Fine-tuning levels of filamins a and b as a specific mechanism sustaining Th2 lymphocyte functions.

Augmenting the portfolio of therapeutics for type 2-driven diseases is crucial to address unmet clinical needs and to design personalized treatment schemes. An attractive therapy for such diseases would consist in targeting the recruitment of T helper 2 (Th2) lymphocytes to inflammatory sites. Herein, we show the degradation of filamins (FLN) a and b by the ASB2α E3 ubiquitin ligase as a mechanism sustaining Th2 lymphocyte functions.

Cerebrospinal fluid proteomic profile of frailty: Results from the PROLIPHYC cohort.

Frailty is a clinical state reflecting a decrease in physiological reserve capacities, known to affect numerous biological pathways and is associated with health issues, including neurodegenerative diseases. However, how global protein expression is affected in the central nervous system in frail subject remains underexplored. In this post hoc cross-sectional biomarker analysis, we included 90 adults (52-85 years) suspected of normal pressure hydrocephalus (NPH) and presenting with markers of neurodegenerative diseases.

TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation.

Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9.

Cerebrospinal fluid proteomics in recent-onset Narcolepsy type 1 reveals activation of the complement system.

Introduction: Narcolepsy type 1 (NT1) is a rare, chronic and disabling neurological disease causing excessive daytime sleepiness and cataplexy. NT1 is characterized pathologically by an almost complete loss of neurons producing the orexin neuropeptides in the lateral hypothalamus. Genetic and environmental factors strongly suggest the involvement of the immune system in the loss of orexin neurons.

Kinetic proofreading through the multi-step activation of the ZAP70 kinase underlies early T cell ligand discrimination.

T cells recognize a few high-affinity antigens among a vast array of lower affinity antigens. According to the kinetic proofreading model, antigen discrimination properties could be explained by the gradual amplification of small differences in binding affinities as the signal is transduced downstream of the T cell receptor. Which early molecular events are affected by ligand affinity, and how, has not been fully resolved.

Cis interactions between CD2 and its ligands on T cells are required for T cell activation.

CD2 is largely described to promote T cell activation when engaged by its ligands, CD48 in mice and CD58 in humans, that are present on antigen-presenting cells (APCs). However, both CD48 and CD58 are also expressed on T cells. By generating new knockout mouse strains lacking CD2 or CD48 in the C57BL/6 background, we determined that whereas CD2 was necessary on T cells for T cell activation, its ligand CD48 was not required on APCs.

IL-33 acts as a costimulatory signal to generate alloreactive Th1 cells in graft-versus-host disease.

Antigen-presenting cells (APCs) integrate signals emanating from local pathology and program appropriate T cell responses. In allogeneic hematopoietic stem cell transplantation (alloHCT), recipient conditioning releases damage-associated molecular patterns (DAMPs) that generate proinflammatory APCs that secrete IL-12, which is a driver of donor Th1 responses, causing graft-versus-host disease (GVHD). Nevertheless, other mechanisms exist to initiate alloreactive T cell responses, as recipients with disrupted DAMP signaling or lacking IL-12 develop GVHD.

A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics.

In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design.

ProMetIS, deep phenotyping of mouse models by combined proteomics and metabolomics analysis.

Genes are pleiotropic and getting a better knowledge of their function requires a comprehensive characterization of their mutants. Here, we generated multi-level data combining phenomic, proteomic and metabolomic acquisitions from plasma and liver tissues of two C57BL/6 N mouse models lacking the Lat (linker for activation of T cells) and the Mx2 (MX dynamin-like GTPase 2) genes, respectively. Our dataset consists of 9 assays (1 preclinical, 2 proteomics and 6 metabolomics) generated with a fully non-targeted and standardized approach.

The T cell CD6 receptor operates a multitask signalosome with opposite functions in T cell activation.

To determine the respective contribution of the LAT transmembrane adaptor and CD5 and CD6 transmembrane receptors to early TCR signal propagation, diversification, and termination, we describe a CRISPR/Cas9-based platform that uses primary mouse T cells and permits establishment of the composition of their LAT, CD5, and CD6 signalosomes in only 4 mo using quantitative mass spectrometry. We confirmed that positive and negative functions can be solely assigned to the LAT and CD5 signalosomes, respectively. In contrast, the TCR-inducible CD6 signalosome comprised both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation.

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN2 as a regulator of effector functions.

T-cell receptor (TCR) ligation-mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time-resolved high-resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T-cell activation.

Proline: an efficient and user-friendly software suite for large-scale proteomics.

Motivation: The proteomics field requires the production and publication of reliable mass spectrometry-based identification and quantification results. Although many tools or algorithms exist, very few consider the importance of combining, in a unique software environment, efficient processing algorithms and a data management system to process and curate hundreds of datasets associated with a single proteomics study.

Results: Here, we present Proline, a robust software suite for analysis of MS-based proteomics data, which collects, processes and allows visualization and publication of proteomics datasets.

Resistance of melanoma to immune checkpoint inhibitors is overcome by targeting the sphingosine kinase-1.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment. Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment. Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity.

Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4 T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement.

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression.

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1.

Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells.

The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity.

CD5, an Undercover Regulator of TCR Signaling.

T cells are critical components of adaptive immunity. As such, their activation is regulated by the T cell receptor (TCR) that constantly scan peptides associated with major histocompatibility complexes (MHC). TCR engagement initiates a series of molecular events leading to cytokine secretion, proliferation, and differentiation of T cells.

The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4 T cells.

The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in which a tag for affinity purification was attached to endogenous VAV1 molecules, we analyzed by quantitative mass spectrometry the signaling complex that assembles around activated VAV1. Fifty VAV1-binding partners were identified, most of which had not been previously reported to participate in VAV1 signaling.

Environmental allergens induce allergic inflammation through proteolytic maturation of IL-33.

Allergic inflammation has crucial roles in allergic diseases such as asthma. It is therefore important to understand why and how the immune system responds to allergens. Here we found that full-length interleukin 33 (IL-33), an alarmin cytokine with critical roles in type 2 immunity and asthma, functioned as a protease sensor that detected proteolytic activities associated with various environmental allergens across four kingdoms, including fungi, house dust mites, bacteria and pollens.

Asb2α-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development.

Rationale: Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation.

Objective: The Asb2α (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins).

Proteomic Analysis of Regulatory T Cells Reveals the Importance of Themis1 in the Control of Their Suppressive Function.

Regulatory T cells (Treg) represent a minor subpopulation of T lymphocytes that is crucial for the maintenance of immune homeostasis. Here, we present a large-scale quantitative mass spectrometry study that defines a specific proteomic "signature" of Treg. Treg and conventional T lymphocyte (Tconv) subpopulations were sorted by flow cytometry and subjected to global proteomic analysis by single-run nanoLC-MS/MS on a fast-sequencing Q-Exactive mass spectrometer.

Identification of a Tissue-Restricted Isoform of SIRT1 Defines a Regulatory Domain that Encodes Specificity.

The conserved NAD-dependent deacylase SIRT1 plays pivotal, sometimes contrasting, roles in diverse physiological and pathophysiological conditions. In this study, we uncover a tissue-restricted isoform of SIRT1 (SIRT1-ΔE2) that lacks exon 2 (E2). Candidate-based screening of SIRT1 substrates demonstrated that the domain encoded by this exon plays a key role in specifying SIRT1 protein-protein interactions.