Project description:CD8+ T cells in chronic viral infections like HIV develop functional defects such as loss of IL-2 secretion and decreased proliferative potential that are collectively termed exhaustion1. Exhausted T cells express increased levels of multiple inhibitory receptors, such as Programmed Death 1 (PD-1). PD-1 inhibition contributes to impaired virus-specific T cell function in chronic infection because antibody-mediated blockade of its ligand, Programmed Death Ligand 1 (PD-L1) is sufficient to improve T cell function and reduce viral replication in animal models. Reversing PD-1 inhibition is therefore an attractive therapeutic target, but the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling. It is not known whether PD-1 ligation also acts by upregulating genes in exhausted T cells that impair their function. Here, we analyzed gene-expression profiles from HIV-specific CD8+ T cells in patients with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8+ T cells from humans and mice. This program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, while BATF knockdown reduced PD-1 inhibition. Silencing BATF in CD4+ and CD8+ T cells from chronic viremic patients rescued HIV-specific T cell function. Thus inhibitory receptors can cause T cell exhaustion by upregulating genes – such as BATF – that inhibit T cell function. PD-1 expressing Jurkat cells were cultured for 18 hours with beads coated with antibodies to CD3 and CD28, with our without an antibody to PD-1.
Project description:This SuperSeries is composed of the following subset Series: GSE24026: Comparison of gene expression profiles of Jurkat cells with or without PD-1 ligation GSE24081: Comparison of gene expression profiles of HIV-specific CD8 T cells from controllers and progressors Refer to individual Series
Project description:Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach (available at: https://github.com/mpc-bioinformatics/FiPSPi), identified a discriminative peptide panel, and confirmed our panel using a public data set.
Project description:T-cell receptor (TCR) signaling is essential for the function of T cells. Here we combine mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the composition and dynamics of the signaling complexes that assemble around 15 nodes of the TCR signaling cascade of primary CD4+ T cells. This dataset contains the experiments performed with 14 different bait proteins • Cbl • Cblb • Fyb • Inpp5d • Itk • Lck • Lcp2 • Nck1 • Nfatc2 • Plcg1 • Ptpn22 • Ptpn6 • Themis • Vav1 Each of them contains mass spectrometry results from the analysis of AP-MS experiments, based on the endogenous expression of One-Strep-tagged (OST) bait proteins in engineered mice models, and affinity purification of these proteins from primary CD4+ T cells, using Streptactin beads. Purification of OST proteins was performed at 5 different time points of stimulation of CD4+ T cells with anti-CD3 and anti-CD4 antibodies (0s; 30s; 120s; 300s; 600s). Each AP-MS purification of an OST- protein is associated with a corresponding control (purification from WT CD4+ T cells) at the same time point of stimulation. Several biological replicate experiments (time course OST series + associated WT controls) were performed for each bait. Several MS replicates were acquired for each sample. In addition, we analyzed the total proteome of CD4+ T cells isolated from each engineered mice model (14 different mice expressing an OST bait) and from WT mice, in order to calculate copy numbers of the baits and their associated proteins.
Project description:The metabotropic glutamate receptor 5 (mGluR5) is a G-protein coupled receptor with an important role in synaptic function at multiple levels. In physiological conditions, mGluR5 is an essential modulator of synaptic plasticity, learning and memory; whereas in pathological conditions, it is an acknowledged therapeutic target that has been implicated in multiple brain disorders. However, the underlying mechanisms by which mGluR5 acts in these diverse synaptic functions and processes is only partly understood. In this study, we dissected the molecular synaptic modulation mediated by mGluR5 using genetic and pharmacological mouse models to chronically and acutely reduce mGluR5 activity. Using SWATH proteomic analysis, we found that next to dysregulation of synaptic proteins, the major regulation in protein expression in both models concerned specific processes in mitochondria, such as oxidative phosphorylation. Second, we observed morphological alterations in shape and area of specifically postsynaptic mitochondria in mGluR5 KO synapses using electron microscopy. Third, computational and biochemical assays suggested an increase of mitochondrial function in neurons, with increased level of NADP/H and oxidative damage in mGluR5 KO. Altogether, our observations provide diverse lines of evidence of the modulation of synaptic mitochondrial function by mGluR5. This connection suggests a role for mGluR5 as a mediator between synaptic activity and mitochondrial function, a finding which might be a relevant for the improvement of the clinical potential of mGluR5.
Project description:This SuperSeries is composed of the following subset Series: GSE40512: Gene expression profile of human T-ALL cell line KOPTK1 treated with vehicle or PD 0332991 GSE40513: Gene expression profile of mouse breast cancer V720 cells treated with vehicle or PD 0332991 Refer to individual Series
Project description:Label-free absolute quantitative proteomics is commonly used for absolute quantification of the proteome or specific proteins of interest in various biological samples. Current label-free absolute protein quantification (APQ) methods determine MS1 intensities, MS2 spectral counts or intensities to absolutely quantify protein concentrations from data obtained from data-dependent acquisition (DDA). In recent years, label-free data-independent acquisition (DIA) has seen increasing use as a powerful tool for relative protein quantification. Here we present a novel label-free DIA-based absolute protein quantification (DIA-APQ) method for the absolute quantification of protein expressions from DIA data. To validate this method, both DDA and DIA experiments were performed on 36 individual human liver microsome and S9 samples. The DIA-APQ assay was able to quantify approximately twice as many proteins as the DDA MS1-based APQ method whereas protein concentrations determined by the two methods were comparable. To evaluate the accuracy of the DIA-APQ method, we absolutely quantified carboxylesterase 1 concentrations in human liver samples using an established SILAC internal standard-based proteomic assay; the SILAC results were consistent with those obtained from DIA-APQ analysis. Finally, we employed a unique algorithm in DIA-APQ to distribute the MS signals from shared peptides to different protein isoforms and successfully applied the DIA-APQ method to the absolute quantification of several drug-metabolizing enzyme isoforms in human liver microsomes. This novel DIA-based APQ method not only provides a powerful approach for absolutely quantifying entire proteomes and specific candidate proteins, but also has with the capacity differentiating protein isoforms.