Pivotal role for S-nitrosylation of DNA methyltransferase 3B in epigenetic regulation of tumorigenesis.

DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-L-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 ≤ 100 nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B.

S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein.

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

Attenuation of Macrophage Migration Inhibitory Factor-Stimulated Signaling via S-Nitrosylation.

Nitric oxide (NO) is a key signaling molecule that has various effects via S-nitrosylation, a reversible post-translational modification that affects the enzymatic activity, localization, and metabolism of target proteins. As chronic nitrosative stress correlates with neurodegeneration, the targets have received focused attention. Macrophage migration inhibitory factor (MIF) plays a pivotal role in the induction of gene expression to control inflammatory responses. MIF acts as a ligand for CD74 receptor and activates the Src-p38 mitogen-activated protein kinase (MAPK) cascade. MIF also elevates the expression of brain-derived neurotrophic factor (BDNF), which contributes to the viability of neurons. Here, we show that MIF is S-nitrosylated by a physiological NO donor. Interestingly, the induction of S-nitrosylation resulted in a loss of MIF activity following stimulation of the Src and p38 MAPK signaling pathways and the induction of BDNF expression. Our results shed light on the pathogenic mechanisms of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

The Emerging Role of Electrophiles as a Key Regulator for Endoplasmic Reticulum (ER) Stress.

The unfolded protein response (UPR) is activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which is called ER stress. ER stress sensors PERK, IRE1, and ATF6 play a central role in the initiation and regulation of the UPR; they inhibit novel protein synthesis and upregulate ER chaperones, such as protein disulfide isomerase, to remove unfolded proteins. However, when recovery from ER stress is difficult, the UPR pathway is activated to eliminate unhealthy cells. This signaling transition is the key event of many human diseases. However, the precise mechanisms are largely unknown. Intriguingly, reactive electrophilic species (RES), which exist in the environment or are produced through cellular metabolism, have been identified as a key player of this transition. In this review, we focused on the function of representative RES: nitric oxide (NO) as a gaseous RES, 4-hydroxynonenal (HNE) as a lipid RES, and methylmercury (MeHg) as an environmental organic compound RES, to outline the relationship between ER stress and RES. Modulation by RES might be a target for the development of next-generation therapy for ER stress-associated diseases.