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Protein S-Nitrosylation in Inflammation and Cancer

Final Report Summary - NO-CANCER (Protein S-Nitrosylation in Inflammation and Cancer)

Chronic inflammation represents a major pathologic basis for many human diseases including cancer. Nitric oxide (NO) production is a hallmark of inflammation, and may play a significant role in inflammation-associated cancers. S-nitrosylation, the covalent attachment of an NO group to the thiol side chain of cysteine is a common mechanism for dynamic, post-translational regulation of most or all main classes of protein. Indeed, protein S-nitrosylation and denitrosylation have emerged as integral components of signal transduction pathways, and accumulating evidence suggests that deregulated S-nitrosylation contributes to a range of human pathologies. Yet, the involvement of protein S-nitrosylation/denitrosylation in inflammation-related cancer remains unclear. We hypothesize that inflammation-associated cancer is promoted by the deregulation of protein S-nitrosylation.

To examine this hypothesis we defined the following specific aims:

1. Identify S-nitrosylated proteins in lung tumor cells and in lung macrophages.
2. Study the regulation by S-nitrosylation of key inflammatory mediators.
3. Characterize denitrosylation mechanisms in lung cancer cells.

During the past four years, we have developed a redox-based proteomics approach to address these specific aims. The approach employs the oxidoreductase, thioredoxin (Trx). Trx is an important redox protein that is ubiquitously distributed. Recent studies by us and others have shown that mammalian Trx is physiological cysteine denitrosylase. However, the scope and functional significance of Trx-mediated denitrosylation remains largely undetermined. We therefore developed a proteomic approach for large scale identification of S-nitrosylated targets of Trx. Applying this approach to human monocytes and mouse macrophages we identified a large number (over 500) of novel putative nitrosylated proteins. We identified multiple known nitrosylation targets (e.g. caspase-3 and GAPDH) supporting the validity of our approach. Yet, the majority of identified proteins (over 400) represent potentially novel nitrosylation targets. These targets are implicated in a wide range of cellular pathways and processes. Bioinformatics analysis applied to these target proteins uncovered several overrepresented cellular processes, such as protein translation and folding, cell division and proliferation, and ubiquitin-mediated protein degradation. In addition, many important signaling proteins were identified, including NF-kappaB, STAT3 and MEK1.

We extensively validated the results of the novel proteomics approach. We focused on a subset of identified nitrosylation targets, including proteins implicated in the regulation of inflammation. For these targets, we verified whether they are indeed subject to Trx-regulated S-nitrosylation. Noteworthy, we demonstrated that several key inflammatory mediators are regulated by reversible S-nitrosylation, including, STAT3, MEK1 and iNOS. In particular, we obtained evidence that MEK1 and iNOS are regulated by S-nitrosylation and Trx-mediated denitrosylation. Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the role of Trx-mediated denitrosylation in NO signaling. We expect that our novel proteomic approach will be generally useful for the identification and exploration of nitroso-proteomes under various physiological and pathophysiological conditions, including those of cancer cells. As such, we feel that our findings achieved our research objectives, in particular Aims 1 and 2. The extent to which the results obtained in monocytes/macrophages apply to lung cancer cells (Aim 3) will require additional experimentation.

In the context of Aim 2, we also investigated how Trx might regulate the proinflammatory NLRP3 inflammasome/interleukin-1beta (IL-1beta) pathway. Using global transcriptional profiling we found that macrophage treatment with the Trx reductase (TrxR) inhibitor auranofin exerted a selective effect on Toll-like receptor 4 (TLR4)-induced gene expression, suppressing the induction of a small number of genes. Interestingly, among these suppressed genes were three members of the interleukin-1 (IL-1) family of genes, among which IL-1beta was most affected. qPCR analyses confirmed the repressive effects of auranofin on IL-1 genes. In addition, qPCR and Western blot analyses showed that auranofin impaired TLR4-dependent induction of the inflammasome receptor NLRP3, which plays a critical role in IL-1beta processing. Consistent with these findings, inflammasome-dependent release of IL-1beta from stimulated macrophages was suppressed by auranofin as was inflammasome-mediated cell death. These findings suggest a regulatory role for the Trx system in macrophage inflammatory signaling. Inhibition of the Trx system in macrophages exerts an anti-inflammatory effect by repressing the activation of the NLRP3/IL-1beta pathway.

In related area of research, we discovered that nitrosylation regulates the activity of the thioredoxin-peroxiredoxin system. The key findings of this research were published in 2013, see: Engelman, R. et al. Multilevel Regulation of 2-Cys Peroxiredoxin Reaction Cycle by S-Nitrosylation. J. Biol. Chem. 288:11312-24. These findings may have significant relevance for understanding the impact of nitrosylation on macrophage function and the response of tumor cells to nitrosative stress.

In summary, our findings contribute to a better understanding of how S-nitrosylation and Trx regulate inflammatory signaling in macrophages. Our research revealed novel mechanisms for regulating inflammatory responses, particularly the redox regulation of the NLRP3 inflammasome/ IL-1beta pathway. Furthermore, our studies expand our knowledge and insight into the nitrosoproteome of the inflammatory cell and its regulation by Trx. As such, this project lays a foundation for future exploration of the roles of nitrosylation/denitrosylation in regulating the function of many proteins, in healthy and diseased cells. We anticipate that the proteomic trapping strategy may be useful for profiling and characterizing nitrosoproteomes in diverse biological contexts.

From a broader perspective, we propose the present findings and the new proteomic tools described will advance our understanding of the role on NO and S-nitrosylation in inflammation and cancer.