BUSCADOR RECOLECTA

Methods used for generation of data are described under the Material and Methods section of the manuscript and the Supplementary Methods of the Supplementary Information Text., Dataset files of this paper will be shared by the corresponding authors upon reasonable request to the e-mail addresses jmserrador@cbm.csic.es and mainiguez@cbm.csic.es. The petitioner should compromise to neither copy, modify or distribute any file without the consent of the authors., El conjunto de datos primarios de esta publicación podrá ser compartido por los autores de correspondencia una vez solicitado a las direcciones de correo jmserrador@cbm.csic.es y mainiguez@cbm.csic.es y efectuado el compromiso de no distribución, copia o modificación de archivos sin la autorización de los autores., Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inflammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modification of nucleophilic amino-acid residues. Several regulatory proteins have been identified as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inflammatory effects. Herein, we report the effects of nitro-oleic acid (NO2-OA) on pro-inflammatory T cell functions, showing that 9- and 10-nitro-oleic acid, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane and IL-2, IL-4 and IFN-γ cytokine gene expression. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates calcineurin A (CaNA) on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on
Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results underscore new mechanisms by which NO2-FAs exert their anti-inflammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses., RTI2018-100815-B-I00 (MICIU/FEDER) and the accompanying 2021 CSIC Exceptional Grant to M.A.I. and J.M.S. R01GM125944-05 and DK112854-04 to F.J.S., and Universidad de la República CSIC Grupos_2018, EI_2020 to R.R., MAIN FIGURES

Figure 1:

Figure 1A:
Exp1: “Histogram_Exp1” and corresponding FACSfiles.
Exp2: “Histogram_Exp2”, “Hig resolution”, “LowResolution_Exp2” and corresponding FACSfiles.
Exp3: “Histogram Exp3” and corresponding FACSfiles.
Exp4: “Histogram Exp4” and corresponding FACSfiles.
“Fig1A_Graph”.
“Fig1A_Histogram”.
“Graph_Fig1A”.
Figure 1B:
Exp1: FACS files corresponding to CD69 and CD71.
Exp2: FACS files corresponding to CD69 and CD71.
Exp3: FACS files corresponding to CD69 and CD71.
Fig1B_HistogramsCD71 and CD69: FACS files corresponding to Figure1B.
“Fig1B_Graph”.
“Graph_Fig1B”.
Figure 1C:
CTV CD4 CD8: FACS files corresponding to cell proliferation.
CTV CD4 CD8II: FACS files corresponding to cell proliferation.
Histogram_Fig 1C: FACS files and Histogram Figure 1C.
Figure 1D:
“Graph_Fig1D”.
“Fig1D_Graph”.
Figure 1E_F:
Exp1: FACS files corresponding to Figure 1E and F.
Exp2: FACS files corresponding to Figure 1E and F.
Exp3: FACS files corresponding to Figure 1E and F.
“Fig1E_Graph”
“Fig1F_Graph”
“Graph_Fig1E_F”
“Layout Th2”
“Layout Th1”

Figure 2

Figure 2A:
“Data RT-PCR IL2IFNGIL4vsGAPDH Fig 2A”
“RT-PCR IFNg vs GAPDH 100%”
“RT-PCR IL2 vs GAPDH 100%”
“RT-PCR IL4 vs GAPDH 100%”
Figure 2B:
IFNgData: “Data Expts IFNgLUC” and “Raw Data Fig 2B IFNgLuc”
IL2Data: “Data Expts IL2LUC” and “Raw Data Fig 2B IL2Luc”
IL4Data: “Data Expts IL4LUC” and “Raw Data Fig 2B IL4Luc”

Figure 3

Figure 3A:
NFATLucPMA+Ion: “Graph_Fig3A_PMAI” and “GraphFig3A_PMAI”.
NFATLucRaji+SEB: “Graph_Fig3A_SEB” and “GraphFig3A_SEB”.
Figure 3B:
“Fig3B_Graph_white” and “Graph_Fig3B”
Figure 3C:
“Fig3C_Graph” and “Graph_Fig3C”
Figure 3D:
Exp:
b-actin: 2 files WB b-actin.
RCAN: 4 files WB inducible and constitutive RCAN.
Quantification: 3 quantifications (3 image files, 3 graphs files and 3 excel files).
Excel “Densitometric analysis_bActin”.
Fig 3D.
Figure 4

Figure 4A:
Photomicrographs_Fig4A: imaging files.
Quantitative imaging: imaging files analysed.
“Nuclear_NFAT percentage”.
“Quantification” Excel file.
Figure 4B:
Exp:
Cytosol: 2 files WB dynamin II and 2 files WB NFATC2.
Nucleus: 2 files WB LaminB1 and 2 files WB NFATC2.
“Figure 4B”.
Figure 4C:
Exp:
DynaminII: 3 files WB Dynamin II.
NFAT: 4 files WB NFATC2.
“Fig4C”
Figure 4D: “CaNA_DCAM domains”.
Figure 4E:
“Graph_Fig4E” and “Fig4E_Graph”.
Figure 4F:
“Graph_Fig4F” and “Fig4F_Graph”.
Figure 4G:
“Graph_Fig4G” and “Fig4G_Graph”.
Figure 4H:
Exp: 5 FACSfiles and “Fig4H”.

Figure 5

Figure 5A:
Exp: 2 files WB nitroalkylation and 1 file WB Coomassie CaNA.
“Fig5A”.
Figure 5B:
Exp: 2 files WB nitroalkylation and 1 file WB Coomassie DCAM- AI.
“Fig 5B”.
Figure 5C: “NitroalkylAA_Sequence_CaNA”.
Figure 5D:
“Graph_Fig5D”.
“Dots Fig 5D”.
“Fig5D”
Figure 5E: ”Graph_Fig5E” and “Fig5E_Graph”.
Figure 5F:
Exp: 4 files WB Biotin_Nitroalkylation and 1 file Coomassie GST-DCAM.
“Fig 5F”.
“Quantification”.

Figure 6

Figure 6A:
Exp:
CoIPCnB: 2 files WB CaNB.
InputCnA: 4 files WB input CaNA.
Input CaNB: 1 file WB input CaNB.
IPCnA: 1 file WB IP CaNA.
“Fig 6A”.
“Quantification co_IP Fig6A”.
Figure 6B:
Exp:
2 files WB CaNB.
1 file WB input CaNB.
1 file Coomassie GST-DCAM-AI.
“Fig 6B”.
“quantification pull_down_Fig6B”.
Figure 6C:
Exp:
2 files WB CaNB.
1 file Input CaNB.
1 file Coomassie GST-DCAM-AI.
“Fig 6C”.
“Quantification Fig6C”.
Figure 6D:
Exp:
CoIP_CaNB: 3 files WB CaNB.
Input_CaNB: 1 file WB input CaNB.
Input_DCAM_GFP: 2 files WB GFP-DCAM-AI.
IP_DCAM_GFP: 2 files WB GFP-DCAM-AI.
“Fig6D”.
“Quantification Co_IPFig6D”., Peer reviewed

Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inflammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modification of nucleophilic amino-acid residues. Several regulatory proteins have been identified as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inflammatory effects. Herein, we report the effects of nitro-oleic acid (NO2-OA) on pro-inflammatory T cell functions, showing that 9- and 10-NOA, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane, and IL-2, IL-4, and IFN-γ cytokine gene expressions. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates CaNA on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results provide evidence for an additional mechanism by which NO2-FAs exert their anti-inflammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses., This work was supported by grant RTI2018-100815-B-I00 Ministerio de Ciencia e Innovación (MICIU)/Fondo Europeo de Desarrollo Regional (FEDER) and the accompanying 2021 CSIC Exceptional Grant to M.A.Í. and J.M.S. Additional funding was obtained from grants PID2021-122348NB-I00 (MICIU) and “La Caixa” Banking Foundation (HR22-00253) to E.C. and J.V., R01GM125944-05 and DK112854-04 to F.J.S., and from Universidad de la República Comisión Intersectorial de Investigación Científica Grupos_2018, Espacio Interdisciplinario_2020 to R.R. The CBMSO receives an institutional grant from the Fundación Ramón Areces., Peer reviewed