Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier

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Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development

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Abnormal Mitochondrial Genomes in Yeast Restored to Respiratory Competence

When crosses are performed between newly arisen, spontaneous petite mutants of Saccharomyces cerevisiae, respiratory competent (restored) colonies can form. Some of the restored colonies are highly sectored and produce large numbers of petite mutants. The high-frequency petite formation trait is inherited in a non-Mendelian manner, and elimination of mitochondrial DNA from these strains results in the loss of the trait. These results indicate that abnormal mitochondrial genomes are sometimes formed during restoration of respiratory competence. It is hypothesized that these abnormalities result either from recombination between mitochondrial DNA fragments to produce molecules having partial duplications contained on inverted or transposed sequences, or else recombinational "hot spots" have been expanded.     

Glucose Modulates Respiratory Complex I Activity in Response to Acute Mitochondrial Dysfunction

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.     

Stress Activated Protein Kinase Pathway Modulates Homologous Recombination in Fission Yeast

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.     

丙酮酸脱氢酶活性调控在线粒体代谢与生长平衡中的作用

丙酮酸脱氢酶多酶复合体(PDC)催化丙酮酸生成乙酰辅酶 A(acetyl-CoA)的反应是线粒体代谢与生长的调控枢纽.丙酮酸脱氢酶激酶 (PDK)/丙酮酸脱氢酶磷酸酶(PDP)对丙酮酸脱氢酶(PDH)的磷酸化 /脱磷酸化作用以及丙酮酸/乙酰辅酶A对PDH底物产物水平的调控是线粒体适应不同生理环境的代谢调节方式,而调控 PDK基因转录的上游信号恰好也是线粒体生长或生物发生的调控机制.过氧化物酶体增殖物激活受体 (PPAR)/过氧化物酶体增殖物激活受体g共激活因子-1(PGC-1) 信号通路可能是线粒体代谢与生长在基因转录水平的共同调控通路.线粒体代谢与生长经共同通路调节可维持线粒体功能与结构之间的平衡.     

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Background

An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.

Methodology/Principal Findings

We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H₂O₂-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.

Conclusions/Significance

With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.     

Recombination Among Three Mitochondrial Genes in Yeast (Saccharomyces cerevisiae)

Eight crosses involving three different mitochondrial genes were made in Saccharomyces cerevisiae. All three genes were linked, but an unambiguous linkage map could not be constructed.     

Anterograde and Retrograde Regulation of Nuclear Genes Encoding Mitochondrial Proteins during Growth,Development, and Stress

Mitochondrial biogenesis and function in plants require the expression of over 1000 nuclear genes encoding mitochondrial proteins （NGEMPs）. The expression of these genes is regulated by tissue-specific, developmental, internal, and external stimuli that result in a dynamic organelle involved in both metabolic and a variety of signaling processes. Although the metabolic and biosynthetic machinery of mitochondria is relatively well understood, the factors that regu- late these processes and the various signaling pathways involved are only beginning to be identified at a molecular level. The molecular components of anterograde （nuclear to mitochondrial） and retrograde （mitochondrial to nuclear） signaling pathways that regulate the expression of NGEMPs interact with chloroplast-, growth-, and stress-signaling pathways in the cell at a variety of levels, with common components involved in transmission and execution of these signals. This positions mitochondria as important hubs for signaling in the cell, not only in direct signaling of mitochondrial function per se, but also in sensing and/or integrating a variety of other internal and external signals. This integrates and optimizes growth with energy metabolism and stress responses, which is required in both photosynthetic and non-photosynthetic cells.     

植物抗寒性与抗寒基因的表达和调控

综合概述了国内外有关植物抗寒机理的研究动态,主要讨论了抗寒基因的表达与调控在植物抗寒性中的反应。此外,亦提出了有关植物抗寒机制研究领域值得深入研讨的问题。     

Regulation by Magnesium of Potato Tuber Mitochondrial Respiratory Activities

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg²⁺. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg²⁺ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg²⁺. However, respiration of malate, citrate, and -ketoglutarate requires at least 4-mM Mg²⁺ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg²⁺ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or -ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg²⁺. Respiration of succinate is active without external and matrix Mg²⁺, although stimulated by the cation. Respiration of -ketoglutarate was strictly dependent on external Mg²⁺ required for substrate transport into mitochondria, and internal Mg²⁺ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg²⁺ but, unlike -ketoglutarate, some activity still remains without external Mg²⁺. All the substrates revealed insensitive to external and internal mitochondrial Ca²⁺, except the exogenous NADH dehydrogenase, which requires either external Ca²⁺ or Mg²⁺ for detectable activity. Calcium is more efficient than Mg²⁺, both having cumulative stimulation. Unlike Ca²⁺, Mn²⁺ could substitute for Mg²⁺, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg²⁺.     

Analysis of Mitochondrial Proteins in the Surviving Myocardium after Ischemia Identifies Mitochondrial Pyruvate Carrier Expression as Possible Mediator of Tissue Viability

The endogenous mechanisms contributing to tissue survival following myocardial infarction are not fully understood. We investigated the alterations in the mitochondrial proteome after ischemia-reperfusion (I/R) and its possible implications on cell survival. Mitochondrial proteomic analysis of cardiac tissue from an in vivo porcine I/R model found that surviving tissue in the peri-infarct border zone showed increased expression of several proteins. Notably, these included subunits of the mitochondrial pyruvate carrier (MPC), namely MPC1 and MPC2. Western blot, immunohistochemistry, and mRNA analysis corroborated the elevated expression of MPC in the surviving tissue. Furthermore, MPC1 and MPC2 protein levels were found to be markedly elevated in the myocardium of ischemic cardiomyopathy patients. These findings led to the hypothesis that increased MPC expression is cardioprotective due to enhancement of mitochondrial pyruvate uptake in the energy-starved heart following I/R. To test this, isolated mouse hearts perfused with a modified Krebs buffer (containing glucose, pyruvate, and octanoate as metabolic substrates) were subjected to I/R with or without the MPC transport inhibitor UK5099. UK5099 increased myocardial infarction and attenuated post-ischemic recovery of left ventricular end-diastolic pressure. However, aerobically perfused control hearts that were exposed to UK5099 did not modulate contractile function, although pyruvate uptake was blocked as evidenced by increased cytosolic lactate and pyruvate levels. Our findings indicate that increased expression of MPC leads to enhanced uptake and utilization of pyruvate during I/R. We propose this as a putative endogenous mechanism that promotes myocardial survival to limit infarct size.Ischemic heart disease resulting from the obstruction of coronary arteries commonly culminates in myocardial infarction. Although restoring blood flow is necessary to re-establish oxygen and nutrients to the ischemic myocardium, reperfusion itself may promote damage due to exacerbated oxidative stress and inflammation (1). Even when patients survive, the damage caused to the heart typically triggers events leading to heart failure. Unfortunately, there are still no effective interventions for limiting injury after ischemia despite the potential of such therapeutic options to reduce morbidity and mortality.Laboratory and clinical studies suggest that the post-infarction failing heart is characterized by a diminished capacity to convert chemical energy into mechanical work due to mitochondrial imbalances (2). The primary function of mitochondria is to generate ATP via oxidative phosphorylation, which is used by the heart to meet its significant energy demands. In the well perfused healthy heart, ∼70–90% of ATP is via β-oxidation of fatty acids, with most of the remaining amount coming from the oxidation of glucose and lactate (3, 4). However, when oxygen is restricted as occurs during ischemia or sub-optimal reperfusion, there is a metabolic shift with glycolysis becoming the primary mechanism of ATP synthesis (5, 6). This metabolic adaptation is likely to be beneficial because glucose oxidation is more efficient than fatty acid oxidation in terms of oxygen consumption. A complete shift to glucose metabolism reduces oxygen demand by 11–13% (7).A better understanding of the alterations to the mitochondrial proteome that occur following I/R¹ may provide important clues about how to limit injury. Previous studies demonstrated that the myocardial mitochondrial proteome was altered after I/R (8–10), including changes in the expression of electron transport chain (ETC) and energy-producing proteins (11). However, these studies were carried out with small animals and did not address whether changes in protein expression were modulated by proximity to the infarct and probably analyzed the heterogeneous combination of surviving and dying and the necrotic tissue that complicates the interpretation of any alterations to the proteome.Here we utilized a large established animal, a porcine model of I/R (12), and focused specifically on changes to the mitochondrial proteome in the peri-infarct border zone that had survived and remained viable. The rationale was that defining changes to the mitochondrial proteome specifically in the surviving myocardium that had been at risk of necrosis may provide new insight to endogenous adaptive mechanisms that limit tissue injury and infarction. Indeed, this strategy allowed us to identify increased expression of several proteins in the surviving peri-infarct border zone. These included BRP44 and BRP44L, which were recently independently identified as subunits 1 and 2 of the mitochondrial pyruvate carrier (MPC) (13, 14). Enhanced mitochondrial pyruvate uptake capability would potentially provide a mechanism of protection against necrosis (15, 16). Consistent with this, we found pharmacological inhibition of the MPC-dependent pyruvate uptake with UK5099 significantly enhanced infarction and worsened recovery of ventricular function in a murine model of I/R. We conclude that strategies to enhance mitochondrial pyruvate uptake in the post-ischemic heart may have therapeutic value and enhance tissue viability.     

Mitochondrial Respiratory Dysfunction and Oxidative Stress after Chronic Malathion Exposure

Malathion is a pesticide used on a large scale and with high potential risk for human exposure. However, it is reasonable to hypothesize that while the malathion is metabolizing reactive oxygen species (ROS) can be generated and subsequently there is onset of an oxidative stress in central nervous system (CNS) structures: hippocampus, cortex, striatum and cerebellum of intoxicated rats due to mitochondrial respiratory chain disfunctions. The present study was therefore undertaken to evaluate malathion-induced lipid peroxidation (LPO), superoxide production from sub-mitochondrial particles and the activity of complexes II and IV of the mitochondrial respiratory chain. Malathion was administered in doses of 25, 50, 100 and 150 mg malathion/kg. After malathion administration LPO increased in hippocampus and striatum. This was accompanied by an increase in the formation of superoxide in submitochondrial particles in the hippocampus. Complex IV suffered significant inhibition of its activity. We could demonstrate in this study that malathion induces oxidative stress and it could be due to inactivation of mitochondrial respiratory complexes.     

二色补血草LbDREB基因在酵母中的抗逆性分析

利用酵母表达系统研究了二色补血草的DREB基因（LbDREB）对不同胁迫的抗性。将LbDREB构建到酵母表达载体pYES2中,转化到酿酒酵母INVSc1菌株中,并以转空pYES2质粒的酵母INVSc1（pYES2）作为对照,通过比较两种酵母在不同胁迫下的存活率来研究LbDREB基因对NaCl、KH_2PO_4、Na_2CO_3、NaHCO_3、低温、干旱、CuSO_4和CdCl_2胁迫的抗性。结果表明,LbDREB转化的酵母在各种胁迫下的存活率均明显高于转空pYES2的对照酵母,说明LbDREB基因除了具有传统认为的抗旱、耐盐、抗寒的作用外,还具有抗KH_2PO_4、Na_2CO_3、NaHCO_3、CuSO_4和CdCl_2等胁迫的能力。     

Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using ¹³C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival.