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A Quantitative Proteomics Approach to Gain Insight into NRF2-KEAP1 Skeletal Muscle System and Its Cysteine Redox Regulation.

ABSTRACT: Mammalian skeletal muscle (SkM) tissue engages the Nrf2-Keap1-dependent antioxidant defense mechanism to respond adaptively to stress. Redox homeostasis mediated by the reversible modification of selective cysteines is the prevalent mode of regulation. The protein targets of SkM redox regulation are largely unknown. We previously reported the proteomic profiles of soleus (Sol) and extensor digitorum longus (EDL) with Nrf2 or Keap1 gene deletion, using SkM-specific Nrf2 or Keap1 knockout models; iMS-Nrf2flox/flox; and iMS-Keap1flox/flox. Here, we employed these two animal models to understand the global expression profile of red tibialis anterior (RTA) using a label free approach and its redox proteomics using iodoacetyl tandem mass tag (iodoTMT^TM)-labeled cysteine quantitation. We quantified 298 proteins that were significantly altered globally in the RTA with Nrf2 deficiency but only 21 proteins in the Keap1 KO samples. These proteins are involved in four intracellular signaling pathways: sirtuin signaling, Nrf2 mediated oxidative stress response, oxidative phosphorylation, and mitochondrion dysfunction. Moreover, we identified and quantified the cysteine redox peptides of 34 proteins, which are associated with mitochondrial oxidative phosphorylation, energy metabolism, and extracellular matrix. Our findings suggest that Nrf2-deficient RTA is implicated in metabolic myopathy, mitochondrial disorders, and motor dysfunction, possibly due to an enhanced oxidative modification of the structure and functional proteins in skeletal myocytes.

SUBMITTER: Abu R

PROVIDER: S-EPMC8622432 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Key pointsNrf2 is a master regulator of endogenous cellular defences, governing the expression of more than 200 cytoprotective proteins, including a panel of antioxidant enzymes. Nrf2 plays an important role in redox haemostasis of skeletal muscle in response to the increased generation of reactive oxygen species during contraction. Employing skeletal muscle-specific transgenic mouse models with unbiased-omic approaches, we uncovered new target proteins, downstream pathways and molecular networks of Nrf2 in skeletal muscle following Nrf2 or Keap1 deletion. Based on the findings, we proposed a two-way model to understand Nrf2 function: a tonic effect through a Keap1-independent mechanism under basal conditions and an induced effect through a Keap1-dependent mechanism in response to oxidative and other stresses.AbstractAlthough Nrf2 has been recognized as a master regulator of cytoprotection, its functional significance remains to be completely defined. We hypothesized that proteomic/bioinformatic analyses from Nrf2-deficient or overexpressed skeletal muscle tissues will provide a broader spectrum of Nrf2 targets and downstream pathways than are currently known. To this end, we created two transgenic mouse models; the iMS-Nrf2flox/flox and iMS-Keap1flox/flox , employing which we demonstrated that selective deletion of skeletal muscle Nrf2 or Keap1 separately impaired or improved skeletal muscle function. Mass spectrometry revealed that Nrf2-KO changed expression of 114 proteins while Keap1-KO changed expression of 117 proteins with 10 proteins in common between the groups. Gene ontology analysis suggested that Nrf2 KO-changed proteins are involved in metabolism of oxidoreduction coenzymes, purine ribonucleoside triphosphate, ATP and propanoate, which are considered as the basal function of Nrf2, while Keap1 KO-changed proteins are involved in cellular detoxification, NADP metabolism, glutathione metabolism and the electron transport chain, which belong to the induced effect of Nrf2. Canonical pathway analysis suggested that Keap1-KO activated four pathways, whereas Nrf2-KO did not. Ingenuity pathway analysis further revealed that Nrf2-KO and Keap1-KO impacted different signal proteins and functions. Finally, we validated the proteomic and bioinformatics data by analysing glutathione metabolism and mitochondrial function. In conclusion, we found that Nrf2-targeted proteins are assigned to two groups: one mediates the tonic effects evoked by a low level of Nrf2 at basal condition; the other is responsible for the inducible effects evoked by a surge of Nrf2 that is dependent on a Keap1 mechanism.

Project description:Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) andNad(p)hquinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.

Project description:AimsDistinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this end, we have now developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based Cys-reactive isobaric tags (irreversible isobaric iodoacetyl Cys-reactive tandem mass tag [iodoTMT]) endowed with unique irreversible Cys-reactivities.ResultsWe have established a sequential iodoTMT-switch procedure coupled with efficient immunoenrichment and advanced shotgun liquid chromatography-MS/MS analysis. This workflow allows us to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of S-nitrosoglutathione (GSNO), indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect.InnovationWe demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels.ConclusionWe have not only developed a new approach to map global Cys-redoxomic regulation in vivo, but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection.

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A Quantitative Proteomics Approach to Gain Insight into NRF2-KEAP1 Skeletal Muscle System and Its Cysteine Redox Regulation.

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