Redox Regulation of Phosphatase and Tensin Homolog by Bicarbonate and Hydrogen Peroxide: Implication of Peroxymonocarbonate in Cell Signaling.

Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) signaling pathway. Notably, its active site contains a cysteine residue that is susceptible to oxidation by hydrogen peroxide (H2O2). This oxidation inhibits the phosphatase function of PTEN, critically contributing to the activation of the PI3K/AKT pathway. Upon the stimulation of cell surface receptors, the activity of NADPH oxidase (NOX) generates a transient amount of H2O2, serving as a mediator in this pathway by oxidizing PTEN. The mechanism underlying this oxidation, occurring despite the presence of highly efficient and abundant cellular oxidant-protecting and reducing systems, continues to pose a perplexing conundrum. Here, we demonstrate that the presence of bicarbonate (HCO3-) promoted the rate of H2O2-mediated PTEN oxidation, probably through the formation of peroxymonocarbonate (HCO4-), and consequently potentiated the phosphorylation of AKT. Acetazolamide (ATZ), a carbonic anhydrase (CA) inhibitor, was shown to diminish the oxidation of PTEN. Thus, CA can also be considered as a modulator in this context. In essence, our findings consolidate the crucial role of HCO3- in the redox regulation of PTEN by H2O2, leading to the presumption that HCO4- is a signaling molecule during cellular physiological processes.

Redox Regulation of PTEN by Reactive Oxygen Species: Its Role in Physiological Processes.

Phosphatase and tensin homolog (PTEN) is a tumor suppressor due to its ability to regulate cell survival, growth, and proliferation by downregulating the PI3K/AKT signaling pathway. In addition, PTEN plays an essential role in other physiological events associated with cell growth demands, such as ischemia-reperfusion, nerve injury, and immune responsiveness. Therefore, recently, PTEN inhibition has emerged as a potential therapeutic intervention in these situations. Increasing evidence demonstrates that reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), are produced and required for the signaling in many important cellular processes under such physiological conditions. ROS have been shown to oxidize PTEN at the cysteine residue of its active site, consequently inhibiting its function. Herein, we provide an overview of studies that highlight the role of the oxidative inhibition of PTEN in physiological processes.

What matters in aging is signaling for responsiveness.

Biological responsiveness refers to the capacity of living organisms to adapt to changes in both their internal and external environments through physiological and behavioral mechanisms. One of the prominent aspects of aging is the decline in this responsiveness, which can lead to a deterioration in the processes required for maintenance, survival, and growth. The vital link between physiological responsiveness and the essential life processes lies within the signaling systems. To devise effective strategies for controlling the aging process, a comprehensive reevaluation of this connecting loop is imperative. This review aims to explore the impact of aging on signaling systems responsible for responsiveness and introduce a novel perspective on intervening in the aging process by restoring the compromised responsiveness. These innovative mechanistic approaches for modulating altered responsiveness hold the potential to illuminate the development of action plans aimed at controlling the aging process and treating age-related disorders.

The Role of Oxidative Inactivation of Phosphatase PTEN and TCPTP in Fatty Liver Disease.

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are becoming increasingly prevalent worldwide. Despite the different etiologies, their spectra and histological feature are similar, from simple steatosis to more advanced stages such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Studies including peroxiredoxin knockout models revealed that oxidative stress is crucial in these diseases, which present as consequences of redox imbalance. Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes that are major targets of reactive oxygen species (ROS) because of an oxidation-susceptible nucleophilic cysteine in their active site. Herein, we review the oxidative inactivation of two tumor suppressor PTPs, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and T-cell protein tyrosine phosphatase (TCPTP), and their contribution to the pathogenicity of ALD and NAFLD, respectively. This review might provide a better understanding of the pathogenic mechanisms of these diseases and help develop new therapeutic strategies to treat fatty liver disease.

Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins.

Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Redox Regulation of PTEN by Peroxiredoxins.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known as a tumor suppressor gene that is frequently mutated in numerous human cancers and inherited syndromes. PTEN functions as a negative regulator of PI3K/Akt signaling pathway by dephosphorylating phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) to phosphatidylinositol (4, 5)-bisphosphate (PIP2), which leads to the inhibition of cell growth, proliferation, cell survival, and protein synthesis. PTEN contains a cysteine residue in the active site that can be oxidized by peroxides, forming an intramolecular disulfide bond between Cys124 and Cys71. Redox regulation of PTEN by reactive oxygen species (ROS) plays a crucial role in cellular signaling. Peroxiredoxins (Prxs) are a superfamily of peroxidase that catalyzes reduction of peroxides and maintains redox homeostasis. Mammalian Prxs have 6 isoforms (I-VI) and can scavenge cellular peroxides. It has been demonstrated that Prx I can preserve and promote the tumor-suppressive function of PTEN by preventing oxidation of PTEN under benign oxidative stress via direct interaction. Also, Prx II-deficient cells increased PTEN oxidation and insulin sensitivity. Furthermore, Prx III has been shown to protect PTEN from oxidation induced by 15s-HpETE and 12s-HpETE, these are potent inflammatory and pro-oxidant mediators. Understanding the tight connection between PTEN and Prxs is important for providing novel therapies. Herein, we summarized recent studies focusing on the relationship of Prxs and the redox regulation of PTEN.

The critical role of redox regulation of PTEN and peroxiredoxin III in alcoholic fatty liver.

Hepatic steatosis and subsequent fatty liver disease are developed in response to alcohol consumption. Reactive oxygen species (ROS) are thought to play an important role in the alcoholic fatty liver disease (AFLD). However, the molecular targets of ROS and the underlying cellular mechanisms are unknown. Here, we investigate roles of peroxiredoxin III and redox regulation of phosphatase and tension homolog deleted on chromosome 10 (PTEN) in the alcoholic fatty liver. Alcohol-induced mitochondrial oxidative stress was found to contribute to reversible oxidation of PTEN, which results in Akt and MAPK hyperactivation with elevated levels of the lipogenesis regulators SREBP1c and PPARγ. Moreover, mitochondrial peroxiredoxin III was found to have antagonistic effects on lipogenesis via the redox regulation of PTEN by removing ROS, upon alcohol exposure. This study demonstrated that redox regulation of PTEN and peroxiredoxin III play crucial roles in the development of AFLD.

Role of Selenoproteins in Redox Regulation of Signaling and the Antioxidant System: A Review.

Selenium is a vital trace element present as selenocysteine (Sec) in proteins that are, thus, known as selenoproteins. Humans have 25 selenoproteins, most of which are functionally characterized as oxidoreductases, where the Sec residue plays a catalytic role in redox regulation and antioxidant activity. Glutathione peroxidase plays a pivotal role in scavenging and inactivating hydrogen and lipid peroxides, whereas thioredoxin reductase reduces oxidized thioredoxins as well as non-disulfide substrates, such as lipid hydroperoxides and hydrogen peroxide. Selenoprotein R protects the cell against oxidative damage by reducing methionine-R-sulfoxide back to methionine. Selenoprotein O regulates redox homeostasis with catalytic activity of protein AMPylation. Moreover, endoplasmic reticulum (ER) membrane selenoproteins (SelI, K, N, S, and Sel15) are involved in ER membrane stress regulation. Selenoproteins containing the CXXU motif (SelH, M, T, V, and W) are putative oxidoreductases that participate in various cellular processes depending on redox regulation. Herein, we review the recent studies on the role of selenoproteins in redox regulation and their physiological functions in humans, as well as their role in various diseases.

Redox regulation of tumor suppressor PTEN in cell signaling.

Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) is a potent tumor suppressor and often dysregulated in cancers. Cellular PTEN activity is restrained by the oxidation of active-site cysteine by reactive oxygen species (ROS). Recovery of its enzymatic activity predominantly depends on the availability of cellular thioredoxin (Trx) and peroxiredoxins (Prx), both are important players in cell signaling. Trx and Prx undergo redox-dependent conformational changes through the oxidation of cysteine residues at their active sites. Their dynamics are essential for protein functionality and regulation. In this review, we summarized the recent advances regarding the redox regulation of PTEN, with a specific focus on our current state-of-the-art understanding of the redox regulation of PTEN. We also proposed a tight association of the redox regulation of PTEN with Trx dimerization and Prx hyperoxidation, providing guidance for the identification of novel therapeutic targets.

Peroxiredoxin III Protects Tumor Suppressor PTEN from Oxidation by 15-Hydroperoxy-eicosatetraenoic Acid.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase that coordinates various cellular processes. Its activity is regulated by the reversible oxidation of an active-site cysteine residue by H2O2 and thioredoxin. However, the potential role of lipid peroxides in the redox regulation of PTEN remains obscure. To evaluate this, 15-hydroperoxy-eicosatetraenoic acid (15s-HpETE), a lipid peroxide, was employed to investigate its effect on PTEN using molecular and cellular-based assays. Exposure to 15s-HpETE resulted in the oxidation of recombinant PTEN. Reversible oxidation of PTEN was also observed in mouse embryonic fibroblast (MEF) cells treated with a 15s-HpETE and Lipofectamine mixture. The oxidative dimerization of thioredoxin was found simultaneously. In addition, the absence of peroxiredoxin III aggravated 15s-HpETE-induced PTEN oxidation in MEF cells. Our study provides novel insight into the mechanism linking lipid peroxidation to the etiology of tumorigenesis.

Redox regulation of the tumor suppressor PTEN by the thioredoxin system and cumene hydroperoxide.

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.

Redox Regulation of the Tumor Suppressor PTEN by Hydrogen Peroxide and Tert-Butyl Hydroperoxide.

Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert-butyl hydroperoxide (t-BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation.

Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.

Protective effect of diet supplemented with rice prolamin extract against DNCB-induced atopic dermatitis in BALB/c mice.

BACKGROUND: Rice prolamin has been reported to possess antioxidative, anti-inflammatory and immune-promoting properties. This study is aimed to examine the protective effects of dietary rice prolamin extract (RPE) against dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like skin lesions in mice. METHODS: BALB/c mice were fed diet supplemented with 0-0.1 % RPE for 6 weeks. For the last 2 weeks, 1 % or 0.2 % DNCB was applied repeatedly to the back skin of mice to induce AD-like lesions. Following AD induction, the severity of skin lesions was examined macroscopically and histologically. In addition, the serum levels of IgE, IgG1 and IgG2a were determined by ELISA, and the mRNA expression of IL-4 and IFN-γ in the skin was determined by real-time PCR. RESULTS: Dietary RPE suppressed the clinical symptoms of DNCB-induced dermatitis as well as its associated histopathological changes such as epidermal hyperplasia and infiltration of mast cells and eosinophils in the dermis. RPE treatment also suppressed the DNCB-induced increase in transepidermal water loss. Dietary RPE inhibited the DNCB-induced enhancement of serum IgE and IgG1 levels, whereas it increased the serum IgG2a level in DNCB-treated mice. In addition, dietary RPE upregulated the IFN-γ mRNA expression and downregulated the IL-4 mRNA expression in the skin of DNCB-treated mice. CONCLUSIONS: The above results suggest that dietary RPE exerts a protective effect against DNCB-induced AD in mice via upregulation of Th1 immunity and that RPE may be useful for the treatment of AD.

Assay of the redox state of the tumor suppressor PTEN by mobility shift.

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.

A unified nomenclature and amino acid numbering for human PTEN.

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

Characterization of mammalian selenoprotein o: a redox-active mitochondrial protein.

Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells with 75Se, and it was abolished when selenocysteine was replaced with serine. A CxxU motif was identified in the C-terminal region of SelO. This protein was reversibly oxidized in a time- and concentration-dependent manner in HEK 293T cells when cells were treated with hydrogen peroxide. This treatment led to the formation of a transient 88 kDa SelO-containing complex. The formation of this complex was enhanced by replacing the CxxU motif with SxxC, but abolished when it was replaced with SxxS, suggesting a redox interaction of SelO with another protein through its Sec residue. SelO was localized to mitochondria and expressed across mouse tissues. Its expression was little affected by selenium deficiency, suggesting it has a high priority for selenium supply. Taken together, these results show that SelO is a redox-active mitochondrial selenoprotein.

Pro178 and Pro183 of selenoprotein S are essential residues for interaction with p97(VCP) during endoplasmic reticulum-associated degradation.

During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro(178) and Pro(183) of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor.

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFßR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFßR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFßR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFßR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4.

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.