14 research outputs found
Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses [version 1; peer review: 2 approved]
Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple 'glycogenes' that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion
Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2
The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.</p
The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway
A hierarchical cascade activated by non-canonical Notch signaling and the mTOR-Rictor complex regulates neglect-induced death in mammalian cells
The regulation of cellular metabolism and survival by trophic factors is not completely understood. Here, we describe a signaling cascade activated by the developmental regulator Notch, which inhibits apoptosis triggered by neglect in mammalian cells. In this pathway, the Notch intracellular domain (NIC), which is released after interaction with ligand, converges on the kinase mammalian target of rapamycin (mTOR) and the substrate-defining protein rapamycin independent companion of mTOR (Rictor), culminating in the activation of the kinase Akt/PKB. Biochemical and molecular approaches using site-directed mutants identified AktS473 as a key downstream target in the antiapoptotic pathway activated by NIC. Despite the demonstrated requirement for Notch processing and its predominant nuclear localization, NIC function was independent of CBF1/RBP-J, an essential DNA-binding component required for canonical signaling. In experiments that placed spatial constraints on NIC, enforced nuclear retention abrogated antiapoptotic activity and a membrane-anchored form of NIC-blocked apoptosis through mTOR, Rictor and Akt-dependent signaling. We show that the NIC-mTORC2-Akt cascade blocks the apoptotic response triggered by removal of medium or serum deprivation. Consistently, membrane-tethered NIC, and AktS473 inhibited apoptosis triggered by cytokine deprivation in activated T cells. Thus, this study identifies a non-canonical signaling cascade wherein NIC integrates with multiple pathways to regulate cell survival
HLA Transgenic Mice Provide Evidence for a Direct and Dominant Role of HLA Class II Variation in Modulating the Severity of Streptococcal Sepsis
Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2
Abstract The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation