Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics

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生物合成谷胱甘肽种间耦合ATP再生系统的构建

利用重组大肠杆菌Ⅱ 1中的谷胱甘肽合成酶系和面包酵母WSH J7中的ATP生物合成酶系 ,构建了一个以葡萄糖为能源的种间耦合ATP再生系统。经过通透性处理的酵母细胞几乎不能消耗葡萄糖。在反应体系中添加 1mmol/LAMP和 0 0 5mmol/LNADH ,即可启动酵母的酵解途径。提高耦合系统中的葡萄糖浓度 ,可促进GSH的合成。当葡萄糖浓度为 40 0mmol/L时 ,系统内GSH浓度达到 1 0 4mmol/L(3 2 g/L)。Mg2 +缺乏时 ,耦合系统和外加ATP的非耦合系统均不能合成谷胱甘肽。耦合系统中Mg2 +与ATP形成螯合物 ,可能是导致耦合系统中GSH产量较低的原因。在耦合系统中补加Mg2 +,反应 6h时GSH浓度达到 1 4 3mmol/L(4 4g/L)。     

Tissue malondialdehyde and adenosine triphosphatase level after experimental liver ischaemia-reperfusion damage

Functional irregularities due to damage after ischaemia-reperfusion vary depending upon the organs affected. High energy phosphates such as ATP and ADP are destroyed after ischaemia-reperfusion damage. Subsequently, protons and inorganic phosphates accumulate within the cells and the proton pumps such as adenosine triphosphatase (ATPase), which maintain intracellular ion balance are damaged. In the present study, malondialdehyde (MDA), a product of lipid peroxidation, was measured as an indicator of tissue damage. Additionally, we measured sodium-potassium-ATPase levels and determined the interactions between MDA and Na+-K+ ATPase levels. A total of 31 female guinea pigs were divided into four groups: sham operated guinea pigs (group 1), ischaemia-reperfusion (group 2), ischaemia-reperfusion + superoxide dismutase (SOD) (group 3), ischaemia-reperfusion + allopurinol (group 4). Following reperfusion, the livers of guinea pigs in each group were removed for histopathological examination and the levels of MDA and Na+-K+ ATPase were determined in homogenized tissue samples. There was a statistically significant (p < 0.05) reduction in tissue MDA levels in group 2 when compared with group 1. The level of tissue MDA in groups 3 and 4 was significantly lower than tissue MDA levels of group 2. However, there was a statistically significant (p < 0.05) reduction in tissue Na+-K+ ATPase levels of group 2 when compared with group 1. Similarly, the level of tissue Na+-K+ ATPase in groups 3 and 4 was significantly higher than the tissue Na+-K+ ATPase levels of group 2. The results of the histopathologic examination also revealed the beneficial effects of the use of SOD and allopurinol in preventing liver damage in cases of ischaemia-reperfusion. Although the levels of MDA and Na+-K+ ATP ase in group 2 were not equal to the level in group 1, antioxidant therapy significantly improved the tendency to reverse the effects of ischaemia-reperfusion and to protect the liver from damage due to ischaemia-reperfusion.     

Identification of P‐glycoprotein co‐fractionating proteins and specific binding partners in rat brain microvessels

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.

     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Lysosomal dysfunction in Parkinson disease: ATP13A2 gets into the groove

Mutations in ATP13A2 (PARK9) cause an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia called Kufor-Rakeb Syndrome (KRS). The ATP13A2 gene encodes a transmembrane lysosomal P5-type ATPase (ATP13A2) whose physiological function in mammalian cells, and hence its potential role in Parkinson disease (PD), remains elusive. In this context, we have recently shown that KRS-linked mutations in ATP13A2 leads to several lysosomal alterations in ATP13A2 KRS patient-derived fibroblasts, including impaired lysosomal acidification, decreased proteolytic processing of lysosomal enzymes, reduced degradation of lysosomal substrates and diminished lysosomal-mediated clearance of autophagosomes (AP). Similar alterations are observed in stable ATP13A2-knockdown dopaminergic cell lines, which are associated with cell death. Restoration of ATP13A2 levels in ATP13A2-mutant/depleted cells is able to restore lysosomal function and attenuate cell death. Relevant to PD, we have determined that ATP13A2 levels are decreased in dopaminergic nigral neurons from sporadic PD patients. Interestingly in these patients, the main signal of ATP13A2 is detected in the Lewy bodies. Our results unravel an instrumental role of ATP13A2 in lysosomal function and in cell viability. Altogether, our results validate ATP13A2 as a likely therapeutic target against PD degeneration.     

Müller Glia as an Active Compartment Modulating Nervous Activity in the Vertebrate Retina: Neurotransmitters and Trophic Factors

Müller cells represent the main type of glia present in the retina interacting with most, if not all neurons in this tissue. Müller cells have been claimed to function as optic fibers in the retina delivering light to photoreceptors with minimal distortion and low loss [Franze et al (2007) Proc Natl Acad Sci 104:8287–8292]. Most of the mediators found in the brain are also detected in the retinal tissue, and glia cells are active players in the synthesis, release, signaling and uptake of major mediators of synaptic function. Müller glia trophic factors may regulate many different aspects of neuronal circuitry during synaptogenesis, differentiation, neuroprotection and survival of photoreceptors, Retinal Ganglion Cells (RGCs) and other targets in the retina. Here we review the role of several transmitters and trophic factors that participate in the neuron-glia loop in the retina. Special issue article in honor of Dr. Ricardo Tapia.     

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.     

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.