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Colorectal cancer (CRC) is a devastating disease that is highly modulated by dietary nutrients. Mechanistic target of rapamycin complex 1 (mTORC1) contributes to tumor growth and limits therapy responses. Growth factor signaling is a major mechanism of mTORC1 activation. However, compensatory pathways exist to sustain mTORC1 activity after therapies that target oncogenic growth factor signaling. Amino acids potently activate mTORC1 via amino acid-sensing GTPase activity towards Rags (GATOR). The role of amino acid-sensing pathways in CRC is unclear.Human colon cancer cell lines, preclinical intestinal epithelial-specific GATOR1 and GATOR2 knockout mouse subjected to colitis-induced or sporadic colon tumor models, small interfering RNA screening targeting regulators of mTORC1, and tissues of patients with CRC were used to assess the role of amino acid sensing in CRC.We identified loss-of-function mutations of the GATOR1 complex in CRC and showed that altered expression of amino acid-sensing pathways predicted poor patient outcomes. We showed that dysregulated amino acid-sensing induced mTORC1 activation drives colon tumorigenesis in multiple mouse models. We found amino acid-sensing pathways to be essential in the cellular reprogramming of chemoresistance, and chemotherapeutic-resistant patients with colon cancer exhibited de-regulated amino acid sensing. Limiting amino acids in in vitro and in vivo models (low-protein diet) reverted drug resistance, revealing a metabolic vulnerability.Our findings suggest a critical role for amino acid-sensing pathways in driving CRC and highlight the translational implications of dietary protein intervention in CRC.

Exercise is among the most effective interventions for age-associated mobility decline and metabolic dysregulation. Although long-term endurance exercise promotes insulin sensitivity and expands respiratory capacity, genetic components and pathways mediating the metabolic benefits of exercise have remained elusive. Here, we show that Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits. In both fly and mouse models, genetic ablation of Sestrins prevents organisms from acquiring metabolic benefits of exercise and improving their endurance through training. Conversely, Sestrin upregulation mimics both molecular and physiological effects of exercise, suggesting that it could be a major effector of exercise metabolism. Among the various targets modulated by Sestrin in response to exercise, AKT and PGC1α are critical for the Sestrin effects in extending endurance. These results indicate that Sestrin is a key integrating factor that drives the benefits of chronic exercise to metabolism and physical endurance., Exercise improves metabolic health and physical condition, particularly important for health in aged individuals. Here, the authors identify that Sestrins, proteins induced by exercise, are key mediators of the metabolic adaptation to exercise and increase endurance through the AKT and PGC1a axes.

By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis.bAbbreviations:/bAMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1.

Spatial barcoding technologies have the potential to reveal histological details of transcriptomic profiles; however, they are currently limited by their low resolution. Here we report Seq-Scope, a spatial barcoding technology with a resolution almost comparable to an optical microscope. Seq-Scope is based on a solid-phase amplification of randomly barcoded single-molecule oligonucleotides using an Illumina sequencing-by-synthesis platform. The resulting clusters annotated with spatial coordinates are processed to expose RNA-capture moiety. These RNA-capturing barcoded clusters define the pixels of Seq-Scope that are approximately 0.5-1 μm apart from each other. From tissue sections, Seq-Scope visualizes spatial transcriptome heterogeneity at multiple histological scales, including tissue zonation according to the portal-central (liver), crypt-surface (colon) and inflammation-fibrosis (injured liver) axes, cellular components including single cell types and subtypes, and subcellular architectures of nucleus, cytoplasm and mitochondria. Seq-scope is quick, straightforward and easy-to-implement, and makes spatial single cell analysis accessible to a wide group of biomedical researchers.

During nutritional overload and obesity, hepatocyte function is grossly altered, and a subset of hepatocytes begins to accumulate fat droplets, leading to nonalcoholic fatty liver disease (NAFLD). Recent single-cell studies revealed how nonparenchymal cells, such as macrophages, hepatic stellate cells, and endothelial cells, heterogeneously respond to NAFLD. However, it remains to be characterized how hepatocytes, the major constituents of the liver, respond to nutritional overload in NAFLD. Here, using droplet-based, single-cell RNA sequencing (Drop-seq), we characterized how the transcriptomic landscape of individual hepatocytes is altered in response to high-fat diet (HFD) and NAFLD. We showed that the entire hepatocyte population undergoes substantial transcriptome changes upon HFD, although the patterns of alteration were highly heterogeneous, with zonation-dependent and -independent effects. Periportal (zone 1) hepatocytes downregulated many zone 1-specific marker genes, whereas a small number of genes mediating gluconeogenesis were upregulated. Pericentral (zone 3) hepatocytes also downregulated many zone 3-specific genes; however, they upregulated several genes that promote HFD-induced fat droplet formation, consistent with findings that zone 3 hepatocytes accumulate more lipid droplets. Zone 3 hepatocytes also upregulated ketogenic pathways as an adaptive mechanism to HFD. Interestingly, many of the top HFD-induced genes, which encode proteins regulating lipid metabolism, were strongly co-expressed with each other in a subset of hepatocytes, producing a variegated pattern of spatial co-localization that is independent of metabolic zonation. In conclusion, our data set provides a useful resource for understanding hepatocellular alteration during NAFLD at single cell level.

During nutritional overload and obesity, hepatocyte function is grossly altered, and a subset of hepatocytes begins to accumulate fat droplets, leading to non-alcoholic fatty liver disease (NAFLD). Recent single cell studies revealed how non-parenchymal cells, such as macrophages, hepatic stellate cells, and endothelial cells, heterogeneously respond to NAFLD. However, it remains to be characterized how hepatocytes, the major constituents of the liver, respond to nutritional overload in NAFLD. Here, using droplet-based single cell RNA-sequencing (Drop-seq), we characterized how the transcriptomic landscape of individual hepatocytes is altered in response to high-fat diet (HFD) and NAFLD. We showed that the entire hepatocytes population undergoes substantial transcriptome changes upon HFD, although the patterns of alteration were highly heterogeneous with zonation-dependent and –independent effects. Periportal (zone 1) hepatocytes downregulated many zone 1-specific marker genes, while a small number of genes mediating gluconeogenesis were upregulated. Pericentral (zone 3) hepatocytes also downregulated many zone 3-specific genes; however, they upregulated several genes that promote HFD-induced fat droplet formation, consistent with findings that zone 3 hepatocytes accumulate more lipid droplets. Zone 3 hepatocytes also upregulated ketogenic pathways as an adaptive mechanism to HFD. Interestingly, many of the top HFD-induced genes, which encode proteins regulating lipid metabolism, were strongly co-expressed with each other in a subset of hepatocytes, producing a variegated pattern of spatial co-localization that is independent of metabolic zonation. In conclusion, our dataset provides a useful resource for understanding hepatocellular alteration during NAFLD at single cell level.

Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause non-alcoholic steatohepatitis (NASH). NASH in turn is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). The formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here we show that upon saturated fatty acid-induced lipotoxicity, Tank-binding protein kinase 1 (TBK1) is activated and phosphorylates p62. The TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and the formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in the lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of the ubiquitin-p62 aggregates, not only in cultured hepatocytes, but also in mouse models of obesity and NASH. These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions.

Spatial barcoding technologies have the potential to reveal histological details of transcriptomic profiles; however, they are currently limited by their low resolution. Here we report Seq-Scope, a spatial barcoding technology with a resolution comparable to an optical microscope. Seq-Scope is based on a solid-phase amplification of randomly barcoded single-molecule oligonucleotides using an Illumina sequencing platform. The resulting clusters annotated with spatial coordinates are processed to expose RNA-capture moiety. These RNA-capturing barcoded clusters define the pixels of Seq-Scope that are approximately 0.5-0.8 μm apart from each other. From tissue sections, Seq-Scope visualizes spatial transcriptome heterogeneity at multiple histological scales, including tissue zonation according to the portal-central (liver), crypt-surface (colon) and inflammation-fibrosis (injured liver) axes, cellular components including single cell types and subtypes, and subcellular architectures of nucleus and cytoplasm. Seq-scope is quick, straightforward, precise and easy-to-implement, and makes spatial single cell analysis accessible to a wide group of biomedical researchers.

mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins, Tsc1 and Depdc5. Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both Tsc1 and Depdc5 (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury.

Summary: DNA damage often induces heterogeneous cell-fate responses, such as cell-cycle arrest and apoptosis. Through single-cell RNA sequencing (scRNA-seq), we characterize the transcriptome response of cultured colon cancer cell lines to 5-fluorouracil (5FU)-induced DNA damage. After 5FU treatment, a single population of colon cancer cells adopts three distinct transcriptome phenotypes, which correspond to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Although some genes are regulated uniformly across all groups of cells, many genes showed group-specific expression patterns mediating DNA damage responses specific to the corresponding cell fate. Some of these observations are reproduced at the protein level by flow cytometry and are replicated in cells treated with other 5FU-unrelated genotoxic drugs, camptothecin and etoposide. This work provides a resource for understanding heterogeneous DNA damage responses involving fractional killing and chemoresistance, which are among the major challenges in current cancer chemotherapy.

Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate aging and age-associated diseases.

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of metabolism that integrates environmental inputs, including nutrients, growth factors, and stress signals. mTORC1 activation upregulates anabolism of diverse macromolecules, such as proteins, lipids, and nucleic acids, while downregulating autolysosomal catabolism. mTORC1 dysregulation is often found in various diseases, including cancer, cardiovascular and neurodegenerative diseases, as well as metabolic syndromes involving obesity and type II diabetes. As an essential metabolic organ, the liver requires proper regulation of mTORC1 for maintaining homeostasis and preventing pathologies. For instance, aberrant hyper- or hypoactivation of mTORC1 disrupts hepatocellular homeostasis and damages the structural and functional integrity of the tissue, leading to prominent liver injury and the development of hepatocellular carcinogenesis. Proper regulation of mTORC1 during liver diseases may be beneficial for restoring liver function and ameliorating the detrimental consequences of liver failure.

Autophagy is one of the major degradative mechanisms that can eliminate excessive nutrients, toxic protein aggregates, damaged organelles and invading microorganisms. In response to obesity and obesity-associated lipotoxic, proteotoxic and oxidative stresses, autophagy plays an essential role in maintaining physiological homeostasis. However, obesity and its associated stress insults can often interfere with the autophagic process through various mechanisms, which result in further aggravation of obesity-related metabolic pathologies in multiple metabolic organs. Paradoxically, inhibition of autophagy, within specific contexts, indirectly produces beneficial effects that can alleviate several detrimental consequences of obesity. In this minireview, we will provide a brief discussion about our current understanding of the impact of obesity on autophagy and the role of autophagy dysregulation in modulating obesity-associated pathological outcomes.

Lipotoxicity induced by saturated fatty acids (SFAs) plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, the exact mechanisms remain to be fully elucidated. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide-dependent deacetylase located primarily inside mitochondria. In this study, we demonstrated that an SFA-rich high-fat diet (HFD) was more detrimental to the liver than an isocaloric unsaturated HFD rich in fatty acids. Unexpectedly, SIRT3 expression and activity were significantly elevated in the livers of mice exposed to the SFA-rich HFD. Using cultured HepG2 and AML-12 hepatocytes, we demonstrated that unlike monounsaturated fatty acids, SFAs up-regulate SIRT3 expression and activity. SIRT3 overexpression renders both the liver and hepatocytes susceptible to palmitate-induced cell death, which can be alleviated by SIRT3 small interfering RNA (siRNA) transfection. In contrast, SIRT3 suppression protects hepatocytes from palmitate cytotoxicity. Further studies revealed that SIRT3 acts as a negative regulator of autophagy, thereby enhancing the susceptibility of hepatocytes to SFA-induced cytotoxicity. Mechanistic investigations revealed that SIRT3 overexpression causes manganese superoxide dismutase deacetylation and activation, which depleted intracellular superoxide contents, leading to adenosine monophosphate-activated protein kinase (AMPK) inhibition and mammalian target of rapamycin C1 activation, resulting in autophagy suppression. In contrast, SIRT3 siRNA gene silencing enhanced autophagy flux. A similar result was observed in the liver tissue of SIRT3 knockout mice.Our data indicate that SIRT3 is a negative regulator of autophagy whose activation by SFAs contributes to lipotoxicity in hepatocytes and suggest that restraining SIRT3 overactivation can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism. (Hepatology 2017;66:936-952).

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery of epithelial cells after inflammatory injury. During colitis-promoted tumorigenesis, Sestrin2 was shown to be an important mediator of p53’s control over mTORC1 signaling and tumor cell growth. These results highlight Sestrin2 as a novel tumor suppressor, whose downregulation can accelerate both colitis and colon carcinogenesis. DOI: http://dx.doi.org/10.7554/eLife.12204.001, eLife digest An organ that is inflamed has an increased risk of developing cancer. Inflammation can be elicited in various ways; and intestinal inflammation and colon cancer development are often associated with a protein complex – called mTORC1 – being overactive in the tissue. This protein complex has been studied in other contexts and is known to instruct cells to produce more proteins. However, when too much protein is made too quickly, cells cannot carry out their routine quality checks. This, in turn, can lead to unfolded proteins accumulating in the cell, which is stressful and damaging, and can cause inflammation. Increased production of proteins and other biomolecules can also allow the uncontrolled growth of cancer cells. Other recently discovered proteins – called sestrins – can counteract the cancer-promoting effects of overactive mTORC1. Sestrins achieve this via several mechanisms, but as yet almost nobody had studied the role of these proteins in intestinal inflammation and colon cancer. Ro, Xue et al. deleted the genes for two members of the sestrin family, called Sestrin2 and Sestrin3, in mice and showed that their colons were more prone to inflammation. Additional analysis showed that people with ulcerative colitis – a condition in which the colon is chronically inflamed – have elevated levels of Sestrin2, whereas very low levels of Sestrin2 could be detected in tissue samples from patients with colon cancers. These data suggested that Sestrin2 might be trying to protect cells from injury and acts as a barrier to cancer formation. Ro, Xue et al. then used biochemical techniques in human cancer cells grown in the laboratory to show that Sestrin2 inhibits mTORC1, making these cells grow less. Colon cancer cells with little or no Sestrin2 were also more resistant to chemotherapy than control cells with normal levels of Sestrin2. Lastly, a type of colon cancer that is associated with inflammation grew faster in mice that lacked the gene for Sestrin2. Taken together these findings represent evidence that Sestrin2 acts as a tumor suppressor in the colon. Future experiments might investigate how losing Sestrin2 makes these cells more resistant to chemotherapy and whether sestrins act as tumor suppressors in other tissues as well. DOI: http://dx.doi.org/10.7554/eLife.12204.002

Chronic redox imbalance in erythrocytes of individuals with sickle cell disease (SCD) contributes to oxidative stress and likely underlies common etiologies of hemolysis. We measured the amounts of six antioxidant enzymes—SOD1, catalase, glutathione peroxidase 1 (GPx1), as well as peroxiredoxins (Prxs) I, II, and VI—in red blood cells (RBCs) of SCD patients and control subjects. The amounts of SOD1 and Prx VI were reduced by about 17% and 20%, respectively, in SCD RBCs compared with control cells. The amounts of Prx II and GPx1 did not differ between SCD and normal RBCs. However, about 18% of Prx II was inactivated in SCD RBCs as a result of oxidation to sulfinic Prx II, whereas inactive Prx II was virtually undetectable in control cells. Furthermore, GPx1 activity was reduced by about 33% in SCD RBCs, and the loss of activity was correlated with hemolysis in SCD patients. RBCs from SCD patients taking hydroxyurea demonstrated 90% higher GPx1 activity than did those from untreated SCD patients, with no differences seen for the other catalytic antioxidants. Hydroxyurea induced GPx1 expression in multiple cultured cell lines in a manner dependent on both p53 and NO-cGMP signaling pathways. GPx1 expression represents a previously unrecognized potential benefit of hydroxyurea treatment in SCD patients. Antioxid. Redox Signal. 13, 1–11.

Catalase, glutathione peroxidase1 (GPx1), and peroxiredoxin (Prx) II are the principal enzymes responsible for peroxide elimination in RBC. We have now evaluated the relative roles of these enzymes by studying inactivation of GPx1 and Prx II in human RBCs. Mass spectrometry revealed that treatment of GPx1 with H2O2 converts the selenocysteine residue at its active site to dehydroalanine (DHA). We developed a blot method for detection of DHA-containing proteins, with which we observed that the amount of DHA-containing GPx1 increases with increasing RBC density, which is correlated with increasing RBC age. Given that the conversion of selenocysteine to DHA is irreversible, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime. Prx II is inactivated by occasional hyperoxidation of its catalytic cysteine to cysteine sulfinic acid during catalysis. We believe that the activity of sulfiredoxin in RBCs is sufficient to counteract the hyperoxidation of Prx II that occurs in the presence of the basal level of H2O2 flux resulting from hemoglobin autoxidation. If the H2O2 flux is increased above the basal level, however, the sulfinic Prx II begins to accumulate. In the presence of an increased H2O2 flux, inhibition of catalase accelerated the accumulation of sulfinic Prx II, indicative of the protective role of catalase. Antioxid. Redox Signal. 12, 1235–1246.

Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix–turn–helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation., Sestrins are conserved stress-inducible metabolic regulators implicated in the prevention of agerelated diseases. Here, Kim et al. report the crystal structure of human Sestrin2 and propose a molecular mechanism for how Sestrin2 functions to prevent ROS accumulation and inhibit mTORC1 activity.

Various proapoptotic stimuli increase the production of superoxide and H(2)O(2) by mitochondria. Whereas superoxide impairs mitochondrial function and is removed by Mn(2+)-dependent superoxide dismutase, the role and metabolism of mitochondrial H(2)O(2) during apoptosis have remained unclear. The effects on apoptotic signaling of depletion of peroxiredoxin (Prx) III, a mitochondrion-specific H(2)O(2)-scavenging enzyme, have now been investigated by RNA interference in HeLa cells. Depletion of Prx III resulted in increased intracellular levels of H(2)O(2) and sensitized cells to induction of apoptosis by staurosporine or TNF-alpha. The rates of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation were increased in Prx III-depleted cells, and these effects were reversed by ectopic expression of Prx III or mitochondrion-targeted catalase. Depletion of Prx III also exacerbated damage to mitochondrial macromolecules induced by the proapoptotic stimuli. Our results suggest that Prx III is a critical regulator of the abundance of mitochondrial H(2)O(2), which itself promotes apoptosis in cooperation with other mediators of apoptotic signaling.

The catalytic cysteine of the typical 2-Cys Prx subfamily of peroxiredoxins is occasionally hyperoxidized to cysteine sulfinic acid during the peroxidase catalytic cycle. Sulfinic Prx (Prx–SO2H) is reduced back to the active form of the enzyme by sulfiredoxin. The abundance of Prx–SO2H was recently shown to oscillate with a period of ∼24 h in human red blood cells (RBCs). We have now investigated the molecular mechanism and physiological relevance of such oscillation in mouse RBCs. Poisoning of RBCs with CO abolished Prx–SO2H formation, implicating H2O2 produced from hemoglobin autoxidation in Prx hyperoxidation. RBCs express the closely related PrxI and PrxII isoforms, and analysis of RBCs deficient in either isoform identified PrxII as the hyperoxidized Prx in these cells. Unexpectedly, RBCs from sulfiredoxin-deficient mice also exhibited circadian oscillation of Prx–SO2H. Analysis of the effects of protease inhibitors together with the observation that the purified 20S proteasome degraded PrxII–SO2H selectively over nonhyperoxidized PrxII suggested that the 20S proteasome is responsible for the decay phase of PrxII–SO2H oscillation. About 1% of total PrxII undergoes daily oscillation, resulting in a gradual loss of PrxII during the life span of RBCs. PrxII–SO2H was detected in cytosolic and ghost membrane fractions of RBCs, and the amount of membrane-bound PrxII–SO2H oscillated in a phase opposite to that of total PrxII–SO2H. Our results suggest that membrane association of PrxII–SO2H is a tightly controlled process and might play a role in the tuning of RBC function to environmental changes.

Glutamate racemase (MurI) is responsible for the synthesis of D-glutamate, an essential building block of the peptidoglycan layer in bacterial cell walls. The crystal structure of glutamate racemase from Aquifex pyrophilus, determined at 2.3 A resolution, reveals that the enzyme forms a dimer and each monomer consists of two α/β fold domains, a unique structure that has not been observed in other racemases or members of an enolase superfamily. A substrate analog, D-glutamine, binds to the deep pocket formed by conserved residues from two monomers. The structural and mutational analyses allow us to propose a mechanism of metal cofactor-independent glutamate racemase in which two cysteine residues are involved in catalysis.

A specific and sensitive fluorescence-based method was developed for the imaging of microbe-induced HOCl production. Furthermore, we demonstrate dual oxidase (DUOX)-mediated HOCl generation in the mucosa of live animals providing a novel insight into mucosal innate immunity.

Glutathione peroxidase 1 (GPx1), peroxiredoxin II (Prx II), and catalase are the principal enzymes responsible for peroxide elimination in red blood cells (RBCs). GPx1, which contains a selenocysteine (Sec) residue at its active site, is irreversibly inactivated by its own substrate as the result of the oxidation of selenium atom followed by the conversion of oxidized Sec to dehydroalanine (DHA). Prx II is inactivated when its catalytic cysteine (Cys) is hyperoxidized to cysteine sulfinic acid during catalysis. The hyperoxidation can be reversed by sulfiredoxin. The activity of sulfiredoxin in RBCs is sufficient to counteract the Prx II hyperoxidation that occurs during elimination of H2O2 molecules resulting from hemoglobin (Hb) autoxidation. We developed a blot method for detection of DHA-containing proteins, with the use of which we observed that the amount of DHA-containing GPx1 increases with aging of RBCs as well as in RBCs exposed to H2O2 generated either externally by glucose oxidase or internally as a result of aniline-induced Hb autoxidation. Given that the conversion of Sec to DHA is irreversible and that protein turnover mechanism is lacking in RBCs, the content of DHA–GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime. Therefore, DHA–GPx1 in RBCs might be a suitable surrogate marker for evaluation of oxidative stress in the body.

Dehydroalanine (DHA), alpha,beta-unsaturated amino acid, is found in the position corresponding to the serine, cysteine, and selenocysteine (Sec) residues of various proteins. Proteinaceous Sec is readily oxidized and subsequently undergoes beta-elimination to produce DHA. Glutathione peroxidase (GPx), which contains a Sec at the active site, is irreversibly inactivated by its own substrate as the result of the oxidation of selenium atom followed by the conversion of oxidized Sec to DHA. We developed a convenient method for estimation of the amount of DHA-GPx1 in cell homogenates. This blot-based method depends on specific addition of biotin-conjugated cysteamine to the DHA residue followed by detection of biotinylated protein based on its interaction with streptavidin. The method required an immunoprecipitation of GPx1 before labeling with the cysteamine derivative because many other proteins contain DHA. With the use of this method, we found that conversion of the Sec residue at the active site of GPx1 to DHA occurred during aging of red blood cells (RBCs) in vivo as well as in RBCs exposed to H(2)O(2) generated either externally by glucose oxidase or internally as a result of aniline-induced Hb autoxidation. Accordingly, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime of 120 days. Previous studies suggested that the activity of GPx1 in RBCs is most influenced by lifestyle and environmental factors such as the use of dietary supplements and smoking habit. Therefore, DHA-GPx1 in RBCs might be a suitable surrogate marker for evaluation of oxidative stress in the body. Our blot-based method for the detection of DHA-GPx1 will be very useful for evaluation of such stress. In addition, similar blot detection method can be devised for other proteins for which immunoprecipitating antibodies are available.

Dehydroalanine (DHA), α,β-unsaturated amino acid, is found in the position corresponding to the serine, cysteine, and selenocysteine (Sec) residues of various proteins. Proteinaceous Sec is readily oxidized and subsequently undergoes β-elimination to produce DHA. Glutathione peroxidase (GPx), which contains a Sec at the active site, is irreversibly inactivated by its own substrate as the result of the oxidation of selenium atom followed by the conversion of oxidized Sec to DHA. We developed a convenient method for estimation of the amount of DHA–GPx1 in cell homogenates. This blot-based method depends on specific addition of biotin-conjugated cysteamine to the DHA residue followed by detection of biotinylated protein based on its interaction with streptavidin. The method required an immunoprecipitation of GPx1 before labeling with the cysteamine derivative because many other proteins contain DHA. With the use of this method, we found that conversion of the Sec residue at the active site of GPx1 to DHA occurred during aging of red blood cells (RBCs) in vivo as well as in RBCs exposed to H2O2 generated either externally by glucose oxidase or internally as a result of aniline-induced Hb autoxidation. Accordingly, the content of DHA–GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime of 120 days. Previous studies suggested that the activity of GPx1 in RBCs is most influenced by lifestyle and environmental factors such as the use of dietary supplements and smoking habit. Therefore, DHA–GPx1 in RBCs might be a suitable surrogate marker for evaluation of oxidative stress in the body. Our blot-based method for the detection of DHA–GPx1 will be very useful for evaluation of such stress. In addition, similar blot detection method can be devised for other proteins for which immunoprecipitating antibodies are available.

Glutamate racemase catalyzes the reversible reaction of L-glutamate to D-glutamate, an essential component of the bacterial cell wall. Glutamate racemase from Aquifex pyrophilus has been crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P6122 or P6522 with unit-cell parameters a = b = 72.1, c = 185.02 Å. The asymmetric unit contains one molecule, corresponding to a Vm value of 2.35 Å3 Da−1. Complete data sets from a native and a mercury-derivative crystal have been collected at 2.0 and 2.3 Å resolution, respectively, using a synchrotron-radiation source.

Background: Glutathione peroxidase-1 (GPX1) is the most significant catalytic antioxidant in the red cell. Congenital deficiency of GPX1 has been reported in association with hemolytic anemia due to oxidant stress. Hemolytic rate has been implicated in the development of sickle vasculopathy. Methods: Red cell pellets were collected from 32 patients with sickle cell disease and 17 healthy African American control subjects, and were stored frozen until assayed. A novel microplate immunoaffinity capture assay was used that measures both immunoreactive GPX1 protein and enzyme activity. Results: Patients with SCD had higher levels of red cell GPX1 activity than controls (2.42 ± 0.12 vs. 1.86 ± 0.15 units/mg red cell protein (mean ± SEM), p=0.006). GPX1 activity correlated with low reticulocyte count (r=−0.50, p=0.004)(Fig. 1A), low indirect bilirubin (r=–0.45, p=0.01) and high fetal hemoglobin expression (r=0.39, p=0.03). GPX1 activity in SCD patients on hydroxyurea was much higher than those not on hydroxyurea (2.75 ± 0.12, n=21 vs. 1.79 ± 0.13, n=11, p Conclusions: In addition to fetal hemoglobin, hydroxyurea may also induce expression of GPX1, a potent antioxidant that might have a role in decreasing hemolysis associated with the robust oxidant stress of SCD. This finding is consistent with previously published evidence of GPX1 induction by hydroxyurea in cancer cell lines, with increased antioxidant function. Additional studies are needed to confirm this phenomenon in a larger cohort of patients with SCD and to further evaluate the combined effect of fetal hemoglobin co-induction, but this finding suggests a potential mechanism by which hydroxyurea may produce clinical benefit in SCD even in the absence of significant fetal hemoglobin induction. Figure Figure