Proteome Characterization of Human NK-92 Cells Identifies Novel IFN-α and IL-15 Target Genes

Abstract

Natural killer (NK) cells are important components of innate immune defense. NK cells kill virus-infected cells and secrete cytokines that are involved in activation of other immune cells. Macrophage-derived cytokines interferon-alpha (IFN-alpha) and interleukin-15 (IL-15) are in turn important activators of NK cells, but the receptors and intracellular pathways that are involved in NK cell functions are still incompletely known. Here we have used expression proteomics to find new IFN-alpha and IL-15 regulated proteins in human NK-92 cells, which have the characteristics of activated NK cells. Cells were stimulated with cytokines for 20 h, lysed, and soluble proteins were separated by two-dimensional electrophoresis, and differentially expressed protein spots were identified with mass spectrometry and database searches. A total of 57 protein spots were found to be reproducibly differentially expressed between control and cytokine stimulated gel pairs, 26 spots being more than 2-fold upregulated and 3 spots being at least 2-fold downregulated. The rest 28 spots showed minor, less than 2-fold changes in their expression levels after quantification. From the differentially expressed protein spots we identified 47 different proteins, most of which are new IFN-alpha and IL-15 target proteins. Interestingly, we show that e.g., adenylate kinase 2 is highly upregulated by IFN-alpha and IL-15 stimulation in NK-92 cells. The expression of selected genes with high expression level differences after cytokine stimulation were further studied at mRNA level. Northern blot analysis showed that the genes studied were induced by IFN-alpha, IL-15, and IL-2 already at 3 h time point, suggesting that they are primary target genes of these cytokines.

Enhanced Interferon Signaling Pathway in Oral Cancer Revealed by Quantitative Proteome Analysis of Microdissected Specimens Using 16O/18O Labeling and Integrated Two-dimensional LC-ESI-MALDI Tandem MS

“…Two key downstream regulators of this pathway (STAT1 and UCRP/ISG15) were identified and quantified in all five experiments with fold changes in expression in tumor and nontumor cells ranging from 3.94 to 8.37 (STAT1) and 8.71 to 38.99 (UCRP/ISG15), respectively. Hundreds of ISG proteins have been previously determined using genomics and proteomics approaches (54,55). Our current study showed that 10 of the 53 up-regulated candidate proteins belong to the ISG family (Table I), and increased mRNA or protein levels of seven proteins (SYWC, IFM1, STAT1, TYPH, UCRP, MX1, and GBP2) have been detected in OSCC via previous genomics or proteomics studies (9,10,70).…”

Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

“…Also, a nonstructural protein 4B (NS4B) from hepatitis C virus, which is absolutely required for viral propagation, was found to possess adenylate kinase-like activity [ 263 ]. Adenylate kinase 2 is highly upregulated by INF-alpha and IL-15 stimulation in natural killer (NK) cells suggesting role in innate immune defense [ 264 ]. In this regard, due to unique catalytic properties and stability adenylate kinase is used as an enzyme amplification system in ATP biosensors for detecting bacterial contaminations in food industry, defense and health care that improves sensitivity levels up to several thousands fold (Celsis, AKuScreen).…”

Proteome analysis of human macrophages reveals the upregulation of manganese‐containing superoxide dismutase after toll‐like receptor activation

“…The key techniques in current proteome studies are 2-DE for protein separation and MS for protein identification and characterization. We have previously used these methods to find and identify proteins regulated by different cytokines in human T and NK cells [9][10][11][12][13]. In this study we have used 2-DE combined with MS to identify novel TLR7/8 agonist-induced target proteins in human monocytederived macrophages.…”