Biology subjects

Brunet, M. A.

Publications and source records attributed to Brunet, M. A..

18 recordsLinked to original sources

bioRxiv2026

Inferring protein-induced spontaneous curvature of lipid bilayers from molecular simulation

Proteins and lipids of a biological cell work together to reshape the membranes in which they reside. Membrane reshaping is necessary for vesicular traffic and maintenance of organelle shape and structure. Curvature is the fundamental mathematical description of the local shape of a surface. Through the well-established Helfrich/Canham/Evans model of bilayer shape, curvature sensing and induction are both consequences of a protein's spontaneous curvature, the local curvature at which the Helfrich model energy is a minimum. This paper describes a straightforward approach for estimating the strength and sign of a bilayer-associated molecule's curvature induction from molecular simulation: Correlating the molecule's lateral position with curvature on thermally-fluctuating bilayer modes. Example demonstrations range from a large multi-pass coarse-grained transmembrane protein to an all-atom amphipathic helical segment. Quantification of these systems is placed in the context of experiments seeking the same strength and sign of curvature induction.

biophysics↗

bioRxiv2026

Fusion Peptide Insertion Configuration Determines the Mechanism of Membrane Curvature Induction in Human Parainfluenza Virus Type 3

Viral entry requires remodeling the host membrane with a short fusion peptide (FP) that anchors and deforms the target lipid bilayer. However, the contribution of the FP throughout the full fusion pathway remains unknown. Here, all-atom molecular dynamics simulations define the membrane- deformation mechanisms associated with two distinct insertion configurations of membrane-bound human parainfluenza virus type 3 (HPIV3) FP. Molecular simulations quantifying the linear response of curvature to the peptide are complemented with analyses of lipid organization and FP-lipid interactions to define the underlying membrane-deformation mechanisms. The surface-parallel FP configuration generates positive membrane curvature through deformation of the proximal leaflet, consistent with the classical hydrophobic insertion (wedging) mechanism. In contrast, the transmembrane FP configuration generates curvature through localized deformation of the distal leaflet, a distinct mechanism that would strongly influence later stages of fusion. Cryo-electron tomography shows that target membrane curvature at virus-host contact sites is consistent with the molecular dynamics prediction. In the absence of F-protein activation and FP insertion, the cryo-ET-determined curvature distribution shifts toward negative values, thereby isolating the role of the fusion protein in inducing early-stage curvature that enables the host and virus membranes to approach one another. Modeling key mutational changes isolates how aromatic and electrostatic interactions act together to promote curvature generation. Taken together, these findings establish FP insertion configuration as a determinant of productive membrane curvature across the full fusion pathway.

biophysics↗

bioRxiv2026

Discovery of non-canonical proteins through modification-aware proteogenomics

ShortThe SwissProt database contains a stable 20,418 human protein-coding genes and 42,541 human protein sequences. Ribo-Seq suggests about 7,000 additional, non-canonical Open Reading Frames (ORFs) are present in humans, though only a few of them are confirmed by Mass Spectrometry (MS). Detecting these proteins requires extensive database searches, increasing computational load and inflating False Discovery Rates (FDR). Using the ionbot search engine with the OpenProt database allows for reliable detection of non-canonical proteins while controlling FDR. Ionbot surpasses the Trans-Proteomics Pipeline (TPP) in reproducibility, identifying more peptides and proteins supported by multiple spectra. In addition, open modification searches yield better PSMs compared to closed searches. This work highlights the importance of employing cutting-edge search engines in non-canonical protein research, as well as the value of open modification search in correcting errors in non-canonical protein detection. LongO_ST_ABSBackgroundC_ST_ABSThe SwissProt database reports a quite stable 20,418 human protein-coding genes and 42,541 human protein sequences, figures that have remained stable. New techniques like Ribo-Seq indicate that approximately 7,000 additional, non-canonical Open Reading Frames (ORFs) are translated in humans, few of which have been confirmed by Mass Spectrometry (MS). Detecting these non-canonical proteins requires comprehensive database searches, which increase computational load and False Discovery Rate (FDR). Here, we use the open search engine ionbot in combination with the OpenProt proteogenomics database to reproducibly detect non-canonical proteins while maintaining a well-controlled FDR. ResultsCompared to the current gold standard, the Trans-Proteomics Pipeline (TPP), ionbot shows higher reproducibility, with a higher number of peptides and proteins supported by multiple spectra, and across multiple samples. We observe that PSMs from the open modification search against OpenProt have higher fragment ion intensity correlation compared to PSMs obtained from the closed search, or by only searching canonical proteins. ConclusionsIn this work, we show the potential for open modification searching to correct potential mistakes in non-canonical proteins detection by preventing modified canonical peptides or variants from being incorrectly identified as non-canonical peptides. We also highlight the importance of assessing the FDR of non-canonical identifications separately from canonical ones, as global FDR calculations are biased by the scarcity of non-canonical identifications in each dataset.

molecular biology↗

bioRxiv2026

Loss-of-function phenomics, ncORFs, and ambiguity of mutant phenotypes in Medicago truncatula

Non-canonical open reading frames (ncORFs) are an emerging area of research that is quickly gaining momentum. Many peptides and proteins missed in initial annotation efforts (ncProts) were subsequently shown to be crucial for a wide range of biological processes. The discovery of ncORFs continues to improve the accuracy of loss-of-function studies because they often occupy the same genomic spaces as annotated ORFs. While databases of mutant phenotypes linked to genomic loci exist in a few species, none of these databases integrate the information on ncORFs present in already characterized loci. In this study, we introduce a nearly comprehensive loss-of-function phenomics dataset of Medicago truncatula (673 loci characterized over the past 30 years), which was integrated as a new track into the genome browser of this organism. This dataset helped critically analyze the potential contribution of ncORFs to published phenotypes. We detected mass spectrometry (MS)-validated ncORFs in 10 characterized genes, including major regulators of development and symbiotic relationships. We also found conserved ncORFs in 113 characterized genes, including four genes with highly conserved ncORFs. In some studies, the contribution of these ncORFs can be ruled out, while in others it cannot. Using real examples, we systematized ambiguities associated with ncORFs. Furthermore, we highlighted little-known trans effects of insertional mutagenesis on splicing as contributors to that ambiguity. Finally, our meta-analysis of published phenotypes revealed that different protein classes have significantly different (unique) proportions of unconditional, conditional, and neutral phenotypes, potentially reflecting their relative functional importance. Significance statementThis study is the first to merge a nearly comprehensive inventory of loss-of-function studies in a eukaryotic organism with the information on novel MS-validated and conserved ncORFs.

genetics↗

bioRxiv2026

sf-pediatric: A robust and age-adaptable end-to-end pipeline for pediatric diffusion MRI

Diffusion MRI (dMRI) provides a powerful, non-invasive window into white matter (WM) development. Yet, most existing processing pipelines are not well-suited to the rapidly evolving neurophysiology of the pediatric brain. Here, we introduce sf-pediatric, a scalable, end-to-end, age-adaptable dMRI pipeline that integrates normative models of brain diffusivities to enable optimal subject-specific analysis from birth through 18 years old. Leveraging normative trajectories derived from nearly 2,000 participants from six cohorts, sf-pediatric dynamically calibrates diffusion priors, template selection, segmentation, and WM atlases based on the subjects age. By incorporating automatic quality control into a portable, tested, containerized, open-access, and press-button framework across computing environments, sf-pediatric provides a robust pipeline for large-scale pediatric dMRI studies. We validated this approach by showing improved local modeling and cortical fanning while preserving reproducibility and the ability to derive brain-behavior relationships. Additionally, we demonstrated robust recovery of known developmental trajectories of WM microstructure and connectome-derived network organization.

neuroscience↗

bioRxiv2025

Regional brain age deviations reveal divergent developmental pathways in youth

BackgroundNormative modeling of brain development has gained traction for quantifying individual deviations in maturation. The brain age gap (BAG), the difference between predicted age from MRI features and chronological age, offers a potential individualized normative metric of neurodevelopment. However, consistent patterns across psychiatric disorders remain elusive, and no studies have examined whether BAG can predict developmental trajectories within an inclusive continuous model of youths cognition and behavior. MethodsUsing longitudinal data from the Adolescent Brain Cognitive Development Study (ages 9-15, n=9,074), we built 8 region-specific brain age models using volumes, thicknesses, and surface areas of parcels from the Brainnetome adolescent atlas. We derived psychiatric diagnoses from a parental questionnaire. Multivariate linear regression was used to assess case-control differences and cross-sectional continuous cognitive/behavioral profiles. We modeled cognitive/behavioral trajectories using a multivariate joint latent-class mixed model and assessed the relationship with BAG values using multinomial logistic regression. ResultsChildren with ADHD showed delayed maturation across multiple regions (Cohens d: - 0.12 to -0.08), while subcortical BAG emerged as a transdiagnostic indicator of delayed development (d: -0.07, pfdr = 0.024). Accelerated maturation characterized the high cognition and low symptom profile, while the inverse was found for the low cognition profile. Three developmental trajectories were identified: stable, towards externalizing behaviors, or internalizing behaviors. Widespread accelerated maturation predicted evolution towards internalizing behaviors but was protective against the externalizing trajectory. ConclusionsIntegrating BAG with continuous cognitive and behavioral profiles yielded a plausible framework for early identification of atypical trajectories, potentially contributing to personalized medicine in psychiatry.

neuroscience↗

bioRxiv2025

An expanded reference catalog of translated open reading frames for biomedical research

Non-canonical (i.e., unannotated) open reading frames (ncORFs) have until recently been omitted from reference genome annotations, despite evidence of their translation, limiting their incorporation into biomedical research. To address this, in 2022, we initiated the TransCODE consortium and built the first community-driven consensus catalog of human ncORFs, which was openly distributed to the research community via Ensembl-GENCODE. While this catalog represented a starting point for reference ncORF annotation, major technical and scientific issues remained. In particular, this initial catalogue had no standardized framework to judge the evidence of translation for individual ncORFs. Here, we present an expanded and refined catalog of the human reference annotation of ncORFs. By incorporating more datasets and by lifting constraints on ORF length and start-codon, we define a comprehensive set of 28,359 ncORFs that is nearly four times the size of the previous catalog. Furthermore, to aid users who wish to work with ncORFs with the strongest and most reproducible signals of translation, we utilized a data-driven framework (i.e. translation signature scores) to assess the accumulated evidence for any individual ncORF. Using this approach, we derive a subset of 7,888 ncORFs with translation evidence on par with canonical protein-coding genes, which we refer to as the Primary set. This set can serve as a reliable reference for downstream analyses and validation, with a particular emphasis on high quality. Overall, this update reflects continual community-driven efforts to make ncORFs accessible and actionable to the broader research public and further iterations of the catalog will continue to expand and refine this resource.

genomics↗

bioRxiv2025

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

genomics↗

bioRxiv2024

Discovery of diverse chimeric peptides in a eukaryotic proteome sets the stage for the experimental proof of the mosaic translation hypothesis

The high complexity of eukaryotic organisms enabled their evolutionary success, which became possible due to the diversification of eukaryotic proteomes. Various mechanisms contributed to this process. Alternative splicing had the largest known impact among these mechanisms: tens or hundreds of protein isoforms produced from a single genetic locus. Earlier, we hypothesized that along with alternative splicing, a different but conceptually similar mechanism creates novel versions of existing proteins in all eukaryotes. However, this mechanism acts at the level of translation, where the novelty of an amino acid sequence is achieved via multiple programmed ribosomal frameshifting. This mechanism, which is termed mosaic translation, is very difficult to demonstrate even with the most up-to-date molecular tools. Thus, it remained unnoticed so far. Using only a portion of all mass spectrometry proteomic data generated from various organs of the model plant Medicago truncatula, we attempted the first step toward the experimental proof of this hypothesis. Our original in silico approach resulted in the discovery of two candidates for mosaic proteins (homologs of EF1 and RuBisCo) and 154 candidates for chimeric peptides. Chimeric peptides and polypeptides are produced in the course of one ribosomal frameshifting event and may correspond to parts of mosaic proteins. In addition, our analysis reveals the possibility of translation of chimeric peptides from five ribosomal RNA transcripts, ten long non-coding RNA transcripts, and one transfer RNA transcript. These findings are very novel and will be the basis for experimental validation in future studies. In this work, we present multiple lines of indirect evidence that support the validity of our in silico data.

biochemistry↗

bioRxiv2024

A Proximity MAP of RAB GTPases

RAB GTPases are the most abundant family of small GTPases and regulate multiple aspects of membrane trafficking events, from cargo sorting to vesicle budding, transport, docking, and fusion. To regulate these processes, RABs are tightly regulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). Activated RABs recruit effector proteins that regulate trafficking. Identifying RAB-associated proteins has proven to be difficult because their association with interacting proteins is often transient. Recent advances in proximity labeling approaches that allow for the covalent labeling of neighbors of proteins of interest now permit the cataloging of proteins in the vicinity of RAB GTPases. Here, we report APEX2 proximity labeling of 23 human RABs and their neighboring proteomes. We have used bioinformatic analyses to map specific proximal proteins for an extensive array of RAB GTPases, and RAB localization can be inferred from their adjacent proteins. Focusing on specific examples, we identified a physical interaction between RAB25 and DENND6A, which affects cell migration. We also show functional relationships between RAB14 and the EARP complex, or between RAB14 and SHIP164 and its close ortholog UHRF1BP1. Our dataset provides an extensive resource to the community and helps define novel functional connections between RAB GTPases and their neighboring proteins.

cell biology↗

bioRxiv2024

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

molecular biology↗

bioRxiv2024

More is better: A simple antibody-based strategy for recovering all major mouse brain cell types from multiplexed single-cell RNAseq samples.

Single-cell RNA sequencing (scRNAseq) is a powerful yet costly technique for studying cellular diversity within the complexity of organs and tissues. Here, we sought to establish an effective multiplexing strategy for the adult mouse brain that could allow multiple experimental groups to be pooled into a single sample for sequencing, reducing costs, increasing data yield, and eliminating batch effects. We first describe an optimized cold temperature single-cell dissociation protocol that permits isolation of a high yield and viability of brain cells from the adult mouse. Cells isolated using this protocol were then screened by flow cytometry using a panel of antibodies, allowing identification of a single antibody, anti-Thy1.2, that can tag the vast majority of isolated mouse brain cells. We then used this primary antibody against a "universal" neural target, together with secondary antibodies carrying sample-specific oligonucleotides and the BD Rhapsody single-cell system and show that multiple adult mouse brain samples can be pooled into a single multiplexed run for scRNAseq. Bioinformatic analyses enable efficient demultiplexing of the sequenced pooled brain sample, with high tagging efficiency and precise annotation and clustering of brain cell populations. The efficiency and flexibility of the cell dissociation protocol and the two-step multiplexing strategy simplifies experimental design, optimizes reagent usage, eliminates sequencing batch effects and reduces overall experimental costs.

molecular biology↗

bioRxiv2023

Tracking the progression of Alzheimer's disease with peripheral blood monocytes

BackgroundAlzheimers disease (AD) is the most common form of dementia with the symptoms gradually worsening over the years. However, the driving pathological processes occur well before the appearance of symptoms. AD patients display signs of systemic inflammation, suggesting that it could precede the well-established AD hallmarks. We recently showed that the innate immune response in the form of monocyte activation is detectable at the pre-clinical stage. ObjectivesOur goal here is to characterize changes of gene expression in peripheral blood monocytes from patients at different stages of AD progression and validate potential biomarkers for a better prognosis and diagnosis of AD clinical spectrum. ResultsWe performed a whole transcriptome analysis on monocytes purified from healthy subjects, Mild Cognitive Impairment (MCI) and AD patients, and established the list of genes differentially expressed in monocytes during the disease evolution. We observed that, in the top 500 genes differentially expressed, a majority of these genes were upregulated (65%) during AD progression. These genes are mainly involved in chemokine/cytokine-mediated signaling pathways. We further confirmed several biomarkers by quantitative PCR and immunoblotting and showed that they are often deregulated at pre-clinical stages of the disease (MCI stage), supporting the hyperactivation of monocytes in MCI patients. PerspectivesOur findings provide evidence that the pre-clinical stage of AD can be detected in monocytes using a specific set of biomarkers, highlighting the importance to study the early innate immune response in AD. Our results open the possibility to use these biomarkers with different diagnostic methodologies to better predict and efficiently treat AD.

neuroscience↗

bioRxiv2022

Functional characterization of a PHF8 processed pseudogene in the mouse genome

The majority of pseudogenes are generated when an RNA transcript is reverse-transcribed and integrated into the genome at a new location. Pseudogenes are often considered as an imperfect and silent copy of a functional gene because of the accumulation of numerous mutations in their sequence. Here we report the presence of PHF8-ps, a PHF8 retrotransposed pseudogene in the mouse genome, which has no disruptions in its coding sequence. We showed that this pseudogene was specifically transcribed in testis and can produce a Phf8-ps protein in vivo. As Phf8-ps protein has a well-conserved JmjC domain, we characterized its enzymatic activity and showed that Phf8-ps did not have the intrinsic capability to demethylate H3K9me2 in vitro compared to the parental Phf8 protein. Surprisingly, Phf8-ps did not localize in the nucleus like Phf8 but rather was mostly located at the cytoplasm. Finally, our proteomic analysis of Phf8-ps associated proteins revealed that Phf8-ps interacted with mitochondrial proteins but also with prefoldin subunits (PFDN proteins) that deliver unfolded proteins to the cytosolic chaperonin complex implicated the folding of cytosolic proteins. Together, our findings highlighted Phf8-ps as a new pseudogene-derived protein with distinct molecular functions from Phf8.

biochemistry↗

bioRxiv2022

Decoupling of mRNA and protein expression in aging brains reveals the age-dependent adaptation of specific gene subsets

During aging, changes in gene expression are associated with decline in physical and cognitive abilities. Here, we investigated the connection between changes of mRNA and protein expression in the brain by comparing the transcriptome and proteome of the mouse cortex during aging. Our transcriptomic analysis revealed that aging mainly triggers gene activation in the cortex. We showed that increase of mRNA expression correlates with protein expression, specifically in the anterior cingulate cortex where we also observed an increase of cortical thickness during aging. Genes exhibiting an aging-dependent increase of mRNA and protein levels are involved in sensory perception and immune functions. Our proteomic analysis also identified changes in protein abundance in the aging cortex and highlighted a subset of proteins that were differentially enriched but exhibited stable mRNA levels during aging, implying the contribution of aging-related post transcriptional and post-translational mechanisms. These specific genes were associated with general biological processes such as translation, ribosome assembly and protein degradation, but also important brain functions related to neuroplasticity. By decoupling mRNA and protein expression, we have thus characterized distinct subsets of genes that differentially adjust to cellular aging in the cerebral cortex.

biochemistry↗

bioRxiv2021

A community-driven roadmap to advance research on translated open reading frames detected by Ribo-seq

Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational vocabulary of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

genomics↗

bioRxiv2020

Newfound coding potential of transcripts unveils missing members of human protein communities

Recent proteogenomic approaches have led to the discovery that regions of the transcriptome previously annotated as non-coding regions (i.e. UTRs, open reading frames overlapping annotated coding sequences in a different reading frame, and non-coding RNAs) frequently encode proteins (termed alternative proteins). This suggests that previously identified protein-protein interaction networks are partially incomplete since alternative proteins are not present in conventional protein databases. Here we used the proteogenomic resource OpenProt and a combined spectrum- and peptide-centric analysis for the re-analysis of a high throughput human network proteomics dataset thereby revealing the presence of 280 alternative proteins in the network. We found 19 genes encoding both an annotated (reference) and an alternative protein interacting with each other. Of the 136 alternative proteins encoded by pseudogenes, 38 are direct interactors of reference proteins encoded by their respective parental gene. Finally, we experimentally validate several interactions involving alternative proteins. These data improve the blueprints of the human protein-protein interaction network and suggest functional roles for hundreds of alternative proteins.

systems biology↗

bioRxiv2019

FUS gene is dual-coding with both proteins united in molecular hallmarks of amyotrophic lateral sclerosis

Novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame. We discovered an altORF nested in the FUS CDS encoding a conserved 169 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Overexpression of wild-type FUS and/or amyotrophic lateral sclerosis-linked FUS mutants is known to trigger toxic mechanisms in different models. These include an inhibition of autophagy, loss of mitochondrial potential, and accumulation of cytoplasmic aggregates. We show here that altFUS, not FUS, is responsible for the inhibition of autophagy. AltFUS is also pivotal in the mechanisms leading to the mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS-related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet we showed they exert a deleterious effect via their missense consequence on the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggest that both proteins, FUS and altFUS, cooperate in toxic mechanisms.

genetics↗