Presence Of Bongkrekic Acid Research Articles

HIV-1 Vpr disrupts mitochondria axonal transport and accelerates neuronal aging

Disruption of mitochondria axonal transport, essential for the maintenance of synaptic and neuronal integrity and function, has been identified in neurodegenerative diseases. Whether HIV-1 viral proteins affect mitochondria axonal transport is unknown, albeit HIV-associated neurocognitive disorders occur in around half of the patients living with HIV. Therefore, we sought to examine the effect of HIV-1 viral protein R (Vpr) on mitochondria axonal transport. Using mice primary neuronal cultures, we demonstrated that 4-day Vpr treatment reduced the ratio of moving mitochondria associated with (i) less energy (ATP) supply, (ii) reduction in Miro-1 and (iii) increase of α-synuclein which led to loss of microtubule stability as demonstrated by inconsecutive distribution of acetylated α-tubulin along the axons. Interestingly, the effect of Vpr on mitochondria axonal transport was partially restored in the presence of bongkrekic acid, a compound that negatively affected the Vpr-adenine nucleotide translocator (ANT) interaction and totally restored the ATP level in neurons. This indicated Vpr impaired mitochondria axonal transport partially related to its interaction with ANT. The above effect of Vpr was similar to the data obtained from hippocampal tissues isolated from 18-month-old aging mice compared to 5-month-old mice. In accord with previous clinical findings that HIV infection prematurely ages the brain and increases the susceptibility to HAND, we found that Vpr induced aging markers in neurons. Thus, we concluded that instead of causing cell death, low concentration of HIV-1 Vpr altered neuronal function related with inhibition of mitochondria axonal transport which might contribute to the accelerated neuronal aging.

Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier

Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial Apoptosis through Blockage of Caspase-2 Activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Menadione induces a low conductance state of the mitochondrial inner membrane sensitive to bongkrekic acid

When rat liver mitochondria are allowed to cycle Ca 2+ and are incubated in the presence of the pro-oxidant menadione, they undergo swelling, membrane potential (ΔΨ) collapse, and ion release. These effects, which are inhibited by cyclosporin A (CsA), are fully consistent with the opening of the so-called permeability transition pore. However, when Ca 2+ cycling is abolished by EGTA, the mitochondria remain energized (ΔΨ collapse and swelling are avoided), but Ca 2+ efflux, promoted by the chelating agent, is stimulated by menadione. This stimulation goes together with the release of Mg 2+, K +, and adenine nucleotides (AdN) and is inhibited by bongkrekic acid (BKA). The effect of menadione is also characterized by biphasic NAD(P)H oxidation which becomes monophasic in the presence of BKA, CsA, or EGTA and by the oxidation of thiol groups not restrained by the above-mentioned inhibitors. These results suggest that BKA acts indirectly by preserving in the matrix a critical amount of AdN without modifying the monophasic oxidation of pyridine nucleotides by menadione. A critical number of thiol groups also seems to be involved in the phenomenon. Their oxidation most probably causes a conformational change on adenine nucleotide translocase with the opening of the “low-conductance state” of the mitochondrial permeability transition, resulting in ion permeability without ΔΨ disruption and mitochondrial swelling.

Four mutations in transmembrane domains of the mitochondrial ADP/ATP carrier increase resistance to bongkrekic acid.

Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p . BA complex.

Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons.

In cultured cerebrocortical neurons, mild excitotoxic insults or staurosporine result in apoptosis. We show here that N-methyl-d-aspartate (NMDA) receptor-mediated, but not staurosporine-mediated, apoptosis is preceded by depolarization of the mitochondrial membrane potential (Deltapsi(m)) and ATP loss. Both insults, however, release cytochrome c (Cyt c) into the cytoplasm. What prompts mitochondria to release Cyt c and the mechanism of release are as yet unknown. We examined the effect of inhibition of the adenine nucleotide translocator (ANT), a putative component of the mitochondrial permeability transition pore. Inhibition of the mitochondrial ANT with bongkrekic acid (BA) prevented NMDA receptor-mediated apoptosis of cerebrocortical neurons. Concomitantly, BA prevented Deltapsi(m) depolarization, promoted recovery of cellular ATP content, and blocked caspase-3 activation. However, in the presence of BA, Cyt c was still released. Because BA prevented NMDA-induced caspase-3 activation and apoptosis, the presence of Cyt c in the neuronal cytoplasm is not sufficient for the induction of caspase activity or apoptosis. In contrast to these findings, BA was ineffective in preventing staurosporine-induced activation of caspases or apoptosis. Additionally, staurosporine-induced, but not NMDA-induced, apoptosis was associated with activation of caspase-8. These results indicate that, in cerebrocortical cultures, excessive NMDA receptor activation precipitates neuronal apoptosis by means of mitochondrial dysfunction, whereas staurosporine utilizes a distinct pathway.

Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei.

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra-mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2-oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferin/luciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2-oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2-oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria.

Substrate-induced modifications of the intrinsic fluorescence of the isolated adenine nucleotide carrier protein: demonstration of distinct conformational states.

The effects of ATP or ADP and the specific inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) on the conformation of the isolated adenine nucleotide (AdN) carrier protein were studied by fluorescence spectroscopy. The addition of ATP to the AdN carrier resulted in a rapid fluorescence increase of the tryptophanyl residue(s) at 355 nm, which leveled up in less than 1 s at 22 degrees C. Among the natural nucleotides, only ATP and ADP were effective. At 10 degrees C or below, the kinetics of the fluorescence increase induced by ATP were biphasic, consisting of a rapid phase of less than 1 s, followed by a slower phase that lasted for a few seconds and had virtually the same amplitude as the rapid one. Both phases were abolished when CATR was added prior to ATP or fully reversed when CATR was added after the fluorescence response to ATP had been elicited. The number of CATR binding sites present on the carrier protein was determined by CATR specific inhibition of the ATP-induced increase in intrinsic fluorescence. The calculated number of CATR sites was equal to that found by another method based on the use of the same preparation of AdN carrier loaded with fluorescent nucleotide naphthoyl-ATP and on the CATR-induced release of the bound naphthoyl-ATP, demonstrating the reliability of the intrinsic fluorescence assay. Addition of BA prior to or together with ATP nearly doubled the amplitude of the ATP-induced fluorescence signal. At 10 degrees C or below, the fluorescence response to ATP in the presence of BA could also be decomposed into rapid and slow phases.(ABSTRACT TRUNCATED AT 250 WORDS)

The adenine nucleotide carrier of plant mitochondria: contraction induced by adenosine diphosphate

Energised mitochondria show an ADP-induced contraction which is partially resistant to oligomycin, uncouplers or respiratory inhibitors but sensitive to atractyloside. The addition of ADP or ATP, but not AMP, to non-energised corn mitochondria induces a contraction with a K d of approx. 1 μM. Titration of the ADP-induced contraction with atractyloside produces an inhibition curve closely resembling the atractyloside inhibition curve of phosphorylating respiration. Partial recovery of atractyloside-inhibited contraction occurs in the presence of bongkrekic acid. It is suggested that these changes reflect changes in orientation of the adenine nucleotide (AdN) carrier in the inner mitochondrial membrane.

The stimulation of bovine epididymal sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors.

Bovine epididymal spermatozoan motility and respiration are enhanced by cyclic nucleotide phosphodiesterase inhibitors such as caffeine (Garbers et al., 1971). This report deals with additional aspects of the relationship of these cyclic nucleotide phosphodiesterase inhibitors and bovine epididymal sperm metabolism. Caffeine induced elevated cyclic3’,5’-adenosine monophosphate (cyclic AMP) concentrations within 1 mm after addition, with either endogenous substrate or 10 mM fructose as substrate. Although the stimulation of motility was rapid, the maximal respiratory rate with either pyruvate or glucose as substrate did not occur until 1 to 4 mm. After incubation of sperm with caffeine for a period of time, no respiratoly lag occurred on addition of pyruvate. With fructose as substrate, ATP concentrations declined significantly between 3 to 5 mm after addition of caffeine, whereas ATP concentrations were significantly reduced by 2 mm in the absence of exogenous substrate. ADP and AMP concentrations increased as ATP decreased, resulting in a marked decline in the phosphate potential. Bongkrekic acid (BKA), an inhibitor of mitochondrial adenine nucleotide translocase, blocked sperm motility and respiration with acetate or pyruvate as substrate, whereas it had no effect on motility in the presence of glucose. Caffeine was capable of stimulating motility in the presence of glucose and BKA. Caffeine also increased glucose utilization rates, and lactate accumulation from glucose, in the absence or presence of BKA. Pyruvate utilization was either not affected or was reduced by caffeine, whereas lactate accumulation from pyruvate was consistently reduced. These results suggest a general stimulation of sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors. The increase in sperm motility and cyclic AMP is rapid and precedes any significant decline in ATP concentrations or increases in respiratory rate. The data suggest that the fall in ATP concentrations, and therefore the decline in “phosphate potential,” is a causal factor responsible for the glycolytic and respiratory rate enhancement observed in sperm treated with cyclic nucleotide phosphodiesterase inhibitors.