Protein phosphorylations, mediated by pro-inflammatory cytokines IL-32g and IL-17A/F

Global kinase activity induced by cytokines IL-32g and IL-17A/F were determined using peptide arrays representing phophorylation targets The objective of this study was to identify common and unique phosphorylation targets of pro-inflammatory cytokines IL-32 and IL-17. These cytokines are associated with the pathogenesis and severity of chronic inflammatory disorders, therefore signaling intermediates of these cytokines could be beneficial as alternate theraputic targets that may likely influence different inflammatory pathways. Phosphorylation of proteins is a critical mechanism in the regulation of cellular processes. This process is meticulously regulated by enzymes known as kinases, which are increasingly being identified as drug targets for a variety of diseases. In this study we interrogated kinase activities (kinome) induced in the presence of the human recombinant cytokines IL-32g and IL-17A/F employing peptide arrays representing 300 peptides, printed in triplicate, representing select phosphorylation events. Human macrophage-like THP-1 cells were used for this comparative kinome analysis. Macrophage-like THP-1 cells were stimulated with either IL-32g (20 ng/ml) or IL-17A/F (20 ng/ml) for 15 min, and the peptide arrays were used to comprehensively analyze protein phosphorylation profiles in the presence of these cytokines. Two independet biological experiments were performed and the kinoem analysis was done in triplicates for each. The phosphorylations of the peptides on the array were quantified in the cytokine-treated cells relative to the un-stimulated control cells. Differentially phosphorylated targets were defined as greater than 1.5 fold increase or decrease (p < 0.06) in phosphorylation compared to un-stimulated control cells.

本研究通过覆盖磷酸化靶点的肽芯片（peptide arrays），测定了细胞因子IL-32g与IL-17A/F诱导的整体激酶活性（kinome）。本研究旨在识别促炎细胞因子IL-32与IL-17共有的及独有的磷酸化靶点。此类细胞因子与慢性炎症性疾病的发病机制及严重程度密切相关，因此其信号转导通路的中间分子有望成为新型治疗靶点，进而调控不同炎症通路。蛋白质磷酸化是调控细胞生命进程的关键机制，该过程由激酶（kinases）精密调控；目前激酶已被广泛认定为多种疾病的药物研发靶点。本研究采用覆盖300条磷酸化事件相关肽段、以三次重复点样的肽芯片，分析了人重组细胞因子IL-32g与IL-17A/F诱导的激酶活性（kinome）。本研究选用人巨噬细胞样THP-1细胞开展对比性激酶组分析：将巨噬细胞样THP-1以20 ng/ml的IL-32g或20 ng/ml的IL-17A/F刺激15分钟，随后通过肽芯片全面分析两种细胞因子处理下的蛋白质磷酸化谱。本研究共开展2次独立生物学重复，每次实验均设置3次技术重复以进行激酶组分析。以未刺激的对照组细胞为参照，对细胞因子处理组细胞的芯片肽段磷酸化水平进行定量；差异磷酸化靶点的判定标准为：与对照组相比，磷酸化水平上调或下调幅度达1.5倍以上（p < 0.06）。