In this study we demonstrate that HHcy upregulated the

In this study, we demonstrate that HHcy upregulated the expression of GSNOR in T cells. As a result, GSNOR induced denitrosylation of Akt in Hcy-activated T AM1241 in vivo and in vitro, which contributed to vascular inflammation and susceptibility to atherosclerosis in GSNOR-/-ApoE-/- mice. The translational implication of the current study builds on the positive correlation between GSNOR-dependent denitrosylation in T cells and the development of HHcy-induced coronary heart disease (CAD) in humans.
Material and methods
Results
Discussion HHcy accelerates atherosclerosis, due in part to, HHcy promotion of T-cell proliferation and secretion of proinflammatory cytokines (IL-2 and IFN-γ) [4], [5], [6], [7]. GSNOR reduces cellular levels of protein S-nitrosylation by regulating the equilibrium between GSNO and SNO-proteins [14]. Despite the reported roles of GSNOR in T-cell development and negative selection [11], whether and how GSNOR participates in T-cell activation in terms to HHcy-related atherosclerosis remain largely unknown. Aiming at studying the mechanism by which HHcy induces T-cell activation, we demonstrated significantly increased GSNOR expression in T cells in response to HHcy in vivo. GSNOR-/-ApoE-/- double knock-out and adoptive transfer of GSNOR-/- T cells into ApoE-/- mouse models showed a reduction in the HHcy-induced T-cell activation and atherosclerosis. The role of GSNOR in the HHcy-induced T-cell activation and T-cell-driven cardiovascular disease were further verified in human CAD patients with HHcy. The level of S-nitrosylation in T cells should involve systematic regulations among an array of enzymes including NOS and denitrosylases. HHcy would disturb the homeostasis of this regulation in T cells. Our present study shows that Hcy stimulation induced GSNOR expression in T cells in vitro, with a marked decline in the overall protein S-nitrosylation levels and no apparent alterations in iNOS and Trx expression. Elevated level of SNOs with an impaired immune system in GSNOR-/- mice [15], [36] indicates that such exacerbated accumulation of SNOs may have inhibitory effects on T-cell activation. Consistently, in our study, gsnor ablation in GSNOR-/- mice represented an in vivo HHcy model, which recapitulated findings from the in vitro study showing HHcy-induced T-cell activation suppressed in GSNOR-/- mice. Evidence from these in vitro and in vivo studies suggest that GSNOR is a major regulator of Hcy-induced denitrosylation in T cells. The Hcy-Akt signaling axis has been found involved in T-cell activation [21], [22], [23], [33], [37]. For example, Hcy-elicited Akt phosphorylation is linked to the enhanced secretion of proinflammatory cytokines [6]. In accordance with these findings, our study demonstrates that Hcy caused the phosphorylation of Akt at Ser473, and GSNOR deficiency or GSNO supplementation reversed this phosphorylation. Therefore, the HHcy-induced Akt activation in T cells is GSNOR-dependent. S-nitrosylation of Akt in various cell types, including C2C12 myotubes, skeletal cells and esophageal squamous cells, is implicated in the pathogenesis of diabetes, burn injuries and Barrett's esophagus, respectively [25], [26], [27]. Given that Hcy activation of Akt is GSNOR-dependent, Akt may be a key target for GSNOR-mediated denitrosylation in T-cell activation. Indeed, our data in Fig. 3 showed that the level of SNO-Akt in T cells was lower, along with T-cell activation on HHcy stimulation. In terms of the underlying mechanism, we showed that GSNOR-dependent Akt denitrosylation was associated with increased Akt activity and Akt phosphorylation, in particular, at Ser473. The crosstalk between S-nitrosylation and other post-translational modifications such as phosphorylation, ubiquitination and acetylation has been extensively studied [38]. In our study, GSNO exerted an inhibitory effect on the Hcy-induced phosphorylation of Akt at Ser473, which could be restored by the S-nitrosylation inhibitors NAc and DTT. Conserved Cys residues within 8 Å to annotated active sites of Akt1 (i.e., 179 K, 274D) are Cys224, Cys296 and Cys310, as indicated by using PyMOL software. The two reported S-nitrosylation sites of Akt, namely, Cys224 and Cys296 [25], [26], were mutated with Ser in our study. In accordance with Kaneki et al. findings [26], we showed that Cys224 but not Cys296 was the putative residue for S-nitrosylation. However, by using nano-LC interfaced with tandem quadrupole time-of-flight mass spectrometry (Q-TOF)micro tandem mass spectrometry, Fischman et al. showed that S-nitrosylation at Cys296 accelerates its interaction with Cys310 to form a disulfide bond, thus leading to the dephosphorylation at Thr308 [25]. One possible explanation for the lack of S-nitrosylation at Akt Cys296 in our study was the transient existence of S-nitrosylated Cys296, which had switched to a disulfide bond with Cys310 by the time of detection. Instead, our results revealed that S-nitrosylated Cys224 of Akt impaired Akt phosphorylation at Ser473. As previously reported [39], Akt activation by phosphorylation at Ser473 requires a disorder to order transition of α-helix to reconstruct the activation segments. Consistently, our CD spectra for Akt revealed S-nitrosylated Cys224 with decreased α-helical content, so a less-ordered α-helix conformation might interfere with phosphorylation of Ser473. However, further investigations are needed to confirm the exact molecular structural changes in Akt.