RESUMO
Interferon gamma (IFNγ) is a potent inflammatory and immune cytokine. IFNγ signals via the interferon gamma receptor (IFNGR), which is constitutively bound to Janus Kinase (JAK) 1 and JAK2 via its intracellular domain. These two JAK proteins then initiate the inflammatory signaling cascade. The most potent inhibitor of IFNγ signaling is Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 negatively regulates IFNγ signaling pathway (and other pathways) by directly inhibiting JAKs. Here, we describe a protocol for the recombinant production and purification of the JAK1 kinase domain and its inhibitor SOCS1, for structural and biochemical studies.
Assuntos
Janus Quinase 1/isolamento & purificação , Janus Quinase 1/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/isolamento & purificação , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Células Cultivadas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Janus Quinase 1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/genéticaRESUMO
Type 1 diabetes is characterized by the autoimmune destruction of pancreatic ß-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 ß-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant K(d)-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206â214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by ß-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP206â214 revealed low-level presentation by K(d) (~25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed K(d)-restricted peptide Janus kinase-1355â363 (~15,000 copies/cell). We have successfully sequenced peptides from NIT-1 ß-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.