Quantification of nuclear factors in hMADS adipocytes
Ontology highlight
ABSTRACT: We performed unbiased quantification of nuclear factors in hMADS adipocytes. To do so, we performed subcellular fractionnation of differentiated hMADS cell followed by mass spectrometry analysis of nuclear extracts.
Project description:Analysis of samples with peptides from different species in known amounts to compare performances of data analysis software tools within the ProteoBench platform.
Project description:This study investigated the effect of Vagus Nerve Stimulation (VNS) on innate neuroinflammation and remyelination in lysolecithin (LPC) induced demyelination, a preclinical model for Multiple Sclerosis (MS). In a first experiment (demyelination experiment), LPC was injected in the corpus callosum of 33 Lewis rats, inducing a demyelinated lesion, and rats were treated with either continuously-cycled VNS (cVNS) or one-minute per day VNS (1minVNS) or sham VNS, from two days before the injection until three days post-injection (dpi), when they were killed for immunohistochemistry and proteomics analysis. This timepoint corresponded with a demyelinated lesion and peak inflammation. In a second experiment (remyelination experiment), 13 rats were analogously treated with either cVNS or sham from two days before LPC injection until 11 dpi, when they were killed for tissue prelevation for immunohistochemistry and proteomics. This timepoints corresponded with partial remyelination of the lesion. For proteomics analysis, 20 rats were randomly selected, namely five cVNS and five sham rats of the demyelination experiment, and five cVNS and five sham rats of the remyelination experiment.
Project description:ASB2α is an E3 ubiquitin ligase which is upregulated during Th2 differentiation of naive CD4+ T lymphocyte, and which is known to trigger ubiquitylation and proteasomal degradation of filamins (FLN) a and b. Because E3 ubiquitin ligases often ubiquitylate several substrates, we used an unbiased and broad mass spectrometry approach to identify ASB2α substrates in Th2 lymphocytes generated from naïve CD4+ T lymphocytes of control or ASB2 KO mice.
Project description:In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation-IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes. This study aims to evaluate the effect of CAPA-IVM on equine oocyte quality and developmental competence. Immature cumulus-oocyte complexes (COCs) were retrieved from slaughterhouse ovaries and matured in vitro either in CAPA-IVM (short or long) or standard IVM. Matured oocytes from each group were analyzed for calcium-releasing potential and single-oocyte proteomics, and embryo development was assessed after fertilization with Piezo-drilled intracytoplasmic sperm injection (ICSI). Genetic analysis of developed blastocysts was performed to detect chromosomal aberrations. Our findings demonstrate that CAPA-IVM of equine COCs yields significantly higher maturation rates than controls. Moreover, short CAPA-IVM with six hours pre-maturation culture showed substantially higher embryo development potential than the control group. Genetic analysis revealed a high euploidy rate in equine blastocysts regardless of the maturation conditions. Live calcium imaging of the fertilized oocytes demonstrated the majority of oocytes with non-continuous calcium oscillation patterns, irrespective of maturation conditions. Single oocyte proteomics reveals a comparable proteomic landscape between matured oocytes from short CAPA-IVM and standard IVM. However, a trend of differential expression was observed in specific proteins related to cytoskeleton, cell cycle, and hemostasis in the short CAPA-IVM group. Our findings indicate that CAPA-IVM holds the potential to improve oocyte quality and competence in horses. However, further fine-tuning of culture conditions based on omics analysis would benefit the effective use of these IVM systems. Moreover, given that the mare serves as an excellent model for human reproduction, the molecular trends identified in this study could provide valuable insights for advancing human artificial reproductive technologies.
Project description:Atherosclerosis is an important pathological factor in the development of cardiovascular diseases. In addition to increased plasma lipid concentrations, irregular/oscillatory shear stress and inflammatory processes trigger pathophysiological changes. Inhibitors of the transcription modulatory bromo- and extra-terminal domain (BET) protein family (BETi) could offer a possible therapeutic approach due to their anti-inflammatory properties. In this study, the influence of laminar shear stress, inflammation and BETi on human endothelial cells in an atherosclerosis in vitro model was investigated using global protein expression profiling. For this purpose, human umbilical cord derived vascular endothelial cells (HUVEC) were treated with TNFα to mimic the inflammatory condition and were exposed to 24h laminar shear stress in the presence or absence of a BRD4 inhibitor, JQ1. Data-independent acquisition mass spectrometry (DIA-MS) alloqed us to quantify 3316 proteins for further statistical analysis. Differentially regulated proteins indicate a clear influence of inflammation and shear stress on human endothelial cells. Overall, application of JQ1 is led to significant changes in the proteome, including a strong anti-inflammatory response as well as a potentially negative impact on atherosclerosis formation. To our knowledge, this is the first proteomics study on HUVEC which investigates the influence of shear stress and BET inhibition in TNFα inflammatory endothelial cell culture model.
Project description:Metronidazole represents a front line-drug against Helicobacter pylori. To unravel its largely unknown cellular targets, we performed activity-based protein profiling (ABPP). Surprisingly, an alkynylated ether probe (Metro-P3) exhibited a 60-fold enhanced potency. Chaperonin HpGroEL and thiol peroxidase HpTpx were identified as prominent Metro-P3 targets, the latter of which is essential for H. pylori survival under oxidative stress. Biological assays validated HpTpx inhibition as the origin of the activity boost, demonstrated by covalent modification of the catalytic cysteine and co-crystallization. Metro-P3 and refined ether analogs exhibited favorable pharmacological profiles with no cytotoxicity. The activity boost translated to an in vivo H. pylori mouse model demonstrating full eradication at 50-fold reduced dosing compared to metronidazole triple therapy. Our results highlight 5-nitroimidazole ethers as dual mode of action antibiotics which induce oxidative stress and inhibit the stress response leading to bacterial death.
Project description:Platelets, traditionally recognized for their involvement in hemostasis and wound healing, also play a central role in immune regulation and inflammation. Their function and production adapt in response to inflammatory cues such as cytokines and danger-associated molecular patterns. Interleukin-33 (IL-33), an alarmin released during tissue damage, particularly in lung inflammation, has been implicated in influencing platelet biology, though its exact effects remain poorly understood. To clarify IL-33’s role, we examined its impact on platelet proteome. We first compared the proteome of platelets purified by FACS from IL-33-deficient (IL-33KO) mice versus WT mice (n=5 biological replicates in each group, 10 raw MS files). We also compared the proteome of platelets from WT mice which were either non stimulated (n= 8 biological replicates) or treated intranasally with recombinant IL-33 (n= 9 biological replicates).
Project description:We designed an experimental setup to investigate the transcriptomic and proteomic responses of the hyperthermophilic archaeon Pyrococcus furiosus to heat and cold shock. P. furiosus is a model organism for studying microbial adaptation to extreme environments, including deep-sea hydrothermal vents with temperature gradients ranging from 1°C to 400°C. We aimed to simulate critical conditions where P. furiosus cannot grow and to examine the immediate response to thermal stress as well as the recovery process.
Project description:Approaches for the discovery of molecular glues remain limited. Here we report a phenotypic screening approach in which cytotoxins whose mechanisms require ubiquitination show gain of viability following pharmacological inhibition of the Ubiquitin-life modifier activating enzyme (UBA1/UAE). This approach revealed PRLX-93936 and BMS-214662 as molecular glues that directly target the E3 ligase TRIM21 to induce degradation of nucleoporin proteins and inhibit nuclear trafficking. The cytotoxicity of these agents correlates strongly with TRIM21 expression, suggesting re-evaluation of these clinically-tested agents in patients with TRIM21-high cancers. Relative to recently-disclosed TRIM21-targeting glues, PRLX-93936 and newly-synthesized analogs represent a distinct structural series, lack known cellular off-targets, and offer greatly enhanced potency. Additionally, we have elaborated PRLX-93936 to the first ‘TRIMTAC’ that uses wild-type TRIM21 to degrade a multimeric protein. Together our work creates new opportunities for targeted protein degradation and enables the design of additional TRIM21-targeting glues and TRIMTACs.
Project description:Spatial tissue proteomics combining microscopy-based cell phenotyping with ultrasensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell-type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultrasensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research.