Biology subjects

Willems, P.

Publications and source records attributed to Willems, P..

16 recordsLinked to original sources

bioRxiv2026

Antagonistic functions of the two oxidative pentose phosphate pathway dehydrogenases in shaping oxidative stress responses

The oxidative pentose phosphate pathway (OPPP) is a source of cellular NADPH, generated through the sequential activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Using the catalase-deficient cat2 background as a model for H2O2-triggered salicylic acid (SA) signaling we identified the cytosolic G6PDH isoform G6PD5 as a key determinant of redox homeostasis and SA-dependent defense activation (Tremulot et al., companion manuscript). However, the mechanisms underlying this function remain enigmatic. In this work, genetic and transcriptomic analyses show that the role of G6PD5 cannot be explained solely by altered NADPH generation for either NADPH oxidases or the ascorbate-glutathione pathway, suggesting other possible links. To identify such links, a forward genetic screen was employed. We searched for mutations that modulate the suppressed lesion phenotype in cat2 g6pd5 in a photorespiration-dependent manner. This screen identified a mutation in PGD2, encoding the cytosolic 6PGDH. Strikingly, functional analyses of mutants and overexpression lines revealed that PGD2 exerts effects opposite to those of G6PD5 in SA signaling. Our observations uncover an unexpected antagonism between the two cytosolic NADPH-producing steps within the OPPP. Pharmacological analyses support a signaling role for the metabolic intermediate 6-phosphogluconolactone in linking the OPPP to SA signaling. These findings indicate that the OPPP is not solely a source of reducing power during oxidative stress but also acts as a signaling module in which metabolic intermediates contribute to the control of stress-induced immune responses.

plant biology↗

bioRxiv2026

A platform for high-throughput and ultrasensitive immunopeptidomics

Mass spectrometry (MS)-based immunopeptidomics is a powerful approach for untargeted discovery of peptides presented on major histocompatibility complex (MHC) molecules, which can guide the selection of vaccine antigens and immunotherapy targets. First-generation immunopeptidomics workflows require processing of hundreds of millions of cells using lengthy, manual procedures. More recent approaches focus on increasing either sensitivity or throughput, but rarely combine both aspects. Here, we describe a semi-automated immunopeptidomics platform that combines high sensitivity with high throughput by implementing highly optimized conditions for immunoprecipitation, elution and purification of MHC class I and II peptides on a 96-well positive-pressure device. Upon analysis of 25% of the eluate from 16 million cells, our workflow identified over 13,500 MHC I and 6,000 MHC II peptides on a timsTOF SCP mass spectrometer, operating in DDA-PASEF mode. Exploring the sensitivity limits of our platform, we identified over 1,000 MHC I peptides from as few as 20,000 JY cells. Validating the platforms performance for quantitative biological discovery, we report the identification of known and novel bacterial immunopeptides from U937 macrophages infected with Listeria monocytogenes or Bacillus Calmette-Guerin (BCG). Together, our optimized immunopeptidomics platform enables robust immunopeptide detection from lower-input samples in a high-throughput fashion, enabling its use for biological applications where sample amounts are limiting.

immunology↗

bioRxiv2025

Fish Show Genetic Evolutionary Responses to River Regulation

Eco-hydraulics traditionally aims at managing riverine systems in a semi-natural state while meeting human demands, assuming aquatic species are static. However, evidence of rapid evolution suggests that ignoring evolutionary dynamics of fish species might limit long-term effectiveness of eco-hydraulics frameworks. It remains unclear how freshwater fish adapt to human perturbation. Why are some fish populations more resilient to human perturbation than others? What are the genetic mechanisms behind it? To answer these questions, we genotyped eleven populations of three-spined stickleback in a regulated river system and collected data on river morphology, connectivity, flow regimes, physico-chemistry and parasite abundance through a combination of field surveys and modelling. Gene-environment association analysis detected strong signals of genetic divergence associated with hydraulic features. Gene ontology analysis revealed evolutionary responses that primarily involve functions in the nervous and sensory systems. These findings demonstrate that fish can evolve in response to river regulation, highlighting the need to transition from eco-hydraulics toward eco-evo-hydraulics.

evolutionary biology↗

bioRxiv2025

Data-independent immunopeptidomics discovery of low-abundant bacterial epitopes

Mass spectrometry-based immunopeptidomics is a powerful approach to uncover peptides presented by human leukocyte antigen (HLA) molecules that can guide vaccine design and immunotherapies. While data-dependent acquisition (DDA) has been the standard for navigating through the complexity associated with non-enzymatic immunopeptide database searches, data-independent acquisition (DIA) is increasingly adopted in immunopeptidomics research. In this work, we compare diaPASEF to conventional ddaPASEF in terms of global immunopeptidome profiling and bacterial epitope discovery of the model intracellular pathogen Listeria monocytogenes. We show that DIA spectrum-centric workflows that search pseudo-MS/MS spectra complement DDA analysis by uncovering additional human and bacterial immunopeptides. Furthermore, we leveraged DIA-NN for generating and searching proteome-wide predicted HLA class I peptide spectral libraries, scoring approximately 150 million immunopeptide peptide precursors. This approach outperformed other spectrum-based methods in identification of MHC class I peptides and recovered low-abundant peptide precursors missed by other methods. Taken together, our results demonstrate how both DIA spectrum- and peptide-centric immunopeptidomics analysis are promising strategies to identify low-abundant immunopeptides.

molecular biology↗

bioRxiv2024

Maximizing immunopeptidomics-based bacterial epitope discovery by multiple search engines and rescoring

Mass spectrometry-based discovery of bacterial immunopeptides presented by infected cells allows untargeted discovery of bacterial antigens that can serve as vaccine candidates. However, reliable identification of bacterial epitopes is challenged by their extreme low abundance. Here, we describe an optimized bioinformatical framework to enhance the confident identification of bacterial immunopeptides. Immunopeptidomics data of cell cultures infected with Listeria monocytogenes were searched by four different search engines, PEAKS, Comet, Sage and MSFragger, followed by data-driven rescoring with MS2Rescore. Compared to individual search engine results, this integrated workflow boosted immunopeptide identification by an average of 27% and led to the high-confidence detection of 18 additional bacterial peptides (+27%) matching 15 different Listeria proteins (+36%). Despite the strong agreement between the search engines, a small number of spectra (< 1%) had ambiguous matches to multiple peptides and were excluded to ensure high-confident identifications. Finally, we demonstrate our workflow with sensitive timsTOF SCP data acquisition and find that rescoring, now with inclusion of ion mobility features, identifies 76% more peptides compared to Q Exactive HF acquisition. Together, our results demonstrate how integration of multiple search engine results along with data-driven rescoring maximizes immunopeptide identification, boosting the detection of high-confidence bacterial epitopes for vaccine development.

molecular biology↗

bioRxiv2024

Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing

We introduce scywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.

bioinformatics↗

bioRxiv2023

CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

molecular biology↗

bioRxiv2023

Shift in vacuolar to cytosolic regime of infecting Salmonella from a dual proteome perspective

By applying dual proteome profiling to Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters with its epithelial host (here, S. Typhimurium infected human HeLa cells), a detailed interdependent and holistic proteomic perspective on host-pathogen interactions over a time course of infection was obtained. Data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows, especially in identifying the downregulated bacterial proteome response during infection progression infection by permitting quantification of low abundant bacterial proteins at early times of infection at low bacterial infection load. S. Typhimurium invasion and replication specific proteomic signatures in epithelial cells revealed interdependent host/pathogen specific responses besides pointing to putative novel infection markers and signalling responses.

cell biology↗

bioRxiv2022

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (~96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general.

systems biology↗

bioRxiv2022

Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Sm protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, like in sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and enhanced resilience to oxidative stress.

plant biology↗

bioRxiv2020

The use of hybrid data-dependent and -independent acquisition spectral libraries empower dual-proteome profiling

In the context of bacterial infections, it is imperative that physiological responses can be studied in an integrated manner, meaning a simultaneous analysis of both the host and the pathogen responses. To improve the sensitivity of detection, data-independent acquisition (DIA) based proteomics was found to outperform data-dependent acquisition (DDA) workflows in identifying and quantifying low abundant proteins. Here, by making use of representative bacterial pathogen/host proteome samples, we report an optimized hybrid library generation workflow for data-independent acquisition mass spectrometry relying on the use of data-dependent and in silico predicted spectral libraries. When compared to searching DDA experiment-specific libraries only, the use of hybrid libraries significantly improved peptide detection to an extent suggesting that infection relevant host-pathogen conditions could be profiled in sufficient depth without the need of a priori bacterial pathogen enrichment when studying the bacterial proteome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/113340v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1bbc20aorg.highwire.dtl.DTLVardef@1c0455eorg.highwire.dtl.DTLVardef@a2873org.highwire.dtl.DTLVardef@e5b0b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

plant biology↗

bioRxiv2019

Lost and found: re-searching and re-scoring proteomics data aids the discovery of bacterial proteins and improves proteome coverage

Prokaryotic genome annotation is heavily dependent on automated gene annotation pipelines that are prone to propagate errors and underestimate genome complexity. We describe an optimized proteogenomic workflow that uses ribo-seq and proteomic data of Salmonella Typhiumurium to identify unannotated proteins or alternative protein forms raised upon alternative translation initiation (i.e. N-terminal proteoforms). This data analysis encompasses the searching of co-fragmenting peptides and post-processing with extended peptide-to-spectrum quality features including comparison to predicted fragment ion intensities. When applying this strategy, an enhanced proteome-depth is achieved as well as greater confidence for unannotated peptide hits. We demonstrate the general applicability of our pipeline by re-analyzing public Deinococcus radiodurans datasets. Taken together, systematic re-analysis using available prokaryotic (proteome) datasets holds great promise to assist in experimentally-based genome annotation.

microbiology↗

bioRxiv2018

The Plant PTM Viewer, a central resource exploring plant protein modifications. From site-seeing to protein function.

Posttranslational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased number and types of mapped protein modifications, a database is necessary that simultaneouly integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 200,000 PTM sites for 17 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools facilitate to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

systems biology↗

bioRxiv2018

Is faster always better? The walking speed-dependency of gait variability in bilateral vestibulopathy

Study of balance and gait deficits associated with vestibulopathy is important for improving clinical care and is critical to our understanding of the vestibular contributions to gait and balance control. Previous studies report a speed-dependency of the vestibular contributions to gait, so we examined the walking speed effects on gait variability in healthy young and older adults and in adults with bilateral vestibulopathy (BVP). Forty-four people with BVP, 12 healthy young adults and 12 healthy older adults completed walking trials at 0.4m/s to 1.6m/s in 0.2m/s intervals on a dual belt, instrumented treadmill. Using a motion capture system and kinematic data, the means and coefficients of variation for step length, time, width and double support time were calculated. The BVP group also completed a video head impulse test and examinations of ocular and cervical vestibular evoked myogenic potentials and dynamic visual acuity. Walking speed significantly affected all assessed gait parameters. Step length variability at slower speeds and step width variability at faster speeds were the most distinguishing parameters between the healthy participants and people with BVP, and within people with BVP with different locomotor capacities. We observed for step width variability, specifically, an apparent persistent importance of vestibular function at increasing speeds. Gait variability was not associated with the clinical vestibular tests. Our results indicate that gait variability at multiple walking speeds has potential as an assessment tool for vestibular interventions.\n\nNew & NoteworthyWalking speed significantly but differentially affects gait variability in healthy adults and in adults with bilateral vestibulopathy. Gait variability at different speeds distinguishes between participants with and without bilateral vestibulopathy, but also between more and less able walkers with bilateral vestibulopathy. Specifically, for step width variability, an apparent persistent importance of vestibular function at increasing walking speeds was observed. Gait variability was generally not correlated with clinical tests of vestibular function.

neuroscience↗

bioRxiv2018

Stability-normalised walking speed: a new approach for human gait perturbation research

In gait stability research, neither self-selected walking speeds, nor the same prescribed walking speed for all participants, guarantee equivalent gait stability among participants. Furthermore, these options may differentially affect the response to different gait perturbations, which is problematic when comparing groups with different capacities. We present a method for decreasing inter-individual differences in gait stability by adjusting walking speed to equivalent margins of stability (MoS). Eighteen healthy adults walked on a split-belt treadmill for two-minute bouts at 0.4m/s up to 1.8m/s in 0.2m/s intervals. The stability-normalised walking speed (MoS=0.05m) was calculated using the mean MoS at touchdown of the final 10 steps of each speed. Participants then walked for three minutes at this speed and were subsequently exposed to a treadmill belt acceleration perturbation. A further 12 healthy adults were exposed to the same perturbation while walking at 1.3m/s: the average of the previous group. Large ranges in MoS were observed during the prescribed speeds (6-10cm across speeds) and walking speed significantly (P<0.001) affected MoS. The stability-normalised walking speeds resulted in MoS equal or very close to the desired 0.05m and reduced between-participant variability in MoS. The second group of participants walking at 1.3m/s had greater inter-individual variation in MoS during both unperturbed and perturbed walking compared to 12 sex, height and leg length-matched participants from the stability-normalised walking speed group. The current method decreases inter-individual differences in gait stability which may be beneficial for gait perturbation and stability research, in particular for studies on populations with different locomotor capacities.

bioengineering↗