The project consists of two subjects: 1. T-cell receptor signaling - quantification of tyrosine phosphorylation. Recognition of foreign antigens by T lymphocytes is an important step in the initiation of the immune response. Unlike growth factor receptors, TCR doesnt have intrinsic enzymatic activity, which is enhanced by tyrosine phosphorylation. Upon foreign antigen recognition via MHC/TCR, Src family phosphotyrosine kinase (PTK) activity results in immunoreceptor tyrosine-based activation motive (ITAM) phosphorylation and recruitment of ZAP-70 PTK, this allows TCR complex to function as active PTK. ZAP-70 phosphorylates adaptor proteins such as LAT and SLP-76 results in activation of phospholipase C (PLC) γ1 leading to Ras/Raf1/ERK activation and Ca++ flux to cytoplasm. We use T lymphocytes isolated from 5C.C7 mouse strain and activated in vitro with the antigen presenting cells (line P13.9) as the experimental model system. 2. Quantification of tyrosine, serine and threonine phosphorylation in the Toll receptor signaling pathways. The Toll-like receptors (TLRs) are a family of pathogen recognition receptors that alert the host to the presence of pathogens by recognizing their molecular signatures, termed pathogen-associated molecular patterns (PAMPs). They act as the first step in the induction of the protective innate and adaptive immune responses. There are 11 human TLR homologues and they are activated by a specific PAMP ligand. TLRs are all transmembrane proteins and their signaling is mediated by association of their internal domains. Classically, the TLR signaling cascade involves the myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-activated kinase (IRAK), and tumor-necrosis factor receptor-associated factor 6 (TRAF6), leading to the activation of Nuclear Factor kappaB (NF-κB). Among the most important genes to be regulated by TLR signaling are those of the cytokines. Given the key role of cytokines in the orchestration of the inflammatory response, mechanisms of modulating their production has garnered a lot of interest, in particular in the area of the development of therapies for the treatment of chronic inflammatory diseases. A clearer understanding of the TLR pathway leading to the cytokine production is required for a successful pharmacological intervention. We are using a well characterized cell line, murine RAW 264.7 macrophages, stimulated with LPS, ligand for TLR4, to decipher the temporal dynamics of protein phosphorylation in the TLR signaling cascade. The second mass spectometer (Orbitrap Velos from Thermo) was installed in January 2011. We have optimized the protocol (cell culture, lysis, lysate preparation, phosphoprotein enrichment) and calibrated, tuned and tested the instrument, finding the parameters optimal for this project. We got initial mass spectrometry data in both projects. Qualitative, non-labeled runs are used to identify proteins and phosphorylation sites of interest. Time-resolved, stimulus-dependent quantitative changes of phosphorylation of individual sites will be obtained using sensitive nanospray-based mass spectrometry combined with Multiple Reaction Monitoring. The dynamics of selected individual phosphorylation sites is being confirmed by Western blotting.
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