NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells.

Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease.

High-Throughput Cellular RNA Sequencing (HiCAR-Seq): Cost-Effective, High-Throughput 3' mRNA-Seq Method Enabling Individual Sample Quality Control.

High-throughput screening is one of the pillars of drug development. Unbiased transcriptome profiling is now widely used for a deeper understanding of a drug's mechanisms of action, off target effects, and cytotoxicity. Although currently available high-throughput RNA-Seq (HT RNA-Seq) methods such as PLATE-Seq, DRUG-Seq, and BRB-Seq serve these purposes, the inherent nature of these methods does not allow sample-wise sequencing library quality control. Here, we describe an HTR method called High-throughput CellulAr RNA Sequencing (HiCAR-Seq). HiCAR-Seq was optimized to work directly on cultured cells (as little as 1,000 cells) or 10 ng of total RNA. HiCAR-Seq involves reverse transcription from cultured cells or total RNA using oligo-dT primers followed by the PCR amplification of full-length cDNAs using sample-specific barcode primers in individual plate wells. Amplification of cDNA from every sample can be verified using Bioanalyzer. This step not only reveals cDNA amplification but also provides greater precision for pooling equal concentrations of cDNA from different samples. A single pooled cDNA library is made suitable for sequencing on Illumina sequencers using a tagmentation kit. Because HiCAR-Seq targets a small region at the 3' of the mRNAs, as little as 3 to 4 million reads/sample are enough to infer changes in gene expression in human or mouse cells. We believe that HiCAR-Seq represents a robust and competitive addition to the existing set of transcriptome-based high-throughput screening methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: cDNA synthesis and barcoding/enrichment PCR Basic Protocol 2: Nextera tagmentation/amplification, quantification, and sequencing.

O-GlcNAcylation stabilizes ß-catenin through direct competition with phosphorylation at threonine 41.

Dysfunctions in Wnt signaling increase ß-catenin stability and are associated with cancers, including colorectal cancer. In addition, ß-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of ß-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of ß-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of ß-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of ß-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for ß-catenin stability. Analyses of ß-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the ß-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the ß-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of ß-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of ß-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.

The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts.

P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12/P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met.