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Identification of candidate proteins involved in interferon gamma cell regulation in macrophages Leon, Carlos Gustavo

Abstract

Interferon-y (IFN-y) plays an important role in host defense against infection and cancer. Many of its biological effects are mediated through the IFN-y receptor (IFN-yR)- which is linked to the cytoplasmic tyrosine kinases Jakl and Jak2 and to the transcription factor Statl. However, regulation of IFN-y receptor signalling is not fully understood and not all responses to IFN-y are Statl-dependent. Research described in this thesis used two strategies to identify novel components of IFN-y signalling. First, a biochemical approach was used to enrich for phosphotyrosine containing proteins regulated by IFN-y. This involved sub-cellular fractionation, Poly (Glu, Tyr) affinity chromatography, preparative SDS-PAGE, followed by irrrmunoprecipitation of eluted proteins based upon reactivity with antiphosphotyrosine antibodies and microsequencing by mass spectrometry. Using this approach, the transcriptional co-activator TIP 120a was identified as a pi25 IFN-y-inducible tyrosine phosphoryIated protein in IFN-y-treated U937 cells, a human monocytic cell line. The finding that tyrosine phosphorylation of TIP 120a occurs in IFN-y treated cells suggests that this post-translational modification may influence its function and that TIP 120a itself may be involved in regulating gene expression in response to IFN-y. A second, genetic approach was used to identify novel proteins that interact with the IFN-y receptor. The cytoplasmic domains of the human IFN-yRl and IFN-yR2 chains were used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This screen identified two novel interactions. S100A9, a calcium-regulated, EF-hand type protein was found to bind to the IFN-yRl chain and annexin A5 (AxV), also a calcium regulated and phospholipid binding protein, was identified as a IFN-yR2 binding partner. These interactions were confirmed in pull-down experiments in which lysates of the human monocytic cell line THP-1 were incubated with the RI and R2 cytoplasmic domains expressed as glutathione-S-transferase (GST)-fusion proteins. Additional verification of these interactions was obtained when the IFN-yRl and IFN-yR2 subunits were immunoprecipitated from lysates of THP-1 cells. In this analysis, S100A9 and AxV were found to co-immunoprecipitate with their respective binding partners from the two hybrid screen and in both cases, these associations were shown to be ligand-dependent. To examine the role of AxV in IFN-y signalling, small interfering RNA (siRNA) was used to reduce AxV expression in human embryonic kidney cells (293T cells), an IFN-y responsive cell line. In cells where AxV protein levels were reduced to < 20% of control cells (AXVLO cells), activation of Jak2 and Statl in response to IFN-y, as assessed by tyrosine phosphorylation, was markedly enhanced. The impact of enhanced tyrosine phosphorylation of Statl in AXVL0 cells was assessed by two approaches. First, analysis of gene transcription by RT-PCR showed that IFN-y treatment of AXVL0 cells was associated with increased expression of the IFN-y-inducible genes Egr-1 and IFN-yR2. Second, when IFN-y-induced growth inhibition, a hallmark of the IFN-y response was examined, the anti-proliferative effect of IFN-y was found to be significantly enhanced in AXVLO cells. Taken together, results suggest that AxV negatively regulates IFN-y signalling through an inducible association with the IFN-yR2 subunit that controls the levels of activation of Jak2 and Statl.

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