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Discovery and Biological Characterization of Potent MEK inhibitors in melanoma

MEK inhibitor

Results from one of two independent experiments are shown

Posted on April 4, 2022 By scienzaunder18

Results from one of two independent experiments are shown. We also demonstrate that pharmacological inhibition of PRC2 in vivo greatly attenuates the severe T cellCdriven autoimmunity caused by regulatory T cell depletion. Our data reveal cytoplasmic PRC2 is one of the most potent regulators of T cell activation and point toward the therapeutic potential of Nalmefene hydrochloride PRC2 inhibitors for the treatment of T cellCdriven autoimmune diseases. Introduction Polycomb repressive complex 2 (PRC2) is a multiprotein complex that is best known for its contribution to transcriptional gene silencing (Margueron and Reinberg, 2011). This function of PRC2 is mediated by the lysine methyltransferases Ezh1 or Ezh2, which catalyze the di/tri-methylation of lysine 27 of histone H3 (H3K27me3; Cao and Zhang, 2004; Margueron and Reinberg, 2011). In T cells, the relative contribution of Ezh1 and Ezh2 to PRC2 function differs between resting and dividing cells. Ezh1 expression levels are very similar in resting and dividing T cells, whereas Ezh2 expression significantly increases after mitotic stimulation (Fig. 1, G and H). The gene regulatory function of PRC2 has been implicated in many aspects of T cell development, differentiation, and activation (Dobenecker et Nalmefene hydrochloride al., 2015; Yang et al., 2015). However, the interpretation of these findings is rather controversial because of the multiplicity of the histone H3Cindependent Ezh2 protein substrates (He et Nalmefene hydrochloride al., 2012; Lee et al., 2012; Kim et al., 2013b; Gunawan et al., 2015). One of the least understood aspects of the histone H3Cindependent PRC2 functions concerns Ezh2s role in signaling (Su et al., 2005; Su and Tarakhovsky, 2006). Our earlier studies Rabbit polyclonal to CDC25C showed the presence of Ezh2 in the T cell cytosol, where it contributes to TCR-driven actin polymerization (Su et al., 2005). The signaling capacity of Ezh2 was further underscored by the identification of the membrane associated protein talin-1, which plays an important role in adhesion, as a cytosolic Ezh2 substrate in dendritic cells (Gunawan et al., 2015). Here we describe the composition of the cytoplasmic PRC2 (cPRC2) complex in T cells. We show that although the cytoplasmic and nuclear PRC2 share common subunits, cPRC2 is uniquely associated with key signaling proteins that control TCR signaling and T cell activation. Using short-term pharmacological PRC2 suppression, we show that cPRC2 is required for TCR-mediated activation of MAPK/Erk and expression of IL2 and IL2RA, which support T cell proliferation. We also show that pharmacological suppression of PRC2 in vivo leads to immunosuppression, characterized by greatly diminished T cell responses. We demonstrate that pharmacological PRC2 inhibition could be used for the treatment of severe autoimmune inflammation caused by excessive T cell activation. Open in a separate window Figure 1. Composition of the cytoplasmic PRC2 complex. (A) Expression levels of the individual PRC2 components in T cell nuclei and cytosol in naive and TCR-activated splenic T cells were measured by Western blotting. Lamin B or cofilin were used as loading controls for the nuclear and cytoplasmic extracts, respectively. The asterisk indicates an unspecific band. Results from one of more than three independent experiments are shown. (B) Ezh2 is present in the cytosol of activated T cells. Cells were stained with fluorescently labeled antibodies against Ezh2 (green) and TCR (red), and chromatin was stained with DAPI (blue). Experiments were performed twice. (C and D) Ezh2 binds to the core PRC2 components in T cell cytosol. Ezh2 was immunoprecipitated from nuclear or cytoplasmic extracts derived from naive or activated T cells. Western blotting of the immunoprecipitates revealed the indicated Ezh2-associated proteins. Immunoprecipitation with IgG was used as control. Lamin B and tubulin or histone 3 (H3) were used as loading controls for the nuclear and cytoplasmic extracts, respectively. Results from one of more than three independent experiments are shown. (E) Nck1 is associated with Ezh2 and Vav1 in naive and activated CD4+ T cells. The cytosolic lysates were immunoprecipitated using an Nck-specific antibody followed by Western blot analysis of Nck, Vav1, and Ezh2. Tubulin was used as a loading control for the input. Results from one of more than three independent experiments are shown. (F) Ezh2 and Suz12 as well as Vav1 and Nck1 bind to CD3 before and after TCR stimulation. CD3 was immunoprecipitated with SA beads before and after TCR cross-linking for 2 min. Western blotting of the immunoprecipitates revealed the indicated associated proteins. Results from one of more than three independent experiments are shown. (G and H) Expression levels of Ezh1 and Ezh2 in whole cell lysate from naive and TCR- (G) or PMA/ionomycinC (H) activated CD4+T cells were measured by Western blotting. Ezh1 and Ezh2 levels were normalized to lamin B, and numbers.

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