Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H 2 O 2 and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H 2 S can be generated from persulfide degradation, much of the reported biological activity associated with H 2 S may actually be that of persulfides. That is, H 2 S may act primarily as a marker for the biologically active of persulfide species.thiol redox | hydrogen sulfide | electrophilic signaling | polysulfidomics H ydrogen sulfide (H 2 S) has been suggested to be an endogenous small molecule signaling species (1) by unknown mechanisms. Our laboratory recently showed that the presence of hydrogen sulfide anion (HS − ) may be responsible for the regulation and metabolism of various important electrophilic species [e.g., 8-nitroguanosine 3′,5′-cyclic GMP (8-nitro-cGMP)] (2). However, these studies also indicated that reactive intermediates other than HS − likely react with the electrophiles of interest. These previous studies alluded to the generation of a more reactive sulfur species capable of reacting with electrophiles, such as 8-nitro-cGMP. As reported herein, it was determined that reactive sulfur intermediates, such as hydropersulfides (RSSH) and polysulfides [RS(S) n H and RS(S) n SR], are formed in appreciable amounts during sulfur amino acid metabolism and possess important chemical and biological properties. Some of these sulfide species have long been known as sulfane sulfur compounds, which were suggested to exist endogenously in mammalian systems (1,(3)(4)(5). Reports also indicated that a hydropersulfide moiety with the general molecular formula RSSH may be formed on specific protein cysteine (Cys) residues, most typically of sulfur-transferring enzymes (i.e., sulfurtransferases) during enzymatic reactions (1, 5). Although such persulfide chemical reactivity is thought to be involved in the catalytic activity of particular enzymes (e.g., rhodanese, Cys desulfurases, and sulfide:quinone oxidoreductase) (6, 7), the more general physiological function and occurrence of Cys persulfides (CysSSH) and related species in cells and tissues, especially mammals, were unclear. Moreover, the exact chemical nature ...

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Mammalian HSCs are kept quiescent in the endosteal niche, a hypoxic zone of the bone marrow (BM). In this study, we show that normal HSCs maintain intracellular hypoxia and stabilize hypoxia-inducible factor-1alpha (HIF-1alpha) protein. In HIF-1alpha-deficient mice, the HSCs lost their cell cycle quiescence and HSC numbers decreased during various stress settings including bone marrow transplantation, myelosuppression, or aging, in a p16(Ink4a)/p19(Arf)-dependent manner. Overstabilization of HIF-1alpha by biallelic loss of an E3 ubiquitin ligase for HIF-1alpha (VHL) induced cell cycle quiescence in HSCs and their progenitors but resulted in an impairment in transplantation capacity. In contrast, monoallelic loss of VHL induced cell cycle quiescence and improved BM engraftment during bone marrow transplantation. These data indicate that HSCs maintain cell cycle quiescence through the precise regulation of HIF-1alpha levels.

Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

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Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with ␥-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.An excess dose of acetaminophen (AAP), 4 the most commonly used analgesic, can lead to possibly fatal hepatitis and more than 100 such deaths occur in the United States annually. AAP is normally detoxified by sulfation or glucoronidation followed by elimination (1). In high doses, it is metabolized by P450 cytochromes to generate the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) (2), which is further inactivated by conjugation with glutathione (GSH) before excretion. This results in a sudden drop in GSH levels (3). In absence of sufficient GSH, the reactive NAPQI can cause toxic and covalent protein modifications that lead to cell death and tissue injury (4 -7).Recent transcriptomic and proteomic studies showed that AAP can cause numerous changes in gene and protein expression levels in pathways related to cellular stress response, mitochondrial function, and metabolism, as well as in cell cycle, structural, signaling, and apoptotic proteins (8, 9). However, little is known about global changes in metabolites. Global information about when and where metabolite levels increase or decrease can reveal connections in biological networks and provide a system level understanding of the cell (10 -14). However, unlike other functional genomic approaches, metabolome analysis methods are still under development. Current large scale metabolite analysis methods are based on gas chromatography mass spectrometry (15), liquid chromatography mass spectrometry (LC-MS) (16), NMR (17), Fourier transform ion cyclotron resonance mass spectrometry) (18), and capillary electrophoresis mass spectrometry (CE-MS) (19). Whereas these analytical tools allow global metabolite profiling, the exploration and identification of changes in compounds am...

Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.