Reduced S-nitrosylation of TGFβ1 elevates its binding affinity toward the receptor and promotes fibrogenic signaling in the breast.

Transforming Growth Factor β (TGFβ) is a pleiotropic cytokine closely linked to tumors. Previously, we pharmacologically inhibited basal nitric oxide (NO) production in healthy mammary glands and found that this induced precancerous progression accompanied by upregulation of TGFβ and desmoplasia. In the present study, we tested whether NO directly S-nitrosylates (forms an NO-adduct at a cysteine residue) TGFβ for inhibition, whereas reduction of NO denitrosylates TGFβ for de-repression. We introduced mutations to 3 C-terminal cysteines of TGFβ1 which were predicted to be S-nitrosylated. We found that these mutations indeed impaired S-nitrosylation of TGFβ1 and shifted the binding affinity towards the receptor from the latent complex. Furthermore, in silico structural analyses predicted that these S-nitrosylation-defective mutations strengthen the dimerization of mature protein, whereas S-nitrosylation-mimetic mutations weaken the dimerization. Such differences in dimerization dynamics of TGFβ1 by denitrosylation/S-nitrosylation likely account for the shift of the binding affinities toward the receptor versus latent complex. Our findings, for the first time, unravel a novel mode of TGFβ regulation based on S-nitrosylation or denitrosylation of the protein.