identify these subsets in primary human samples limits translation. We developed a single-cell ELISA to enrich human Th17s, enabling transcriptomic and flow-cytometric classification. nTh17 cells predominated in Type 2 diabetes and exhibited signatures of acylcarnitine synthesis, while knockdown of CPT1A demonstrated that acylcarnitine metabolism regulates Th17 pathogenicity.

immunology↗

bioRxiv2026

A High-Confidence Atlas of Protein Methylation Enables AI-Driven Detection of Methylated Peptides

Lysine and arginine methylation regulate chromatin dynamics, transcription, and cellular signaling, however confident mass spectrometry (MS)-based detection and localization of this modification remain challenging. We reanalyzed eight public human methylation-enriched datasets using an open and standardized workflow that integrates database searching via the Trans-Proteomic Pipeline with a decoy-based statistical method for the independent estimation of false localization rates (FLR). This yielded a high-confidence Human Methylation Atlas of 1,828 sites (57 methyl-lysine, 1,771 methyl-arginine) across 1,021 proteins, classified into Gold, Silver, and Bronze confidence tiers. This is far fewer sites than reported in previous studies, reflecting the application of stringent FLR control, and what we hypothesise is potential high-false discovery in previous analyses. We then leveraged this resource to adapt a deep learning-based methodology for the improved detection of methylated peptides. Three mouse methylation-enriched datasets were reanalysed to augment training, and the phosphoproteomics-trained AHLF (ad hoc learning of peptide fragmentation) model was fine-tuned by transfer learning to create AHLF-Methylation. The model achieved mean ROC-AUC values of 0.824 on human spectra, and 0.829 on combined human-mouse spectra. The atlas is available through PTMeXchange and PRIDE, with curated site evidence integrated into UniProt and PeptideAtlas.

bioinformatics↗

bioRxiv2026

A High-Quality Acetylation Dataset Reveals Modest Data Requirements for Transfer Learning to Identify Little Studied Post-Translational Modifications

Dysregulation of post-translational modifications (PTMs) is associated with severe pathologies, including cancers and Alzheimers disease. Despite their biological importance, identifying modified peptides remains challenging due to the immense combinatorial search space. While searches benefit from prior knowledge of a peptides modification status, the data scarcity for most PTMs hinders the development of accurate deep learning classifiers like AHLF (ad hoc learning of peptide fragmentation). Here, we overcome this data bottle-neck for acetylation and ubiquitination. We harmonised a dataset with about 500,000 high quality acetylated peptide-spectrum matches (PSMs) from nine publicly available acetylation-enriched datasets. We fine-tuned AHLF with the acetylation and a 2-million spectra strong ubiquitination dataset separately and assessed the minimum data requirement for training by iteratively downsampling. Training separate models on SILAC and label-free subsets also assessed the impact of data diversity. The resulting acetylation and ubiquitination models achieve an AUC of 0.87 and 0.90 respectively. Beyond 28,500 acetylated spectra, corresponding to roughly 0.3% of the original models training data, additional data just provides minor performance gains. Finally, we show that data diversity is beneficial for generalizability, while models trained on homogeneous data sources tend to overfit to their respective data type. All code, and model weights are available at https://gitlab.com/dacs-hpi/ahlf-ptmai.

bioinformatics↗

bioRxiv2026

MicroAge Mission: Effects of Microgravity and Heat Shock Protein 10 Overexpression on the Proteome of Human Tissue-Engineered Muscle Constructs - Implications for Skeletal Muscle Ageing

Age-related loss of skeletal muscle mass and function, or sarcopenia, presents a growing clinical challenge, mirroring the accelerated muscle atrophy seen in microgravity. This study, part of the UK Space Agencys MicroAge Mission, aimed to investigate microgravity-induced proteomic changes in 3D human skeletal muscle constructs and assess whether mitochondrial Heat Shock Protein 10 (HSP10) overexpression could modulate these responses. Constructs derived from control human AB1167 myoblasts and AB1167 myoblasts that were transduced to overexpress HSP10, were flown to the International Space Station (ISS), with a ground reference experiment (GRE) conducted post-flight. Proteomic analysis using mass spectrometry and bioinformatics revealed significant alterations in metabolic, structural, and mitochondrial protein profiles after microgravity exposure. Microgravity caused downregulation of key proteins involved in energy metabolism, stress responses and structural integrity, while upregulating catabolic and apoptotic enzymes. Many of these modifications parallel previously reported changes in protein composition of muscle with ageing on earth. Overexpression of HSP10 attenuated the effects of microgravity, with fewer proteins showing significant changes and reduced disruption to mitochondrial and cytoskeletal components. Pathway analysis indicated that HSP10 overexpression preserved mitochondrial protein expression, particularly in the matrix, and promoted mitochondrial gene expression and translation under microgravity conditions. Notably, 284 proteins altered by microgravity in unmodified muscle constructs remained stable in HSP10-overexpressing constructs, suggesting a protective effect. MitoCarta 3.0 analysis confirmed that HSP10 expression modulated protein responses at the mitochondrial level, mitigating declines in bioenergetic proteins that are typically associated with microgravity. Collectively, the findings demonstrate that microgravity induces extensive proteomic remodelling in human muscle, which is partially offset by HSP10 overexpression. These results offer insights into muscle atrophy in spaceflight and suggest that targeting mitochondrial stress pathways via chaperone modulation may be a viable strategy to combat sarcopenia and disuse-induced muscle loss on Earth and in space.

physiology↗

bioRxiv2026

A Multi-Objective Scoring (MOS) Framework for Detecting Cross-Modal Spatial Similarity: Conceptual and Direct Formulations

Spatial multi-omics methods allow researchers to study complex biological systems by integrating multiple molecular layers while preserving their spatial organization. However, integrating spatial transcriptomics and mass spectrometry imaging (MSI) remains challenging due to the differences between the two modalities, including sampling geometry, spatial resolution, signal scaling, and measurement principles. For example, 10x Genomics Visium captures transcriptomic data on a discrete hexagonal grid of spots; however, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) produces dense Cartesian pixel maps of molecular distributions. These differences make exact spatial co-registration difficult and limit the effectiveness of single-metric similarity approaches. Here, we introduce a Multi-Objective Scoring (MOS) framework designed to detect cross-modal spatial similarity without requiring exact pixel-to-pixel alignment. The MOS framework integrates multiple complementary spatial descriptors into a unified similarity score, including coordinate-based metrics (value correlation, importance-based Intersection-over-Union (IoU), and importance-map correlation) and descriptor-based metrics that capture higher-order spatial organization, such as spatial histograms, radial profiles, quadrant statistics, and Morans I spatial autocorrelation. These metrics are combined through a weighted ensemble model. This model calculates the weights using synthetic spatial datasets that simulate realistic tissue geometry, sampling differences, and spatial distortions. The framework was applied to a spatial multi-omics dataset from murine brain tissue, integrating spatial transcriptomics with MALDI-MSI lipidomics across young and aged control and Alzheimers disease (AD) models. Synthetic data validation results demonstrated strong pattern-matching performance (96.14% accuracy), and application to experimental data identified several MSI analyte features whose spatial distributions closely matched transcriptomic patterns. In particular, strong and reproducible associations were observed between myelin-related genes (Mbp and Plp1) and multiple analyte features enriched in white matter regions. Overall, whether applied conceptually or directly, the MOS framework provides a strong strategy for cross-modal spatial integration and offers a scalable tool for discovering spatial relationships across diverse multi-omics datasets and facilitating hypothesis generation.

biochemistry↗

bioRxiv2025

Long non-coding RNAs as a novel source of beta cell autoantigens in type 1 diabetes

Aims/hypothesisGenome-wide association studies increasingly highlight the role of non-coding regions in complex diseases such as type 1 diabetes, pointing, in particular, to long non-coding RNAs (lncRNAs) as potentially active molecular players. Emerging evidence suggests that lncRNAs may encode (small) peptides with the potential to modulate cellular processes, including immune responses. In this study, we have investigated whether these (micro)peptides translated from lncRNAs can act as neoantigens capable of activating autoreactive CD4T cells in individuals with type 1 diabetes. MethodsWe have integrated transcriptomic, proteomic and in silico data to identify a subset of lncRNAs with genuine peptide-coding potential in basal or inflamed-condition beta cells. We further assessed the suitability of those candidates to encode (micro)peptides by sequence analysis and in vitro experimentation techniques. Finally, we have predicted HLA-II epitopes within the micropeptide candidates and evaluated their immunogenicity by analyzing T cell activation responses in peripheral blood mononuclear cells from HLA matching individuals with type 1 diabetes. ResultsBy merging ribosome-bound RNA sequencing with nascent peptide mass spectrometry, we identified a total of 30 lncRNAs with potential coding capacity. We verified the peptide-coding ORF translation for three lncRNA candidates: UXT-AS1, RAPGEF4-AS1 and ENSG00000227066. After predicting potential type 1 diabetes risk-associated HLA-DRB1*03:01 and HLA-DRB1*04:01-binding epitopes within translated lncRNA (micro)peptides, we identified several peptides that elicited CD4+ T cell activation. Furthermore, several epitopes elicited T cell activation in multiple donors with type 1 diabetes. T cell lines were isolated and studied to confirm responses restricted by type 1 diabetes risk HLA alleles. ConclusionsThese results reveal a novel class of immunogenic (micro)peptides derived from lncRNAs, supporting their potential role in the autoimmune response that underlies type 1 diabetes. Our findings open new perspectives on the contribution of non-coding genomic elements to autoimmunity and highlight the need to further investigate lncRNA-encoded (micro)peptides as possible targets for future immunotherapies. RESEARCH IN CONTEXTO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIShort proteins can sometimes be translated from lncRNAs. C_LIO_LIType 1 diabetes neoepitopes arising from non-native forms of proteins and their post translational modifications have been described. C_LI What is the key question?O_LICan lncRNA-encoded peptides be recognized by the human immune system and contribute to type 1 diabetes development? C_LI What are the new findings?O_LIWe have identified three candidate lncRNAs with peptide coding capacity. C_LIO_LIEpitopes derived from these peptides drive T cell activation in PBMCs of individuals with type 1 diabetes. C_LIO_LIPresentation of some of these peptides are restricted to type 1 diabetes risk-associated HLA-DRB1*03:01 and *04:01 molecules. C_LI How might this impact on clinical practice in the foreseeable future?O_LIDiscovery of lncRNA-derived peptide reactive T cells might serve as a diagnostic or stratification indicator in the early stages of disease development, aiding the development of more personalized treatments and unlocking a new array of therapeutic targets. C_LI

immunology↗

bioRxiv2025

Environmental dipolar relaxation during excited state proton transfer in Green Fluorescent Protein

Engineered variants of Green Fluorescent Protein (GFP) are in widespread use as genetically-encoded labels for biological imaging. Excitation of its neutral phenolic ground state chromophore populates an emissive anionic phenolate state via excited state proton transfer (ESPT) to E222 along a molecular wire. Computational studies indicate that ESPT fits to an electronically adiabatic rate expression. Although reactions that operate close to the adiabatic limit can be sensitive to environmental dipolar relaxation, little is known about the role of such relaxation during ESPT in GFP. Here, we present the first experimental evidence that dipolar relaxation of the protein matrix occurs during ESPT in response to the change in electron density distribution in the excited state GFP chromophore and is a key determinant of the reaction pathway. Using fluorescence spectroscopy, we excited along the red edge of the neutral phenolic ground state absorption band of several GFP variants with differing chromophore environments. Instead of resulting in a significant red shift of the centre of spectral mass (CSM) of the emission spectra common for biological chromophores such as tryptophan, the CSM of each GFP variant remains almost unchanged as a function of excitation wavelength. This is consistent with each sub-population that is selectively excited along the red edge reaching the same, fully relaxed state before emission of a photon. The kinetics of environmental dipolar relaxation are therefore on the same sub-nanosecond timescale as ESPT, which provides an explanation for its adiabatic nature and could inform the rational design of novel fluorescent proteins with tailored photophysics.

biophysics↗

bioRxiv2025

The functional landscape of the human ubiquitinome

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. To date, hundreds of thousands of ubiquitination sites (ubi-sites) have been identified, however fewer than 1% have known functional roles. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. To pinpoint critical regulatory events requiring ubiquitination at a precise site, we mapped ubi-site conservation across proteomics data from six non-human species. Perturbation proteomics revealed that highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize site-specific ubiquitination relevant for organismal fitness, we constructed a machine learning-based positional importance score for more than 100,000 ubi-sites, which identifies sites regulating diverse protein functions and rationalizes genetic vulnerabilities. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying site-specific protein ubiquitination.

cell biology↗

bioRxiv2025

How the Drosophila Cryptochrome C-terminus mediates magnetosensitivity

The Earths magnetic field plays an important role in the seasonal migrations of many species of animals. A Cryptochrome (CRY)-based radical pair mechanism (RPM) has been suggested to underlie the mechanistic basis of animal magnetosensitivity and navigation. The quantum spin state of a radical pair involving flavin adenine dinucleotide (FAD) bound to CRY in the canonical pocket is sensitive to external magnetic fields that can alter the signalling concentration of activated CRY1-5. However, several experimental observations challenge this model including the finding that the C-terminal fragment of Drosophila CRY (DmCRY), which lacks any canonical FAD binding pocket, and human CRY2, which lacks affinity for FAD, are sufficient to support magnetosensitivity6-9. Here, we use all-atom molecular dynamic (MD) simulations, alongside in vitro and in vivo analyses to reveal that the C-terminus of Drosophila CRY (DmCRY-CT) binds FAD. FAD binding is required for transduction of a magnetic signal within cells, and, in vitro, initiates formation of high molecular weight DmCRY-CT oligomers, including large insoluble aggregates reminiscent of CRY photobodies observed in plants10-14. These results provide a plausible mechanistic basis for several experimental observations that have reported non-canonical magnetosensitivity in animals.

biochemistry↗

bioRxiv2025

Ubiquitin-like SUMO protease expansion in rice (Oryza sativa)

O_LISUMOylation is a protein post-translational modification that is essential for plant growth and response to changing environments. However, past work in this area has mainly focused on simple sequence similarity methods for discovering SUMOylation genes, often using orthologue mapping from yeast or Arabidopsis. C_LIO_LIIn this work, we employed a range of computational techniques and approaches to describe and characterise the SUMOylation machinery in Asian rice (Oryza sativa), as a globally important stable crop, and where the SUMOylation system has been shown to play key roles in responses to biotic and abiotic stresses. We describe and analyse the ULP system at the phylogenetic, transcriptional, and protein structural levels, with a focus on the rice reference genome, a well-annotated Rice Population Reference Panel (RPRP), and wild rice genomes. C_LIO_LIOur analysis revealed the expansion of ULPs in the reference genome and RPRP set (32 - 45 ULPs) compared to wild rice (9-36 ULPs), raising an intriguing hypothesis about the expansion of the ULP family being driven by selective breeding pressure. C_LIO_LIWe provide evidence of potential functional ULPs and their possible roles in biotic and abiotic stress responses in cultivated rice. These insights offer valuable resources for future rice breeding and crop improvement. C_LI

plant biology↗

bioRxiv2025

A pan-gene catalogue of Asian cultivated rice

The rice genome underpins fundamental research and breeding, but the Nipponbare (japonica) reference does not fully encompass the genetic diversity of Asian rice. To address this gap, the Rice Population Reference Panel (RPRP) was developed, comprising high-quality assemblies of 16 rice cultivars to represent japonica, indica, aus, and aromatic varietal groups. The RPRP has been consistently annotated, supported by extensive experimental data and here we report the computational assignment, characterization and dissemination of stably identified pan-genes. We identified 25,178 core pan-genes shared across all cultivars, alongside cultivar-specific and family-enriched genes. Core genes exhibit higher gene expression and proteomic evidence, higher confidence protein domains and AlphaFold structures, while cultivar-specific genes were enriched for domains under selective breeding pressure, such as for disease resistance. This resource, integrated into public databases, enables researchers to explore genetic and functional diversity via a population-aware "reference guide" across rice genomes, advancing both basic and applied research.

genomics↗

bioRxiv2025

Cryptocercus genomes expand knowledge of adaptations to xylophagy and termite sociality

Subsociality and wood-eating or xylophagy are understood as key drivers in the evolution of eusociality in Blattodea (cockroaches and termites), two features observed in the cockroach genus Cryptocercus, the sister group of all termites. We present and analyse two new high-quality genomes from this genus, C. punctulatus from North America and C. meridianus from Southeast Asia, to explore the evolutionary transitions to xylophagy and subsociality within Blattodea. Our analyses reveal evidence of relaxed selection in both Cryptocercus and termites, indicating that a reduction in effective population size may have occurred in their subsocial ancestors. These findings challenge the expected positive correlation between dN/dS ratios and social complexity, as Cryptocercus exhibits elevated dN/dS values that may exceed those of eusocial termites. Additionally, we identify positive selection on mitochondrial ribosomal proteins and components of the NADH dehydrogenase complex, suggesting significant evolutionary changes in energy production. Future studies incorporating additional genomic data from diverse blattodean species are essential to elucidate the molecular mechanisms driving transitions to xylophagy and eusociality.

evolutionary biology↗

bioRxiv2025

Applying a conservation-based approach for predicting novel phosphorylation sites in eukaryotes and evaluating their functional relevance

Protein phosphorylation, a key post-translational modification, is central to cellular signalling and disease pathogenesis. The development of high-throughput proteomics pipelines has led to the discovery of large numbers of phosphorylated protein motifs and sites (phosphosites) across many eukaryotic species. However, the majority of phosphosites are reported from human cellular sources, with most species having only a few experimentally confirmed or computationally predicted phosphosites. Furthermore, only a small fraction of the characterised human phosphoproteome has an annotated functional role. A common method of predicting functionally relevant phosphosites is conservation analysis. However, extensive evolutionary studies involving large numbers of species are scarce. In this study, we explore the conservation of 20,751 confident human phosphosites across 100 eukaryotic species. We link the observed conservation patterns to the functional relevance of phosphosites and investigate the evolution of associated protein domains and kinases. We identify several protein functions that are likely regulated by phosphorylation, including ancient functions conserved across all life and relatively new functions only conserved in species closely related to humans. We demonstrate the importance of conservation analysis in identifying organisms suitable as biological models for studying conserved signalling pathways relevant to human biology and disease. Finally, we use human protein sequences as a reference to apply conservation analysis and predict over 1,000,000 potential phosphosites in analysed eukaryotes. Our results can ultimately be used to improve proteome annotations of several species and direct downstream research surrounding the evolution and functional relevance of eukaryotic phosphosites.

bioinformatics↗

bioRxiv2024

Searching for Sulfotyrosines (sY) in a HA(pY)STACK

Protein sulfation can be crucial in regulating protein-protein interactions but remains largely underexplored. Sulfation is near-isobaric to phosphorylation, making it particularly challenging to investigate using mass spectrometry. The degree to which tyrosine sulfation (sY) is misidentified as phosphorylation (pY) is thus an unresolved concern. This study explores the extent of sY misidentification within the human phosphoproteome by distinguishing between sulfation and phosphorylation based on their mass difference. Using Gaussian mixture models (GMMs), we screened [~]45M peptide-spectrum matches (PSMs) from the PeptideAtlas Human Phosphoproteome build for peptidoforms with mass error shifts indicative of sulfation. This analysis pinpointed 104 candidate sulfated peptidoforms, backed-up by Gene Ontology (GO) terms and custom terms linked to sulfation. False positive filtering by manual annotation resulted in 31 convincing peptidoforms spanning 7 known and 7 novel sY sites. Y47 in Calumenin was particularly intriguing since mass error shifts, acidic motif conservation, and MS2 neutral loss patterns characteristic of sulfation provided strong evidence that this site is sulfated rather than phosphorylated. Overall, although misidentification of sulfation in phosphoproteomics datasets derived from cell and tissue intracellular extracts can occur, it appears relatively rare and should not be considered a substantive confounding factor in high-quality phosphoproteomics datasets.

bioinformatics↗

bioRxiv2024

Integrated View of Baseline Protein Expression in Human Tissues using public Data Independent Acquisition datasets

The PRIDE database is the largest public data repository of mass spectrometry-based proteomics data and currently stores more than 40,000 datasets covering a wide range of organisms, experimental techniques and biological conditions. During the past few years, PRIDE has seen a significant increase in the amount of submitted Data-Independent Acquisition (DIA) proteomics datasets. This provides an excellent opportunity for large scale data reanalysis and reuse. We have reanalysed 15 public label-free DIA datasets across various healthy human tissues, to provide a state-of-the-art view of the human proteome in baseline conditions (without any perturbations). We computed baseline protein abundances and compared them across various tissues, samples and datasets. Our second aim was to compare protein abundances obtained here from the results of previous analyses using human baseline Data-Dependent Acquisition (DDA) datasets. We observed a good correlation across some tissues, especially in liver and colon but weak correlations were found in others, such as lung and pancreas. The reanalysed results including protein abundance values and curated metadata are made available to view and download from the resource Expression Atlas. For TOC Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/613191v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1414dforg.highwire.dtl.DTLVardef@66606aorg.highwire.dtl.DTLVardef@143f862org.highwire.dtl.DTLVardef@1682406_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

ROS responsive Aux/IAA multimerization modulates auxin responses

Reactive oxygen species (ROS) function as key signals in plants to enable adaptation to environmental stresses. Plant roots respond to transient water stress by temporarily ceasing branching using the acclimative response xerobranching1. In this study, we report that a rapid ROS burst regulates Xerobranching by inducing multimerization of auxin repressor protein IAA3/SHY2. Mutations in specific cysteine residues in IAA3/SHY2 disrupt redox-mediated multimerization and interaction with co-repressor TPL, but not with auxin response partner ARF7 and auxin receptor TIR1. ROS-mediated oligomerization of IAA3/SHY2 is required for efficient ARF mediated target gene repression during Xerobranching and lateral root emergence. We demonstrate that AUX/IAA proteins vary in their redox mediated multimerization, revealing a new auxin response regulatory mechanism that directly connects ROS sensing to auxin signalling. Our study reveals how ROS, auxin and water stress intersect to shape acclimative responses in plant roots and maintain their phenotypic plasticity.

plant biology↗

bioRxiv2023

Phosphorylation in the Plasmodium falciparum proteome: A meta-analysis of publicly available data sets

Malaria is a deadly disease caused by Apicomplexan parasites of the Plasmodium genus. Several species of the Plasmodium genus are known to be infectious to human, of which P. falciparum is the most virulent. Post-translational modifications (PTMs) of proteins coordinate cell signalling and hence, regulate many biological processes in P. falciparum homeostasis and host infection, of which the most highly studied is phosphorylation. Phosphosites on proteins can be identified by tandem mass spectrometry (MS) performed on enriched samples (phosphoproteomics), followed by downstream computational analyses. We have performed a large-scale meta-analysis of 11 publicly available phosphoproteomics datasets, to build a comprehensive atlas of phosphosites in the P. falciparum proteome, using robust pipelines aimed at strict control of false identifications. We identified a total of 28,495 phosphorylated sites on P. falciparum proteins at 5% false localisation rate (FLR) and, of those, 18,100 at 1% FLR. We identified significant sequence motifs, likely indicative of different groups of kinases, responsible for different groups of phosphosites. Conservation analysis identified clusters of phosphoproteins that are highly conserved, and others that are evolving faster within the Plasmodium genus, and implicated in different pathways. We were also able to identify over 180,000 phosphosites within Plasmodium species beyond falciparum, based on orthologue mapping. We also explored the structural context of phosphosites, identifying a strong enrichment for phosphosites on fast evolving (low conservation) intrinsically disordered regions (IDRs) of proteins. In other species, IDRs have been shown to have an important role in modulating protein-protein interactions, particularly in signalling, and thus warranting further study for their roles in host- pathogen interactions. All data has made available via UniProtKB, PRIDE and PeptideAtlas, with visualisation interfaces for exploring phosphosites in the context of other data on Plasmodium proteins. Author SummaryPlasmodium parasites continue to pose a significant global health threat, with a high proportion of the world at risk of malaria. It is imperative to gain new insights into cell signalling and regulation of biological processes in these parasites to develop effective treatments. This study focused on post- translational modifications (PTMs) of proteins, specifically phosphorylation. We conducted a meta- analysis of 11 publicly available phosphoproteomics datasets, identifying over 28,000 phosphorylated sites on P. falciparum proteins, using very rigorous statistics to avoid reporting false positives, and mapping to over 180,000 phosphorylation sites on other species of Plasmodium. The analysis revealed distinct sequence motifs associated with different groups of phosphosites (and likely indicative of different upstream kinases), and differences in the downstream pathways regulated. Conservation analysis highlighted clusters of phosphoproteins evolving at different rates within the Plasmodium genus. Notably, phosphorylation was enriched in regions of proteins lacking distinct structural elements, known as intrinsically disordered regions (IDRs), which are poorly conserved across the genus - we speculate that they are important for modulating protein interactions. The findings provide valuable insights into the molecular mechanisms of P. falciparum, with potential implications for understanding host-pathogen interactions. The comprehensive dataset generated is now publicly accessible, serving as a valuable resource for the scientific community through UniProtKB, PRIDE, and PeptideAtlas.

bioinformatics↗

bioRxiv2023

A meta-analysis of rice phosphoproteomics data to understand variation in cell signalling across the rice pan-genome

Phosphorylation is the most studied post-translational modification, and has multiple biological functions. In this study, we have re-analysed publicly available mass spectrometry proteomics datasets enriched for phosphopeptides from Asian rice (Oryza sativa). In total we identified 15,522 phosphosites on serine, threonine and tyrosine residues on rice proteins. We identified sequence motifs for phosphosites, and link motifs to enrichment of different biological processes, indicating different downstream regulation likely caused by different kinase groups. We cross-referenced phosphosites against the rice 3,000 genomes, to identify single amino acid variations (SAAVs) within or proximal to phosphosites that could cause loss of a site in a given rice variety. The data was clustered to identify groups of sites with similar patterns across rice family groups, for example those highly conserved in Japonica, but mostly absent in Aus type rice varieties - known to have different responses to drought. These resources can assist rice researchers to discover alleles with significantly different functional effects across rice varieties. The data has been loaded into UniProt Knowledge-Base - enabling researchers to visualise sites alongside other data on rice proteins e.g. structural models from AlphaFold2, PeptideAtlas and the PRIDE database - enabling visualisation of source evidence, including scores and supporting mass spectra.

bioinformatics↗