New mimetic peptides of the kinase-inhibitory region (KIR) of SOCS1 through focused peptide libraries

2012 ◽  
Vol 443 (1) ◽  
pp. 231-240 ◽  
Author(s):  
Nunzianna Doti ◽  
Pasqualina L. Scognamiglio ◽  
Stefania Madonna ◽  
Claudia Scarponi ◽  
Menotti Ruvo ◽  
...  
Keyword(s):  
Interferon Γ ◽  
Catalytic Site ◽  
Janus Kinase ◽  
Scanning Method ◽  
Expression Levels ◽  
Interferon Regulatory Factor 1 ◽  
Inhibitory Region ◽  
Inflammatory Processes ◽  
Cytokine Stimulation ◽  
Kinase Inhibitory Region

SOCS (suppressor of cytokine signalling) proteins are negative-feedback regulators of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway. Their expression levels are low under physiological conditions, but they are up-regulated in response to cytokine stimulation in many immune and inflammatory processes. Overexpression of SOCS1 in keratinocyte clones abrogates the IFNγ (interferon γ)-induced expression of many pro-inflammatory genes and the release of related chemokines by blocking the JAK/STAT pathway. SOCS1 inhibits JAK2 kinase activity by binding the catalytic site of JAK2, with its KIR (kinase-inhibitory region) acting as a pseudo-substrate of the enzyme. In the present study, we screened a focused combinatorial peptide library of KIR to identify new peptides able to mimic its function with an improved affinity towards the JAK2 catalytic site. Using an alanine-scanning method, KIR residues that are crucial for the interaction with JAK2 were unveiled. In this way, the KIR sequence was restricted to a shorter segment and ‘non-essential’ residues were replaced by different amino acids following a simplified combinatorial approach. We selected a new unnatural sequence able to bind to JAK2 with Kd values in the nanomolar range. This peptide was tested in human keratinocyte cultures and reduced the phosphorylation of STAT1 and the expression levels of IRF-1 (interferon regulatory factor-1).

scholarly journals Isoliquiritigenin Inhibits Interferon-γ-Inducible Genes Expression in Hepatocytes through Down-Regulating Activation of JAK1/STAT1, IRF3/MyD88, ERK/MAPK, JNK/MAPK and PI3K/Akt Signaling Pathways

2015 ◽  
Vol 37 (2) ◽  
pp. 501-514 ◽  
Author(s):  
Shanshan Wu ◽  
Jihua Xue ◽  
Ying Yang ◽  
Haihong Zhu ◽  
Feng Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Interferon Γ ◽  
Akt Signaling ◽  
Janus Kinase ◽  
Expression Levels ◽  
Erk Mapk ◽  
Ifn Γ ◽  
Inducible Genes ◽  
L02 Cells

Background & Aims: The high expression levels of interferon-γ (IFN-γ)-inducible genes correlate positively with liver diseases. The present study aimed to explore the effect of isoliquiritigenin (ISL) on the expression of genes induced by IFN-γ in vitro, and to elucidate the underlying molecular mechanisms. Methods: HepG2 and L02 cells were divided into control, ISL, IFN-γ, and IFN-γ plus ISL groups. The cytotoxicity of compounds to cells was evaluated by Cell Counting Kit 8 (CCK8) assay; the expression levels of chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, CXCL11, and interleukin-6 (IL-6) in cells and supernatant were measured by quantitative real time polymerase chain reaction (qRT-PCR) and ELISA, respectively. Moreover, western blot was used to examine the phosphorylated levels of janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1), nuclear factor (NF)-γB, interferon regulatory factor 3 (IRF3)/myeloid differentiation factor 88 (MyD88), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt) in HepG2 and L02 cells exposed to ISL, IFN-γ and IFN-γ plus ISL. Results: The results showed that IFN-γ treatment induced the expression of CXCL9, CXCL10, CXCL11, and IL-6 in HepG2 and LO2 cells, which could be significantly and dose-dependently inhibited by ISL treatment (P < 0.05 or P < 0.01), but the inhibitory effect of ISL on IL-6 expression was not so good as on CXCL9, CXCL10, and CXCL11 expression. Furthermore, ISL treatment dose-dependently inhibited the activation of JAK1/STAT1, IRF3/MyD88, extracellular signal-regulated kinase (ERK)/MAPK, c-Jun N-terminal kinase (JNK)/MAPK, and PI3K/Akt signaling pathways (P < 0.05), but had no effect on the activation of JAK2/STAT1, NF-γB and p38/MAPK signaling pathways. Conclusion: We demonstrate that ISL inhibits IFN-γ-induced inflammation in hepatocytes via influencing the activation of JAK1/STAT1, IRF3/MyD88, ERK/MAPK, JNK/MAPK, and PI3K/Akt signaling pathways.

scholarly journals Salicylate-enhanced activation of transcription factors induced by interferon-γ

1999 ◽  
Vol 342 (3) ◽  
pp. 503-507 ◽  
Author(s):  
Li-Chun CHEN ◽  
Diane KEPKA-LENHART ◽  
Timothy M. WRIGHT ◽  
Sidney M. MORRIS
Keyword(s):  
Transcription Factors ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Interferon Γ ◽  
Janus Kinase ◽  
Macrophage Cell ◽  
Cellular Targets ◽  
Nuclear Extracts ◽  
Interferon Regulatory Factor 1 ◽  
Activation Of Transcription

Salicylate enhanced the interferon-γ-dependent activation of two transcription factors in a murine macrophage cell line: signal transducer and activator of transcription (STAT)1 and interferon-γ-responsive factor 1. Salicylate alone did not activate these transcription factors. This enhancement was reflected by increased DNA-binding activities and was the consequence of prolonged tyrosine phosphorylation of these transcription factors following interferon-γ treatment. However, salicylate did not directly inhibit protein-tyrosine phosphatase activity in nuclear extracts of interferon-γ-treated cells. The enhanced activation of STAT1 resulted in increased induction of mRNA encoding interferon regulatory factor-1. These results not only demonstrate that aspirin and its metabolite salicylate may have pro-inflammatory as well as anti-inflammatory effects but also raise the possibility that new cellular targets may be identified for modulating the Janus kinase-STAT signalling pathway.

A common epitope is shared by activated signal transducer and activator of transcription-5 (STAT5) and the phosphorylated erythropoietin receptor: implications for the docking model of STAT activation

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2230-2237 ◽  
Author(s):  
Dwayne L. Barber ◽  
Bryan K. Beattie ◽  
Jacqueline M. Mason ◽  
Melody H.-H. Nguyen ◽  
Monique Yoakim ◽  
...  
Keyword(s):  
Structural Features ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Signal Transducer ◽  
Specific Antibodies ◽  
Docking Model ◽  
Receptor Interactions ◽  
Cytokine Stimulation ◽  
Carboxy Terminal

Abstract Erythropoietin (EPO) specifically activates the Janus kinase JAK2 and the transcription factor signal transducer and activator of transcription-5 (STAT5). All members of the STAT family are tyrosine phosphorylated in response to cytokine stimulation at a conserved carboxy-terminal tyrosine, Y694, in the case of STAT5. To determine structural features important for STAT signaling, we generated an activation-specific STAT5 antibody using a phosphopeptide containing amino acids 687 to 698 of STAT5 as antigen. This antibody specifically recognizes tyrosine- phosphorylated STAT5 but not nonphosphorylated STAT5. In immunoprecipitation reactions from cell lines and primary erythroblasts, 2 distinct polyclonal activation-specific STAT5 antibodies selectively immunoprecipitate the tyrosine phosphorylated EPO receptor (EPO-R) in addition to STAT5 under native and denaturing conditions. We propose that the activation-specific STAT5 antibody recognizes the 2 substrates to which the STAT5 SH2 domain interacts, namely, the tyrosine- phosphorylated EPO-R and STAT5 itself. Several studies have implicated EPO-R Y343, Y401, Y431, and Y479 in the recruitment of STAT5. Using a series of EPO-R tyrosine mutants expressed in Ba/F3 cells, we have shown that the activation-specific STAT5 antibody immunoprecipitates an EPO-R containing only 2 tyrosines at positions 343 and 401, confirming the importance of these tyrosines in STAT5 recruitment. These data uncover a novel aspect of STAT SH2 domain recognition and demonstrate the utility of activation-specific antibodies for examining the specificity of STAT–cytokine receptor interactions.

Potentiation of flagellin responses in gut epithelial cells by interferon-γ is associated with STAT-independent regulation of MyD88 expression

2009 ◽  
Vol 423 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Ciara Bannon ◽  
Pam J. Davies ◽  
Andrew Collett ◽  
Geoffrey Warhurst
Keyword(s):  
Epithelial Cells ◽  
Enteric Bacteria ◽  
Interferon Γ ◽  
Janus Kinase ◽  
Cxcl8 Secretion ◽  
Health And Disease ◽  
Toll Like Receptor 5 ◽  
Myeloid Differentiation Factor ◽  
Interfering Rna ◽  
Dose Dependent

Flagellin acting via TLR5 (Toll-like receptor 5) is a key regulator of the host response to the gut microbial flora in both health and disease. The present study has investigated regulation of flagellin–TLR5 signalling in human colonocytes (HT29-19A) by IFNγ (interferon-γ), a cytokine released early in the inflammatory process which has multiple effects on gut epithelial function that may facilitate abnormal responses to enteric bacteria. Flagellin induced a dose-dependent secretion of chemokines CXCL8 and CCL2 in the human colonocyte line, HT29-19A. Exposure to IFNγ did not induce chemokine secretion, but markedly potentiated responses to flagellin, increasing CXL8 gene expression and protein secretion by approx. 4-fold. Potentiation by IFNγ was independent of changes in TLR5 and was associated with a rapid, sustained increase in expression of the downstream adaptor molecule MyD88 (myeloid differentiation factor 88). Knockdown of MyD88 expression using siRNA (small interfering RNA) abolished flagellin-dependent CXCL8 secretion and the potentiating effect of IFNγ. Exposure of non-transformed mouse and human colonocytes to IFNγ also increased MyD88 expression. STAT (signal transducer and activator of transcription) 1 knockdown and use of the broad-spectrum JAK (Janus kinase)-STAT inhibitor AG490 had no effect on IFNγ-mediated up-regulation of MyD88. The findings of the present study suggest that IFNγ sensitizes colonic epithelial cells to bacterial flagellin via a largely STAT-independent up-regulation of MyD88 expression leading to increased secretion of immunomodulatory factors. These results indicate that epithelial responses to flagellin are potentiated by IFNγ, most likely mediated by increased MyD88 expression. The present study adds to our understanding of the spectrum of effects of this cytokine on gut epithelium that may contribute to bacterial-driven inflammation in the gut.

Chromatin bound by survivin regulates the glycolytic switch in interferon-γ producing CD4+ T cells

2021 ◽  
Author(s):  
Malin Erlandsson ◽  
Karin Andersson ◽  
Nina Oparina ◽  
Venkataragavan Chandrasekaran ◽  
Anastasios Damdimopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
Glucose Utilization ◽  
Interferon Γ ◽  
Interferon Regulatory Factor ◽  
Metabolic Adaptation ◽  
Regulatory Factor ◽  
Cell Functions ◽  
Interferon Regulatory Factor 1 ◽  
Direct Binding ◽  
Nuclear Survivin

Abstract Upon activation, CD4+ T cells adapt metabolically to fulfill their effector function in autoimmunity. Here we show that nuclear survivin is essential for transcriptional regulation of glucose utilization. We found that the glycolytic switch in interferon (IFN) g–producing CD4+ cells is dependent on a complex of survivin with interferon regulatory factor 1 (IRF1), and Smad3 and was reversed by survivin inhibition. Transcriptome analysis of CD4+ cells and sequencing of survivin-bound chromatin identified a hub of metabolism regulating genes whose transcription depended on survivin. Direct binding of survivin to IRF1 and SMAD3 promoted IRF1-mediated transcription, repressed SMAD3 activity, and lowered PFKFB3 production. Inhibiting survivin upregulated PFKFB3, restored glycolysis, and reduced glucose uptake, improving control over IFNg-dependent T-cell functions. Thus, IRF1-survivin-SMAD3 interactions are important for metabolic adaptation of CD4+ cells and provide an attractive strategy to counteract IFNg-dependent inflammation.

scholarly journals Molecular Cloning and Expression Analysis of Three Suppressors of Cytokine Signaling Genes (SOCS5, SOCS6, SOCS7) in the Mealworm Beetle Tenebrio molitor

Insects ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 76 ◽  
Author(s):  
Bharat Patnaik ◽  
Bo Kim ◽  
Yong Jo ◽  
In Bang
Keyword(s):  
Functional Characterization ◽  
Tenebrio Molitor ◽  
Janus Kinase ◽  
Cloning And Expression ◽  
Cytokine Signaling ◽  
Type I ◽  
Growth Factor Signaling ◽  
Expression Levels ◽  
Mealworm Beetle ◽  
Suppressors Of Cytokine Signaling

Suppressors of cytokine signaling (SOCS) influence cytokine and growth factor signaling by negatively regulating the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway to maintain homeostasis during immune responses. However, functional characterization of SOCS family members in invertebrates is limited. Here, we identified and evaluated three SOCS genes (type I sub-family) in the mealworm beetle Tenebrio molitor. The full-length open reading frames (ORFs) of TmSOCS5, TmSOCS6, and TmSOCS7 comprised of 1389, 897, and 1458 nucleotides, encoding polypeptides of 462, 297, and 485 amino acids, respectively. The SH2 and SOCS box domains of the TmSOCS C-terminal region were highly conserved. Phylogenetic analysis revealed that these SOCS genes were clustered within the type I subfamily that exhibits the highest amino acid identity with Tribolium castaneum SOCS genes. Contrary to TmSOCS7 expression, the expression levels of TmSOCS5 and TmSOCS6 were lower in the larval, pupal, and adult stages. In larvae and adults, the expression levels of TmSOCS5 and TmSOCS6 were highest in the hemocytes and ovaries, respectively. SOCS transcripts were also highly upregulated in the hemocytes of T. molitor larvae within 3–6 h post-infection with the fungus Candida albicans. Collectively, these results provide valuable information regarding the involvement of TmSOCS type-I subfamily in the host immune response of insects.

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