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Pierre Thibault

Publications and source records attributed to Pierre Thibault.

At least 19 recordsLinked to original sources
PubMed2026

Mice with the mono-allelic p.R37H Dhdds variant show aberrant glycosylation and interneuron deficits.

Developmental delay and seizures with or without movement abnormalities (OMIM 617836) caused by heterozygous pathogenic variants in the DHDDS gene (DHDDS-CDG) is a rare genetic disease that belongs to the progressive encephalopathy spectrum. It results in cognitive delay in affected children, accompanied by myoclonus, seizures, ataxia and tremor, which worsens over time. DHDDS encodes a subunit of a DHDDS/NUS1 cis-prenyltransferase (cis-PTase), a branch point enzyme of the mevalonate pathway essential for N-linked glycosylation. We describe the first mouse model of this disease, DhddsR37H+/- strain, heterozygous for the human recurrent de novo c.110G>A:p.R37H pathogenic variant. DhddsR37H+/- mice present with seizures, myoclonus and memory deficits associated with reduced density or/and maturity of inhibitory interneurons in the cortex. Multiomics analyses of mouse CNS tissues, together with the enzymatic/structural characterization of the R37H DHDDS mutant protein, reveal that the variant produces a catalytically inactive enzyme and results in a brain dolichol deficit, aberrant glycosylation of brain glycoproteins, including those involved in synaptic transmission and major perturbations in the CNS proteome and lipidome. Acetazolamide, a carbonic anhydrase inhibitor clinically approved for treatment of glaucoma, epilepsy, and intracranial hypertension, and successfully used "off-label" to treat genetic movement disorders, reduces seizure susceptibility to pentylenetetrazol in DhddsR37H+/- mice, suggesting potential therapeutic value of using this drug in human DHDDS-CDG patients. Together, our results define cis-PTase as a master regulator of CNS development and function and establish that its monoallelic debilitating variants cause a novel congenital disorder of glycosylation associated with aberrant levels of neuronal proteins and lipids.

DHDDS↗
PubMed2026

Noncanonical Transcription and Splicing Shape the Colorectal Cancer Immunopeptidome in MSI and MSS Tumors.

Treatment with immune checkpoint inhibitors in colorectal cancer (CRC) has largely benefited patients with microsatellite instability-high (MSI-H) and not the larger proportion of patient with microsatellite-stable (MSS) tumors. This clinical dichotomy has fueled the view that high mutational burden is the dominant driver of tumor immunogenicity and that MSS CRC fails to respond because it is "antigen poor". To directly test this premise and define the origins of presented tumor antigens, we integrated HLA class I immunopeptidomics and matched RNA-seq from 26 primary CRC tumors spanning MSI-H and MSS subtypes. Using patient-specific canonical and cancer-specific proteogenomic databases, we identified 115,292 unique major histocompatibility complex (MHC)-associated peptides (MAPs) across 61 HLA alleles, with a mean of 9292 MAPs per tumor and no significant difference in MAP counts between MSI-H and MSS tumors. In toto, we identified 266 tumor antigens, all coded by unmutated genomic sequences, comprising 70 aberrantly expressed tumor-specific antigens (aeTSAs) and 196 tumor-associated antigens (TAAs). In our cohort, MSS tumors presented more TAAs and a comparable number of aeTSAs per tumor relative to MSI-H tumors. In TCGA-COAD stratified analyses (483 tumors), MSS tumors yielded more presentable aeTSAs and TAAs per patient than MSI-H tumors. Across both subtypes, aeTSAs arose predominantly from intronic translation, UTR usage, retroelement activation, and germline-like transcription, including recurrent aeTSAs from PIWIL1, L1TD1, and endogenous retroviral loci. Together, these data demonstrate that MSS CRC is not antigen poor and highlight noncanonical translation as a major, previously underappreciated contributor to the CRC immunopeptidome.

Humans↗
PubMed2025

Studying Mitotic Phosphorylation in Drosophila.

Mitosis is largely controlled by the reversible phosphorylation of effector proteins. The addition or removal of phosphate groups alters the activities of these proteins, resulting in changes in chromosome structure, cytoskeletal dynamics, nuclear envelope integrity, and other transformations that must occur as a cell progresses through mitosis. Drosophila has been instrumental in the elucidation of the molecular mechanisms of mitosis, which are mostly conserved among animals. In this model system, sophisticated genetic tools can be used to study mitosis in different tissues during development in vivo. Drosophila cell culture affords complementary possibilities. In this chapter, we present a phosphoproteomic protocol using Drosophila cell culture to identify phosphorylation sites that depend on mitotic kinases and phosphatases. We also provide protocols to dissect the roles of the identified sites in the regulation of protein interactions and localization during mitosis, using Drosophila embryos. We emphasize the advantages of the selected methods compared to possible alternatives in Drosophila or in other systems.

Animals↗
PubMed2007

aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb.

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.

Amino Acid Sequence↗
PubMed2006

Reconstruction of a yeast cell from X-ray diffraction data.

Details are provided of the algorithm used for the reconstruction of yeast cell images in the recent demonstration of diffraction microscopy by Shapiro, Thibault, Beetz, Elser, Howells, Jacobsen, Kirz, Lima, Miao, Nieman & Sayre [Proc. Natl Acad. Sci. USA (2005), 102, 15343-15346]. Two refinements of the iterative constraint-based scheme are developed to address the current experimental realities of this imaging technique, which include missing central data and noise. A constrained power operator is defined whose eigenmodes allow the identification of a small number of degrees of freedom in the reconstruction that are negligibly constrained as a result of the missing data. To achieve reproducibility in the algorithm's output, a special intervention is required for these modes. Weak incompatibility of the constraints caused by noise in both direct and Fourier space leads to residual phase fluctuations. This problem is addressed by supplementing the algorithm with an averaging method. The effect of averaging may be interpreted in terms of an effective modulation transfer function, as used in optics, to quantify the resolution. The reconstruction details are prefaced with simulations of wave propagation through a model yeast cell. These show that the yeast cell is a strong-phase-contrast object for the conditions in the experiment.

Algorithms↗
PubMed2006

ETO2 coordinates cellular proliferation and differentiation during erythropoiesis.

The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix-loop-helix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroid-specific TAL-1 target genes and of Gfi-1b and p21(Cip), encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation.

Animals↗
PubMed2006

Integrated microfluidic devices with enhanced separation performance: application to phosphoproteome analyses of differentiated cell model systems.

This work reports on the application of a microfluidic device integrating nanoscale LC to nanoelectrospray MS (nano-LC-chip-MS) for the analysis of complex protein digests. Peak profile analyses of more than 700 peptide ions, reproducibly detected across replicate nano-LC-chip-MS runs (n = 5), indicated that the system provided RSD values of 0.24% on retention time, +/- 30 ppm on m/z measurement and +/- 30% variation on intensity over three orders of magnitude. RP adsorbant media with different alkyl chains and particle size packed in both trapping and separation channels were investigated to improve the chromatographic performance of this system. A two-fold improvement in chromatographic peak capacity was achieved using microfluidic devices comprising a 5 mircrom C3 trap with 2.5 microm C18 trap separation channel compared to the traditional 5 microm C18 stationary phase. Enhanced sample selectivity for the identification of phosphopeptides was obtained by combining immobilized metal affinity media prior to peptide separation on the RP microfluidic device. This system was evaluated in the context of differential phosphoproteome analyses to identify changes in signaling events and protein expression of human monocytes following the administration of phorbol ester.

Animals↗
PubMed2006

Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence.

Analysis of the complete flagellin glycosylation locus of Campylobacter jejuni strain 81-176 revealed a less complex genomic organization than the corresponding region in the genome strain, C. jejuni NCTC 11168. Twenty-four of the 45 genes found between Cj1293 and Cj1337 in NCTC 11168 are missing in 81-176. Mutation of six new genes, in addition to three previously reported, resulted in a non-motile phenotype, consistent with a role in synthesis of pseudaminic acid (PseAc) or transfer of PseAc to flagellin. Mutation of Cj1316c or pseA had been shown to result in loss of the acetamidino form of pseudaminic acid (PseAm). Mutation of a second gene also resulted in loss of PseAm, as well as a minor modification that appears to be PseAm extended with N-acetyl-glutamic acid. Previously described mutants in C. jejuni 81-176 and Campylobacter coli VC167 that produced flagella lacking PseAm or PseAc failed to autoagglutinate. This suggests that interactions between modifications on adjacent flagella filaments are required for autoagglutination. Mutants (81-176) defective in autoagglutination showed a modest reduction in adherence and invasion of INT407 cells. However, there was a qualitative difference in binding patterns to INT407 cells using GFP-labelled 81-176 and mutants lacking PseAm. A mutant lacking PseAm was attenuated in the ferret diarrhoeal disease model.

Agglutination↗
PubMed2005

Identification of unusual bacterial glycosylation by tandem mass spectrometry analyses of intact proteins.

The characterization of protein glycosylation can be a complex and time-consuming procedure, especially for prokaryote O-linked glycoproteins, which often comprise unusual oligosaccharide structures with no known glycosylation motif. In this report, we describe a "top-down" approach that provides information on the extent of glycosylation, the molecular masses, and the structure of oligosaccharide residues on bacterial flagella, important structural proteins involved in the motility of pathogenic bacteria. Flagella from four bacterial pathogens, namely, Campylobacter jejuni, Helicobacter pylori, Aeromonas caviae, and Listeria monocytogenes, were analyzed by this top-down mass spectrometry approach. The approach needs minimal sample preparation and can be performed within a few minutes compared to the tedious and often time-consuming "bottom-up" approach involving proteolytic digestion and LC-MS-MS analyses of the suspected glycopeptides. Multiply protonated protein precursor ions subjected to low-energy collisional activation in a quadrupole time-of-flight instrument showed extensive and specific gas-phase deglycosylation resulting in the formation of abundant oxonium ions with very few fragment ions from peptidic bond cleavages. Structural information on individual carbohydrate residues is obtained using a second-generation product ion scan of oxonium ions formed by collisional activation of the intact protein ions in the source region. The four bacterial flagella examined differed not only by the extent of glycosylation but also by the nature of carbohydrate substituents. For example, the flagellin from the Gram-positive bacterium, L. monocytogenes showed O-linked GlcNAc residues at up to 6 sites/protein monomer. In contrast, the three Gram-negative bacterial pathogens C. jejuni, H. pylori and A. caviae displayed up to 19 Ser/Thr O-linked sites modified with residues structurally related to N-acetylpseudaminic acid (Pse5Ac7Ac) and in the case of Campylobacter include a novel N-acetylglutamine substituent on Pse5Am7Ac.

Flagellin↗
PubMed2005

Organ messenger ribonucleic acid and plasma proteome changes in the adjuvant-induced arthritis model: responses to disease induction and therapy with the estrogen receptor-beta selective agonist ERB-041.

Two receptors [estrogen receptor (ER)alpha and ERbeta] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ERalpha in the classical effects of estrogen activity, the physiological role of ERbeta is less well understood. A small-molecule ERbeta selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.

Animals↗
PubMed2005

Biological imaging by soft x-ray diffraction microscopy.

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.

Freeze Drying↗
PubMed2005

Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein.

Oligodendrocytes (OLs) extend arborized processes that are supported by microtubules (MTs) and microfilaments. Little is known about proteins that modulate and interact with the cytoskeleton during myelination. Several lines of evidence suggest a role for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in mediating process formation in OLs. In this study, we report that tubulin is a major CNP-interacting protein. In vitro, CNP binds preferentially to tubulin heterodimers compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces dramatic morphology changes in both glial and nonglial cells, resulting in MT and F-actin reorganization and formation of branched processes. These morphological effects are attributed to CNP MT assembly activity; branched process formation is either substantially reduced or abolished with the expression of loss-of-function mutants. Accordingly, cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. We propose that CNP is an important component of the cytoskeletal machinery that directs process outgrowth in OLs.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2005

Improvement in peptide detection for proteomics analyses using NanoLC-MS and high-field asymmetry waveform ion mobility mass spectrometry.

Sensitive and selective detection of multiply charged peptide ions from complex tryptic digests was achieved using high-field asymmetric waveform ion mobility spectrometry (FAIMS) combined with nanoscale liquid chromatography-mass spectrometry (nanoLC-FAIMS-MS). The combination of FAIMS provided a marked advantage over conventional nanoLC-MS experiments by reducing the extent of chemical noise associated with singly charged ions and enhancing the overall population of detectable tryptic peptides. Such advantages were evidenced by a 6-12-fold improvement in signal-to-noise ratio measurements for a wide range of multiply charged peptide ions. An increase of 20% in the number of detected peptides compared to conventional nanoelectrospray was achieved by transmitting ions of different mobilities at high electric field vs low field while simultaneously recording each ion population in separate mass spectrometry acquisition channels. This method provided excellent reproducibility across replicate nanoLC-FAIMS-MS runs with more than 90% of all detected peptide ions showing less than 30% variation in intensity. The application of this technique in the context of proteomics research is demonstrated for the identification of trace-level proteins showing differential expression in U937 monocyte cell extracts following incubation with phorbol ester.

Animals↗
PubMed2005

Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs.

The present investigation describes the analytical performances of a microfluidic device comprising an enrichment column, a reversed-phase separation channel, and a nanoelectrospray emitter embedded altogether in polyimide layers. This configuration minimizes transfer lines and connections and reduces postcolumn peak broadening and dead volumes. This compact and versatile modular nanoLC-chip system was interfaced to both ion trap and time-of-flight mass spectrometers, and its analytical potentials were evaluated in the context of proteomics applications. The figures of merit of this system in terms of peak capacity, reproducibility, sensitivity, and linear dynamic range of peptide detection were determined using tryptic digests of complex protein extracts including albumin- and immunoglobulin-depleted rat plasma samples. The analysis of peak profiles for more than 600 peptide ions reproducibly detected across replicate nanoLC-chip-MS runs (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity with RSD values of less than 0.5 and 9.1%, respectively. Variation in peptide abundance as low as 2-fold changes was identified for spiked tryptic digests present at levels of 2-5 fmol in plasma samples. Sensitivity measurements were performed on dilution series of protein digests spiked into rat plasma samples and provided a detection limit of 1-5 fmol. The modular concept of the microfluidic systems also facilitated the integration of two-dimensional chromatography (strong cation exchange/C18) thereby increasing the sample loading and selectivity of the nanoLC-chip-MS system. The application of this integrated device was evaluated for complex rat plasma samples to compare the number of protein identifications obtained using one- and two-dimensional nanoLC-chip-MS/MS.

Biomarkers↗
PubMed2005

Multiplex multidimensional nanoLC-MS system for targeted proteomic analyses.

The present work describes a dual-column and dual-sprayer LC-MS system for high-throughput proteomic analyses. This system consists of two precolumns for sample desalting and two analytical columns. Each column is terminated by a nanoelectrospray emitter mounted on a robotic arm enabling their sequential positioning in front of the sampling cone of the mass spectrometer. The effluent from each emitter is recorded in separate acquisition channels without detectable crosstalk. Gradient elution to both nanoLC columns is delivered by a single HPLC system via a flow splitter. The reproducibility of retention time and peak intensity of the present multiplex system were comparable to those obtainable using a single emitter configuration. Replicate injections of complex tryptic digests (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity on both columns with RSD values of less than 0.9 and 18.6%, respectively. The application of this system is demonstrated for the monitoring of protein expression changes in U937 human monocyte cells with and without phorbol ester administration. Furthermore, we also demonstrated the use of this multiplex system in a 2-D LC configuration to increase sample loading and throughput for the analysis of biomarker samples of higher complexity. Variations in peptide abundance down to two-fold change were identified across salt fractions for spiked tryptic digests present at a level of 50 fmol in 1.5 microg of plasma samples.

Animals↗
PubMed2004

Diffractive imaging of nonperiodic materials with future coherent X-ray sources.

Coherent diffractive imaging using a coherent X-ray source promises to be a useful microscopic method for imaging noncrystalline objects at high spatial resolution. In this article a simple method to estimate the coherently scattered signal as a function of resolution is presented, and it is shown that the required X-ray flux or dose scales as the inverse third power of resolution for a specimen of constant volume and density. A simulated case study using the proposed energy-recovery linac source is also presented, which confirms the estimated flux requirement.

Journal Article↗
PubMed2004

Probing genetic variation and glycoform distribution in lectins of the Erythrina genus by mass spectrometry.

Six leguminous lectins from the seeds of plants of the Erythrina genus, namely E. caffra (ECafL), E. cristagalli (ECL), E. flabelliformis (EFL), E. lysistemon (ELysL), E. rubrinerva (ERL), and E. vespertilio (EVL), were examined to establish their sequence homology and to determine the structure and sites of attachment of their glycans. Tryptic digests of these lectins were analyzed by capillary electrophoresis coupled to electrospray mass spectrometry (CE-ESMS). Assignments were made by comparing the molecular masses of the observed tryptic peptides with those of Erythrina corallodendron lectin (ECorL), the sequence of which had been established previously. Glycan structure and genetic variations in the amino acid sequence were probed by tandem mass spectrometry. Small differences were found between the sequences of the various lectins examined and all of them exhibited C-terminal processing resulting in proteins with a C-terminal Asn residue. The major glycan of these glycoproteins was shown to be the heptasaccharide Man(3)XylFucGlcNAc(2), consistent with previous investigations on ECL and ECorL. A minor glycan heterogeneity was observed for most lectins examined except for that of ECafL and ECorL where an extra hexose residue was observed on the reducing GlcNAc residue of the heptasaccharide.

Amino Acid Sequence↗