Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms.For nearly a century it has been appreciated that an organism's intrinsic metabolic rate is an important determinant of life span. This theory, initially known as the "rate of living" hypothesis, has merged with another proposed mechanism for aging first enunciated by Denham Harman (1) and often called the "free radical theory of aging." The basis for combining these two hypotheses came from observations demonstrating that mitochondria determine both cellular and organismal metabolic rate and that these organelles also produce a continuous stream of reactive oxygen species. Although both the rate of living and the "free radical" theories of aging remain viable and attractive explanations for determining the rate of aging in a wide range of species, neither hypothesis has been conclusively proven (2-4).Surprisingly relatively little is known regarding what regulates the intrinsic metabolic rate of an organism. Similarly on a cellular level the molecular regulation of mitochondrial activity is incompletely understood. Resting oxygen consumption presumably is set at a point to meet overall energetic demands. Nonetheless for most cells this basal respiration is considerably below the maximum oxidative capacity of the mitochondria. The maximal oxidative or ATP synthetic capacity can be easily assessed by treating the cell with various chemical uncouplers and thereby producing the maximal degree of respiration. It is imp...
Abstract-Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC).Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca 2ϩ channel ␣1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca 2ϩ -ATPase, ␣-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase ␣1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart. (Circ Res. 2007;101:1155-1163.) Key Words: preconditioning Ⅲ S-nitrosylation Ⅲ cardioprotection I schemic preconditioning (IPC) is a cellular adaptive phenomenon whereby brief episodes of myocardial ischemia and reperfusion (I/R) render the heart resistant to subsequent prolonged ischemic injury. 1 Through activation of a complex cascade of signaling events, IPC has been shown to reduce arrhythmias, infarct size, and postischemic contractile dysfunction. [2][3][4][5] Nitric oxide (NO) has been shown to be an important signal in cardioprotection. 6 -8 In acute IPC, NO has been shown to mediate protection at least in part by activation of guanylyl cyclase, resulting in the production of cyclic guanosine monophosphate (cGMP) and the activation of protein kinase G, which in turn leads to the opening of the mitochondrial K ATP channel. 6 Recently, it has also been shown that protein kinase G results in activation of an endogenous mitochondrial protein kinase C that is involved in activation of the mitochondrial K ATP channel. 9,10 The opening of the mitochondrial K ATP channel is reported to reduce mitochondrial Ca 2ϩ loading 11,12 and also to lead to generation of reactive oxygen species, which activate signaling cascades in a feed-forward manner to elicit cardioprotection. 13,14 In addition to activating cGMP/protein kinase G-dependent signaling pathways, NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which ha...
Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondria-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondria matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca 2+ ) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis/mass spectrometry of Pro-Q Diamond staining while many more Pro-Q Diamond stained proteins were below mass spectrometry detection. Time dependent 32 P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondria matrix is extensive and dynamic. Ca 2+ has previously been shown to activate various dehydrogenases, promote reactive oxygen species (ROS) generation, and initiate apoptosis via cytochrome c release. To evaluate the Ca 2+ signaling network, the effects of a Ca 2+ challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca 2+ -induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca 2+ dose dependent dephosphorylation of MnSOD was associated with a ∼2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca 2+ . These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca 2+ modification of mitochondrial function at the level of MnSOD.Mitochondria are thought to be the result of an early interaction of two lines of cellular life, the bacterium and eukaryotic cell (1;2). At this point in time, mitochondria play a critical role in energy metabolism, apoptosis and cell signaling pathways in the cell. However, the acute and chronic regulatory mechanisms of this organelle remain poorly defined. One approach to assessing the function and regulation of the mitochondrion is an evaluation of the mitochondrial Address correspondence to: Robert S. Balaban, Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive Room B1D416, Bethesda, MD 20892-1061. Tel. 301 496-3658; Fax. 301 402-2389; E-mail: rsb@nih.gov. proteome. Estimates predict up to 3000 proteins (3;4) in mitochondria, however, recent largescale screening studies by Taylor (5) and Mootha (6) identified only about 600 distinct mitochondrial proteins. Many have used proteomic approaches to evaluate differential protein expr...
Postoperative cognitive dysfunction (POCD) is a subtle disorder of thought processes, which may influence isolated domains of cognition and has a significant impact on patient health. The reported incidence of POCD varies enormously due to lack of formal criteria for the assessment and diagnosis of POCD. The significant risk factors of developing POCD mainly include larger and more invasive operations, duration of anesthesia, advanced age, history of alcohol abuse, use of anticholinergic medications, and other factors. The release of cytokines due to the systemic stress response caused by anesthesia and surgical procedures might induce the changes of brain function and be involved in the development of postoperative cognitive dysfunction. The strategies for management of POCD should be a multimodal approach involving close cooperation between the anesthesiologist, surgeon, geriatricians, and family members to promote early rehabilitation and avoid loss of independence in these patients.
Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed ϳ100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial posttranslational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.
Omental adipose tissue plays a central role in insulin resistance in gestational diabetes mellitus (GDM), and the molecular mechanisms leading to GDM remains vague. Evidence demonstrates that maternal hormones, such as estradiol, contribute to insulin resistance in GDM. In this study we determined the differential expression patterns of microRNAs (miRNAs) in omental adipose tissues from GDM patients and pregnant women with normal glucose tolerance using AFFX miRNA expression chips. MiR-222, 1 of 17 identified differentially expressed miRNAs, was found to be significantly up-regulated in GDM by quantitative real-time PCR (P < .01), and its expression was closely related with serum estradiol level (P < .05). Furthermore, miR-222 expression was significantly increased in 3T3-L1 adipocytes with a high concentration of 17β-estradiol stimulation (P < .01), whereas the expressions of estrogen receptor (ER)-α protein and insulin-sensitive membrane transporter glucose transporter 4 (GLUT4) protein (P < .01) were markedly reduced. In addition, ERα was shown to be a direct target of miR-222 in 3T3-L1 adipocytes by using the luciferase assay. Finally, antisense oligonucleotides of miR-222 transfection was used to silence miR-222 in 3T3-L1 adipocytes. The results showed that the expressions of ERα and GLUT4, the insulin-stimulated translocation of GLUT4 from the cytoplasm to the cell membrane and glucose uptake in mature adipocytes were dramatically increased (P < .01). In conclusion, miR-222 is a potential regulator of ERα expression in estrogen-induced insulin resistance in GDM and might be a candidate biomarker and therapeutic target for GDM.
Background: Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age, is characterized by polycystic ovaries, chronic anovulation, hyperandrogenism and insulin resistance. Despite the high prevalence of hyperandrogenemia, a definitive endocrine marker for PCOS has so far not been identified. Circulating miRNAs have recently been shown to serve as diagnostic/prognostic biomarkers in patients with cancers. Our current study focused on the altered expression of serum miRNAs and their correlation with PCOS. Method and Results: We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to identify and validate the expression of serum miRNAs of PCOS patients. The expression levels of three miRNAs (miR-222, miR-146a and miR-30c) were significantly increased in PCOS patients with respect to the controls in our discovery evaluation and followed validation. The area under the receiver operating characteristic (ROC) curve (AUC) is 0.799, 0.706, and 0.688, respectively. The combination of the three miRNAs using multiple logistic regression analysis showed a larger AUC (0.852) that was more efficient for the diagnosis of PCOS. In addition, logistic binary regression analyses show miR-222 is positively associated with serum insulin, while miR-146a is negatively associated with serum testosterone. Furthermore, bioinformatics analysis indicated that the predicted targets function of the three miRNAs mainly involved in the metastasis, cell cycle, apoptosis and endocrine. Conclusion: Serum miRNAs are differentially expressed between PCOS patients and controls. We identified and validated a class of three serum miRNAs that could act as novel non-invasive biomarkers for diagnosis of PCOS. These miRNAs may be involved in the pathogenesis of PCOS.
Pulmonary arterial hypertension (PAH) is emerging as a major complication and independent risk factor for death among adults with sickle cell disease (SCD). Using surface-enhanced laser desorption/ ionization time of flight mass spectrometry (SELDI-TOF MS), we searched for biomarkers of PAH in plasma specimens from 27 homozygous sickle cell anemia (HbSS) patients with PAH and 28 without PAH. In PAH patients, analysis consistently showed lower abundance of a 28.1-kDa peak (P < .001), identified by high-resolution mass spectrometry as the oxidant-scavenging protein apolipoprotein A-I (apoA-I), which correlated with clinical assays of apoA-I (r ؍ .58, P < .001) and high-density lipoprotein (HDL) levels (r ؍ .50, P ؍ .001). Consistent with endothelial dysfunction that may mediate this effect in PAH, HbSS patients with lower apoA-I levels also displayed impaired vasodilatory responses to acetylcholine (mean ؎ SEM, 189% ؎ 34% [n ؍ 13] vs 339% ؎ 51% [n ؍ 13], P < .001). As a group, patients with SCD demonstrated significantly lower apoA-I levels than African-American control subjects. The PAH cohort was further characterized by high levels of apolipoproteins A-II and B and serum amyloid A, and low levels of haptoglobin dimers and plasminogen. These results imply a relationship of apolipoproteins to the development of PAH vasculopathy in SCD, potentially involving an unexpected mechanistic parallel to atherosclerosis, another proliferative vasculopathy. (Blood. 2009;113:1122-1128) IntroductionPulmonary arterial hypertension (PAH) has emerged as a major complication and independent risk factor for sudden death among adults with sickle cell disease (SCD). A prospective study of 195 sickle cell patients revealed a PAH prevalence of 32% with a relative risk of mortality of 10.1 with tricuspid regurgitant jet velocity (TRV) higher than 2.5 m/s on transthoracic Doppler echocardiography, an indirect indicator of elevated pulmonary artery pressure. 1 This high prevalence and mortality rate are confirmed in other retrospective and prospective studies of PAH in SCD. [2][3][4][5][6][7] Markers that correlate significantly with PAH in SCD include increasing age, hemolysis, iron overload, renal dysfunction, and cholestatic liver dysfunction. 1,[6][7][8][9] Patients with SCD exhibit worse levels of exercise performance, maximal oxygen consumption, and mortality rates at milder elevations of pulmonary arterial pressures than do patients with idiopathic PAH. 2,10 These data indicate that PAH in SCD is associated with impaired cardiopulmonary function and early mortality.Recent epidemiologic, pathophysiological, and biochemical findings have linked PAH in SCD to a hemolysis-linked reduced bioavailability of nitric oxide (NO), a potent vasodilatory, antioxidant, and anti-inflammatory molecule produced endogenously in endothelium. PAH in these patients is further characterized by hemolysis-associated activation of platelets, 11 and variable activation of hemostatic proteins. 12 The only identified plasma marker of PAH in patient...
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