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Nitric oxide mediated inhibition of antigen presentation from DCs to CD4⁺ T cells in cancer and measurement of STAT1 nitration.

ABSTRACT: Myeloid derived suppressor cells (MDSC) produce nitric oxide (NO) and inhibit dendritic cell (DC) immune responses in cancer. DCs present cancer cell antigens to CD4⁺ T cells through Jak-STAT signal transduction. In this study, NO donors (SNAP and DETA-NONOate) inhibited DC antigen presentation. As expected, MDSC isolated from peripheral blood mononuclear cells (PBMC) from cancer patients produced high NO levels. We hypothesized that NO producing MDSC in tumor-bearing hosts would inhibit DC antigen presentation. Antigen presentation from DCs to CD4⁺ T cells (T cell receptor transgenic OT-II) was measured via a [³H]-thymidine incorporation proliferation assay. MDSC from melanoma tumor models decreased the levels of proliferation more than pancreatic cancer derived MDSC. T cell proliferation was restored when MDSC were treated with inhibitors of inducible nitric oxide synthase (L-NAME and NCX-4016). A NO donor inhibited OT II T cell receptor recognition of OT II specific tetramers, thus serving as a direct measure of NO inhibition of antigen presentation. Our group has previously demonstrated that STAT1 nitration also mediates MDSC inhibitory effects on immune cells. Therefore, a novel liquid chromatography-tandem mass spectrometry assay demonstrated that nitration of the STAT1-Tyr701 occurs in PBMC derived from both pancreatic cancer and melanoma patients.

SUBMITTER: Markowitz J

PROVIDER: S-EPMC5684213 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Nitric oxide mediated inhibition of antigen presentation from DCs to CD4<sup>+</sup> T cells in cancer and measurement of STAT1 nitration.

Markowitz Joseph J Wang Jiang J Vangundy Zach Z You Jia J Yildiz Vedat V Yu Lianbo L Foote Isaac P IP Branson Owen E OE Stiff Andrew R AR Brooks Taylor R TR Biesiadecki Brandon B Olencki Thomas T Tridandapani Susheela S Freitas Michael A MA Papenfuss Tracey T Phelps Mitch A MA Carson William E WE

Scientific reports 20171113 1

Myeloid derived suppressor cells (MDSC) produce nitric oxide (NO) and inhibit dendritic cell (DC) immune responses in cancer. DCs present cancer cell antigens to CD4<sup>+</sup> T cells through Jak-STAT signal transduction. In this study, NO donors (SNAP and DETA-NONOate) inhibited DC antigen presentation. As expected, MDSC isolated from peripheral blood mononuclear cells (PBMC) from cancer patients produced high NO levels. We hypothesized that NO producing MDSC in tumor-bearing hosts would inhi ...[more]

PMID: 29133913

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Project description:Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.

Dataset Information

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4⁺ T cells in cancer and measurement of STAT1 nitration.

Publications

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4<sup>+</sup> T cells in cancer and measurement of STAT1 nitration.

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Dataset Information

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4+ T cells in cancer and measurement of STAT1 nitration.

Publications

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4<sup>+</sup> T cells in cancer and measurement of STAT1 nitration.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4⁺ T cells in cancer and measurement of STAT1 nitration.