Nobuko Minagawa

Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala

A chemiluminescence study showed that Qi site inhibitors such as antimycin A induce O2 − generation in respiring cyanide‐sensitive mitochondria from the yeast, Hansenula anomala. The O2 − generation was suppressed by radical scavengers such as flavone, butylated hydroxyanisole, and Co0. Induction of cyanide‐resistant respiration in H. anomala cells by Qi site inhibitors was also inhibited by these radical scavengers. Furthermore, antimycin A‐induced synthesis of the mitochondrial 36‐kDa protein, which is thought to be the alternative oxidase functional in the cyanide‐resistant respiratory pathway, was abolished by the addition of flavone. These observations suggest that O2 − is somehow involved in the induction of cyanide‐resistant respiration.

An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei.

Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae, strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei. This inhibition was suggested to be due to inhibition of the mitochondrial electron-transport system, composed of glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) and plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q1-dependent O2 uptake of the mitochondria with respect to ubiquinol (Ki = 2.38 nM). Therefore, the susceptible site is deduced to be the ubiquinone redox machinery which links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Our findings suggest that ascofuranone might be a promising candidate for the chemotherapeutic agents of African trypanosomiasis.

The efficacy of ascofuranone in a consecutive treatment on Trypanosoma brucei brucei in mice.

Consecutive administration of ascofuranone without glycerol was found to have therapeutic efficacy against Trypanosoma brucei brucei infection in mice. A suspension of ascofuranone (25-100 mg/kg) was administrated intraperitoneally every 24 h for 1-4 consecutive days to trypanosome-infected mice and efficacy was compared with oral treatment. With intraperitoneal administration, all mice treated with 100 mg/kg ascofuranone for 4 consecutive days were cured. On contrary, with oral treatment a higher dose of ascofuranone (400 mg/kg) was needed for 8 consecutive days to cure the mice. With intraperitoneal treatment, parasitemia was strongly suppressed, with almost all long slender bloodstream forms of the parasite changed to short stumpy forms by day 3 and the parasites have been eliminated 4 days after the start of treatment. These ascofuranone-induced short stumpy forms were morphologically analogous to the stumpy forms 2 days after peak parasitemia of pleomorphic clone of T. b. brucei GUTat 3.1. However, the properties of ubiquinol oxidase activity, which is the target of ascofuranone, in mitochondria isolated from before and after treatment, were almost same. The enzymatic activities of ubiquinol oxidase were only decreased to approximately 30% within a day after treatment, and then kept at nearly the same level. In the present study, we have improved regimen for administration of ascofuranone without glycerol, and demonstrated that consecutively administrated ascofuranone showed trypanocidal effects in T. b. brucei infected mice. Our present results strongly suggest that consecutive administration of ascofuranone may be an effective chemotherapy for African trypanosomiasis.

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.

Erratum to ``An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei'': [Mol. Biochem. Parasitol. 81 (1996) 127–136]1

Abstract Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae , strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O 2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei . This inhibition was suggested to be due to inhibition of the mitochondria electron-transport system, composed of glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) and plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q 1 -dependent O 2 uptake of the mitochondria with respect to ubiquinol (K i =2.38 nM). Therefore, the susceptible site is deduced to be the ubiquinone redox machinery which links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Our findings suggest that ascofuranone might be a promising candidate for the chemotherapeutic agents of African trypanosomiasis.

Characterization of the alternative oxidase protein in the yeast Hansenula anomala

The cyanide‐resistant respiratory pathway is induced by respiratory inhibitors in the yeast Hansenula anomala. A monoclonal antibody against the alternative oxidase in the higher plant Sauromatum guttatum cross‐reacted with a 36‐kDa mitochondrial protein induced by antimycin A in H. anomala and with a protein encoded by a cDNA which was previously cloned for an antimycin A‐inducible mRNA in the yeast. There was a similarity in the amino acid sequence between the cDNA‐encoded protein and the plant alternative oxidase protein. We propose that the 36‐kDa mitochondrial protein encoded by the cDNA is a component of alternative oxidase in H. anomala.

Purification of active recombinant trypanosome alternative oxidase

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain in long slender bloodstream forms of African trypanosomes. TAO is a cytochrome‐independent, cyanide‐insensitive quinol oxidase. These characteristics are distinct from those of the bacterial quinol oxidases, proteins that belong to the heme‐copper terminal oxidase superfamily. The inability to purify stable TAO has severely hampered biochemical studies of the alternative oxidase family. In the present study, we were able to purify recombinant TAO to homogeneity from Escherichia coli membranes using the detergent digitonin. Kinetic analysis of the purified TAO revealed that the specific inhibitor ascofuranone is a competitive inhibitor of ubiquinol oxidase activity.

Oral and Intraperitoneal treatment of Trypanosoma brucei brucei with a combination of ascofuranone and glycerol in mice

Abstract On the basis of our previous report of ascofuranone, an antibiotic isolated from Ascochyta visiae , which strongly inhibited both the mitochondrial O 2 consumption in mitochondrial preparation and growth of in vitro cultured bloodstream forms of Trypanosoma brucei brucei in combination with glycerol, we investigated the chemotherapeutic efficacy of ascofuranone on experimental African trypanosomiasis in mice. A suspension of ascofuranone (6–200 mg/kg) was given and then glycerol (1 g/kg) was administered orally or intraperitoneally at 30-min intervals to heavily parasitemic mice. Both orally (100 mg/kg) and intraperitoneally (25 mg/kg) administered ascofuranone combined with a total dose of 3 g/kg glycerol showed potent antitrypanosomal activity in infected mice. The trypanocidal activity of ascofuranone was very powerful and all trypanosomes disappeared within 30 and 180 min after final intraperitoneal and oral treatment, respectively. This combination treatment showed high efficacy and low toxicity. Our results strongly suggest that ascofuranone in combination with glycerol may be an effective tool in chemotherapy for African trypanosomiasis.

A 36-kDa mitochondrial protein is responsible for cyanide-resistant respiration in Hansenula anomala

Antimycin A‐dependent induction of cyanide‐resistant respiration in Hansenula anomala was reversibly blocked by carbonylcyanide‐m‐chlorophenylhydrazone (CCCP). When the cells were pulse‐labeled with [35S]methionine in the presence of both antimycin A and CCCP, the radioactivity was incorporated into a 39 kDa mitochondrial protein. Upon removal of CCCP, this protein was processed into a 36 kDa form. The increase in the 36 kDa protein completely paralleled that in cyanide‐resistant respiration activity, suggesting that the 39 kDa protein is the precursor of the 36 kDa protein, which is responsible for cyanide‐resistant respiration.

Strain-specific difference in amino acid sequences of trypanosome alternative oxidase.

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. The cDNA for TAO has been cloned from Trypanosoma brucei brucei EATRO110 strain and has been used for further characterization. In this study, we found amino acid sequence of the C-terminal part of TAO from the strain that we are using, T. b. brucei TC221, is considerably different from that of the EATRO110 strain.