Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (H2O2) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate H2O2, have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner. Compared with its epimer trans-WOOH and H2O2, cis-WOOH reacts slower with the major arterial forms of glutathione peroxidases and peroxiredoxins while it reacts more readily with its target, protein kinase G 1α. Our results indicate a paradigm of redox signaling by H2O2 via its enzymatic conversion to an amino acid-derived hydroperoxide that ‘escapes’ effective reductive inactivation to engage in selective oxidative activation of key target proteins.

Protein kinase G signaling pathway is involved in sympathetically maintained pain by modulating ATP-sensitive potassium channels

BackgroundSympathetically maintained pain (SMP) involves an increased excitability of dorsal root ganglion (DRG) neurons to sympathetic nerve stimulation and circulating norepinephrine. The current treatment of SMP has limited efficacy, and hence more mechanistic insights into this intractable pain condition are urgently needed.MethodsA caudal trunk transection (CTT) model of neuropathic pain was established in mice.Immunofluorescence staining, small interfering RNA, pharmacological and electrophysiological studies were conducted to test the hypothesis that norepinephrine increases the excitability of small-diameter DRG neurons from CTT mice through the activation of cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway.ResultsBehavior study showed that CTT mice developed mechanical and heat hypersensitivities, which were attenuated by intraperitoneal injection of guanethidine. CTT mice also showed an abnormal sprouting of tyrosine hydroxylase-positive nerve fibers in DRG, and an increased excitability of small-diameter DRG neurons to norepinephrine, suggesting that CTT is a useful model to study SMP. Importantly, inhibiting cGMP-PKG pathway with small interfering RNA and KT5823 attenuated the increased sympathetic sensitivity in CTT mice. In contrast, cGMP activators (Sp-cGMP, 8-Br-cGMP) further increased sympathetic sensitivity. Furthermore, phosphorylation of ATP-sensitive potassium channel, which is a downstream target of PKG, may contribute to the adrenergic modulation of DRG neuron excitability.ConclusionsOur findings suggest an important role of cGMP-PKG signaling pathway in the increased excitability of small-diameter DRG neurons to norepinephrine after CTT, which involves an inhibition of the ATP-sensitive potassium currents through PKG-induced phosphorylation. Accordingly, drugs targeting this pathway may help to treat SMP.

Single Serine on TSC2 Exerts Biased Control over mTORC1 Activation by ERK1/2 but Not Akt

The mammalian target of rapamycin complex 1 (mTORC1) is tightly controlled by tuberous sclerosis complex-2 (TSC2), itself regulated by kinase phosphorylation reflecting environmental cues. Among these kinases is protein kinase G that modifies TSC2 at S1365 (S1364, human). This minimally affects basal mTORC1 activity, but upon phosphorylation or with an SE mutation, it blocks mTORC1 co-activation by pathological stress. An SA (phospho-silenced) mutation does the opposite. Here we reveal S1365 exerts biased regulation over mTORC1 activity (S6K phosphorylation). In myocytes and fibroblasts, ERK1/2 stimulated mTORC1 via endothelin-1 (ET-1) is potently and bidirectionally regulated by S1365. By contrast, Akt stimulation of mTORC1 (insulin) is minimally impacted. S1365 phosphorylation rises with ET-1 but not insulin stimulation, supporting intrinsic engagement by one and not the other. Energy and nutrient modulation of mTORC1 are minimally influenced by S1365. Consistent with these findings, knock-in mice with SA or SE mutations develop identical obesity, glucose intolerance, and fatty liver disease. These results reveal an ERK1/2-biased TSC2 regulatory mechanism controlling mTORC1 activation, with implications for suppressing pathological but not physiological mTORC1 stimulation.