Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.     

hXRCC2 enhances ADP/ATP processing and strand exchange by hRAD51

The assembly of bacterial RecA, and its human homolog hRAD51, into an operational ADP/ATP-regulated DNA-protein (nucleoprotein) filament is essential for homologous recombination repair (HRR). Yet hRAD51 lacks the coordinated ADP/ATP processing exhibited by RecA and is less efficient in HRR reactions in vitro. In this study, we demonstrate that hXRCC2, one of five other poorly understood non-redundant human mitotic RecA homologs (hRAD51B, hRAD51C, hRAD51D, hXRCC2, and hXRCC3), stimulates hRAD51 ATP processing. hXRCC2 also increases hRAD51-mediated DNA unwinding and strand exchange activities that are integral for HRR. Although there does not seem to be a long-lived interaction between hXRCC2 and hRAD51, we detail a strong adenosine nucleotide-regulated interaction between the hXRCC2-hRAD51D heterodimer and hRAD51. These observations begin to elucidate the separate and specialized functions of the human mitotic RecA homologs that enable an efficient nucleoprotein filament required for HRR.     

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

     

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.     

家蚕ADP/ATP转运酶(BmANT)抑制BmNPV增殖作用机制研究

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)隶属于杆状病毒,需要借助宿主细胞能量代谢进行自身增殖复制。家蚕ADP/ATP转运酶(Bombyx mori ADP/ATP translocase,BmANT)是线粒体转运蛋白,在BmNPV感染条件下和家蚕热休克蛋白60(heatshockprotein60,BmHSP60)具有直接的相互作用。因此,鉴定Bmant基因在BmNPV感染过程中的功能特征,有助于解析杆状病毒劫持宿主细胞因子促进自身增殖复制机制,完善杆状病毒和宿主相互作用网络。【方法】通过结构域预测BmANT蛋白的结构特征,荧光定量PCR分析Bmant基因在BmNPV感染后的变化特征;并过表达BmANT检测其对病毒DNA复制和病毒蛋白表达变化影响;进一步在转录水平分析Bmant和Bmhsp60基因的调控关系;最后通过流式细胞术等技术鉴定Bmant和Bmhsp60基因共同调控BmNPV增殖复制的机制。【结果】SMART软件预测显示BmANT包含3个线粒体载体结构域,BmNPV感染24 h后Bmant基因持续下调表达。过表达Bmant基因能够显著抑制BmNPV DNA的复制和VP39蛋白表达。荧光定量PCR分析显示Bmant和Bmhsp60基因具有相互拮抗作用,能够相互抑制转录。Bmant和Bmhsp60共同过表达分析显示,BmANT和BmHSP60共同作用BmNPV能够抑制病毒的增殖复制。【结论】结果表明,BmANT是一个线粒体载体蛋白,具有显著的抗病毒作用,能够下调Bmhsp60基因表达,并抑制BmNPV增殖复制。     

Influence of divalent cations on the reconstituted ADP, ATP exchange

1. Divalent cations cause a decrease in the exchange activity of the reconstituted ADP,ATP translocator from beef heart mitochondria. This effect is due to complex formation with the adenine nucleotides. 2. It is confirmed that only the free nucleotides are transported. A possible competition of free adenine nucleotides and the Mg2+-complexes for the binding site at the carrier protein is excluded. 3. The stability constants (Kn) for the cation-nucleotide complexes are derived from these experiments. For Mg2+-ATP, Kn = 0.8 x 10(4) M-1 and for Mg2+-ADP, Kn = 0.8 x 10(3) M-1 is obtained. 4. The carrier system was reconstituted with the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine. Interaction of the divalent cations with these phospholipids seem not to be important for the exchange suppression.     

Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport

The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.     

Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange

Growth factor withdrawal is associated with a metabolic arrest that can result in apoptosis. Cell death is preceded by loss of outer mitochondrial membrane integrity and cytochrome c release. These mitochondrial events appear to follow a relative increase in mitochondrial membrane potential. This change in membrane potential results from the failure of the adenine nucleotide translocator (ANT)/voltage-dependent anion channel (VDAC) complex to maintain ATP/ADP exchange. Bcl-xL expression allows growth factor-deprived cells to maintain sufficient mitochondrial ATP/ADP exchange to sustain coupled respiration. These data demonstrate that mitochondrial adenylate transport is under active regulation. Efficient exchange of ADP for ATP is promoted by Bcl-xL expression permitting oxidative phosphorylation to be regulated by cellular ATP/ADP levels and allowing mitochondria to adapt to changes in metabolic demand.     

Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange.

The mammalian 70-kilodalton heat shock cognate protein (Hsc70) is an abundant, cytosolic molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis. In vitro, purified Hsc70 was found to have a slow, intrinsic ATPase activity in the absence of protein substrates. The addition of an unfolded protein such as apocytochrome c stimulated ATP hydrolysis 2-3-fold. In contrast, the native holoprotein, cytochrome c, did not stimulate the ATPase rate, in accord with recent observations that 70-kilodalton heat shock proteins interact selectively with unfolded proteins. Stimulation of ATP hydrolysis by apocytochrome c was due to an increase in the Vmax, with no effect on the Km for ATP. Following hydrolysis of [3H]ATP, a relatively stable [3H]ADP.Hsc70 complex was formed. Release of [3H]ADP from Hsc70 was most efficient in the presence of other nucleotides such as ADP or ATP, suggesting that ADP release occurs as an ADP/ATP exchange reaction. The loss of radiolabeled ADP from Hsc70 in the presence of exogenous nucleotides followed first-order kinetics. In the presence of nucleotides, apocytochrome c induced a 2-fold increase in the rate of ADP release from Hsc70. Moreover, rate constants of the nucleotide exchange reaction measured in the absence and presence of apocytochrome c (0.16 and 0.34 min-1, respectively) closely matched the kcat values derived from ATP hydrolysis measurements (0.15 and 0.38 min-1, respectively). The results suggest that ADP release in a rate-limiting step in the Hsc70 ATPase reaction and that unfolded proteins stimulate ATP hydrolysis by accelerating the rate of ADP/ATP exchange.     

Temperature dependence of ADP/ATP translocation in mitochondria

The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench-back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time-dependent progress at about 10 points. The exchange rate in liver mitochondria was determined from -10 degrees C to + 10 degrees C in the presence of 20% glycol, from 0 degrees C to 25 degrees C, and from 20 degrees C to 40 degrees C under partial inhibition by carboxyatractylate. The total range between -10 degrees C to + 40 degrees C has only one temperature break at 13 degrees C. From the Arrhenius plot between -10 degrees C to + 13 degrees C, EA approximately equal to 140 kJ and above 13 degrees C, EA approximately equal to 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively. In beef heart mitochondria the exchange rate was measured between 0 degrees C and 20 degrees C, and between 15 degrees C and 30 degrees C under partial inhibition with carboxyatractylate. There is a temperature break around 14 degrees C with EA approximately equal to 143 kJ between 0 degrees C and 14 degrees C and EA approximately equal to 60 kJ from 15 degrees C to 30 degrees C. The extrapolated translocation rates at 37 degrees C are 500 and 1800 mumol min-1 (g protein)-1 for rat liver and for beef heart mitochondria respectively. The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14 degrees C in mitochondria has been reported.     

Multimeric structure enables the acceleration of KaiB-KaiC complex formation induced by ADP/ATP exchange inhibition

Circadian clocks tick a rhythm with a nearly 24-hour period in a variety of organisms. In the clock proteins of cyanobacteria, KaiA, KaiB, and KaiC, known as a minimum circadian clock, the slow KaiB-KaiC complex formation is essential in determining the clock period. This complex formation, occurring when the C1 domain of KaiC hexamer binds ADP molecules produced by the ATPase activity of C1, is considered to be promoted by accumulating ADP molecules in C1 through inhibiting the ADP/ATP exchange (ADP release) rather than activating the ATP hydrolysis (ADP production). Significantly, this ADP/ATP exchange inhibition accelerates the complex formation together with its promotion, implying a potential role in the period robustness under environmental perturbations. However, the molecular mechanism of this simultaneous promotion and acceleration remains elusive because inhibition of a backward process generally slows down the whole process. In this article, to investigate the mechanism, we build several reaction models of the complex formation with the pre-binding process concerning the ATPase activity. In these models, six KaiB monomers cooperatively and rapidly bind to C1 when C1 binds ADP molecules more than a given threshold while stabilizing the binding-competent conformation of C1. Through comparison among the models proposed here, we then extract three requirements for the simultaneous promotion and acceleration: the stabilization of the binding-competent C1 by KaiB binding, slow ADP/ATP exchange in the binding-competent C1, and relatively fast ADP/ATP exchange occurring in the binding-incompetent C1 in the presence of KaiB. The last two requirements oblige KaiC to form a multimer. Moreover, as a natural consequence, the present models can also explain why the binding of KaiB to C1 reduces the ATPase activity of C1.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.     

Valine 181 is critical for the nucleotide exchange activity of human mitochondrial ADP/ATP carriers in yeast

We isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment.     

A third ADP/ATP translocator gene in yeast

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.     

Inhibition of electrical activity in mouse pancreatic beta-cells by the ATP/ADP translocase inhibitor, bongkrekic acid

Bongkrekic acid causes fatal food poisoning which is associated with hyperglycaemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic beta-cell through the stimulation of ATP-sensitive potassium channel (K-ATP-channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycaemia by impairing beta-cell function.     

Deep Origin of Plastid/Parasite ATP/ADP Translocases

Abstract Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.     

Suggested mitochondrial ancestry of nonmitochondrial ATP/ADP carrier

One of the major evolutionary events that transformed an endosymbiotic bacterium into a mitochondrion was the acquisition of the ATP/ADP carrier (AAC) in order to supply the host with respiration-derived ATP. Along with the mitochondrial carrier, an unrelated carrier is known, which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic α-proteobacteria. This nonmitochondrial carrier was recently described in rickettsia-like endosymbionts (RLE), a group of obligate intracellular bacteria classified with the order Rickettsiales, which have diverged after free-living α-proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on nonmitochondrial AAC were re-analyzed in the present work, using both DNA and protein sequences and various methods including Bayesian analysis. The data presented are consistent with the classic endosymbiont theory for the origin of mitochondria and suggest that even the last but one common ancestor of rickettsiae and organelles was an endosymbiotic bacterium, in which AAC first originated.