Trypanosome Alternative Oxidase Research Articles

Structure of the trypanosomal alternative oxidase: Opportunities for rational drug design to treat trypanosomiasis

rates and electron transport efficiency, although membrane integrity, cellular ATP levels and rates of ATP synthesis are unaltered as are bacterial growth rates. However, in vitro infection studies using J774A.1 cells and theM. tuberculosis Rv0561c deletionmutant demonstrated that the mutant is unable to survive in macrophage-like cells. Thus, Rv0561c represents a novel, contextually essential enzyme that is a potential drug target inM. tuberculosis. Funded by NIH/NIAID grant # AI049151.

Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.

The alternative oxidases: simple oxidoreductase proteins with complex functions

The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water.

Structure of the trypanosome cyanide-insensitive alternative oxidase.

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.

Pharmacophore identification of ascofuranone, potent inhibitor of cyanide-insensitive alternative oxidase of Trypanosoma brucei

Trypanosoma brucei is a parasite that causes human African trypanosomiasis (HAT). The parasites depend on the cyanide-insensitive trypanosome alternative oxidase (TAO) for their vital aerobic respiration. Ascofuranone (AF), a potent and specific sub-nanomolar inhibitor of the TAO quinol oxidase, is a potential novel drug with selectivity for HAT, because mammalian hosts lack the enzyme. To elucidate not only the inhibition mechanism but also the inhibitor-enzyme interaction, AF derivatives were designed and synthesized, and the structure-activity relationship was evaluated. Here we identified the pharmacophore of AF that interacts with TAO. The detailed inhibitory profiles indicated that the 1-formyl and 6-hydroxyl groups, which might contribute to intramolecular hydrogen bonding and/or serve as hydrogen-bonding donors, were responsible for direct interaction with the enzyme.

Mitochondria as Targets of Chemotherapy

Living organisms have developed a wide variety of energy metabolism to survive within the specialized environments. There is a remarkable diversity in mitochondrial electron transport system, which might be potential targets for chemotherapy. Atovaquone, clinically used to treat malaria and pneumocystis pneumonia, is a specific inhibitor of Qo site in the cytochrome bc(1) complex of Plasmodium falciparum and Pneumocystis jirovecii. Phytopathogenic fungus, Ascochyta viciae produces two antibiotics, ascochlorin and ascofuranone. Ascochlorin specifically binds to inhibit the electron transport of both Qi and Qo sites in cytochrome bc(1) complex. Besides the unique respiratory inhibition, further investigation is in progress to elucidate the effects on cancer cells. On the other hand, ascofuranone specifically inhibits cyanide-insensitive trypanosome alternative oxidase, which is a sole terminal oxidase in the mitochondrion of Trypanosoma brucei, causative of African trypanosomiasis. In vivo study suggests that ascofuranone is a promising candidate for chemotherapeutic agents to treat African trypanosomiasis.

ChemInform Abstract: A Short and Efficient Total Synthesis of (.+‐.)‐Ascofuranone.

AbstractAscofuranone (V), a potent inhibitor of trypanosome alternative oxidase, is synthesized in seven steps starting from geranyl acetate.

Trypanosome alternative oxidase, a potential therapeutic target for sleeping sickness, is conserved among Trypanosoma brucei subspecies

Trypanosoma brucei rhodesiense and T. b. gambiense are known causes of human African trypanosomiasis (HAT), or “sleeping sickness,” which is deadly if untreated. We previously reported that a specific inhibitor of trypanosome alternative oxidase (TAO), ascofuranone, quickly kills African trypanosomes in vitro and cures mice infected with another subspecies, non-human infective T. b. brucei, in in vivo trials. As an essential factor for trypanosome survival, TAO is a promising drug target due to the absence of alternative oxidases in the mammalian host. This study found TAO expression in HAT-causing trypanosomes; its amino acid sequence was identical to that in non-human infective T. b. brucei. The biochemical understanding of the TAO including its 3 dimensional structure and inhibitory compounds against TAO could therefore be applied to all three T. brucei subspecies in search of a cure for HAT. Our in vitro study using T. b. rhodesiense confirmed the effectiveness of ascofuranone (IC 50 value: 1 nM) to eliminate trypanosomes in human infective strain cultures.

Role of Tob55 on mitochondrial protein biogenesis in Trypanosoma brucei

Mitochondrial outer membrane (MOM) proteins in parasitic protozoa like Trypanosoma brucei are poorly characterized. In fungi and higher eukaryotes, Tob55 is responsible for the assembly of β-barrel proteins in the MOM. Here we show that T. brucei Tob55 (TbTob55) has considerable similarity in its primary and secondary structure to Tob55 from other species. TbTob55 is localized in T. brucei MOM and is essential for procyclic cell survival. Induction of Tob55 RNAi decreased the level of the voltage-dependent anion channel (VDAC) within 48h. Although the primary effect is on VDAC, induction of TbTob55 RNAi for 96h or more also decreased the levels of other nucleus encoded mitochondrial proteins. In addition, the mitochondrial membrane potential was reduced at this later time point possibly due to a reduction in the level of the proteins involved in oxidative phosphorylation. However, mitochondrial structure was not altered due to depletion of Tob55. In vitro protein import of VDAC into mitochondria with a 50–60% reduction of TbTob55 was reduced about 40% in comparison to uninduced control. In addition, the import of presequence-containing proteins such as, cytochrome oxidase subunit 4 (COIV) and trypanosome alternative oxidase (TAO) was affected by about 20% under this condition. Depletion of VDAC levels by RNAi did not affect the import of either COIV or TAO. Furthermore, TbTob55 over expression increased the steady state level of VDAC as well as the level of the assembled protein complex of VDAC, suggesting that similar to other eukaryotes TbTob55 is involved in assembly of MOM β-barrel proteins and plays an indirect role in the biogenesis of mitochondrial preproteins destined for the mitochondrial inner membrane.

A Short and Efficient Total Synthesis of (±)-Ascofuranone

Abstract Ascofuranone is a potent inhibitor of trypanosome alternative oxidase. A short and efficient total synthesis of (±)-ascofuranone was accomplished in only seven steps starting with geranyl acetate. The synthetic concept and methodology described in detail here provide a short and simple synthesis for ascofuranone and its derivatives.

Crystallization and preliminary crystallographic analysis of cyanide-insensitive alternative oxidase from Trypanosoma brucei brucei.

Cyanide-insensitive alternative oxidase (AOX) is a mitochondrial membrane protein and a non-proton-pumping ubiquinol oxidase that catalyzes the four-electron reduction of dioxygen to water. In the African trypanosomes, trypanosome alternative oxidase (TAO) functions as a cytochrome-independent terminal oxidase that is essential for survival in the mammalian host; hence, the enzyme is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in haem-deficient Escherichia coli was purified and crystallized at 293 K by the hanging-drop vapour-diffusion method using polyethylene glycol 400 as a precipitant. X-ray diffraction data were collected at 100 K and were processed to 2.9 A resolution with 93.1% completeness and an overall R(merge) of 9.5%. The TAO crystals belonged to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 63.11, b = 136.44, c = 223.06 A. Assuming the presence of two rTAO molecules in the asymmetric unit (2 x 38 kDa), the calculated Matthews coefficient (V(M)) was 3.2 A(3) Da(-1), which corresponds to a solvent content of 61.0%. This is the first report of a crystal of the membrane-bound diiron proteins, which include AOXs.

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis–Menten kinetics ( K m of 338 μM and V max of 601 μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

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.

Downregulation of Mitochondrial Porin Inhibits Cell Growth and Alters Respiratory Phenotype in Trypanosoma brucei

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a beta-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in T. brucei according to mitochondrial energy demand.

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272β, γ, δ, ɛ and ζ, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272β and ɛ (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272γ, δ, and ζ (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272β and δ, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.

Crystallization and preliminary crystallographic analysis of trypanosome alternative oxidase

Crystallization and preliminary crystallographic analysis of trypanosome alternative oxidase

Trypanosoma brucei: Differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (Δψ) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial Δψ was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.

Stage dependent regulation of trypanosome alternative oxidase (TAO) gene expression in Trypanosoma brucei

African trypanosomes, like Trypanosoma brucei possesses a cytochrome-independent terminal oxidase of mitochondrial electron transport chain, which is known as trypanosome alternative oxidase (TAO). The mammalian bloodstream form of T. brucei depends exclusively on TAO for their respiration. TAO expression is down regulated in the procyclic form, which grows in the insect vector. We have shown previously that TAO expression is regulated primarily at the level of transcript stability and de novo protein synthesis is required for transcript instability in the procyclic form. Here, we showed that the UTRs of the TAO transcript are responsible for its stage specific degradation pattern. TAO contains a long 3′-UTR of about 2.2 Kb and a 5′-UTR that is 125 bp. Deletion mutagenesis of the TAO 3′-UTR revealed the presence of a cis-element within the region of 500–1000 nt downstream of the stop codon that is responsible for destabilization of the luciferase reporter transcript in the procyclic form. Luciferase activity and the steady-state level of the luciferase protein were unaltered in all these mutants. This indicates that possibly the shorter transcript produced by alternative polyadenylation possesses an increased efficiency for translation. This project was supported by MBRS SCORE 3SO6GM08037-30S1 and NIH Minority Predoctoral Fellowship Grant 1F31GM077048

Novel inhibitors of the trypanosome alternative oxidase inhibit Trypanosoma brucei brucei growth and respiration

African trypanosomiasis is a deadly disease for which few chemotherapeutic options are available. The causative agents, Trypanosoma brucei rhodesiense and T. b. gambiense, utilize a non-cytochrome, alternative oxidase (AOX) for their cellular respiration. The absence of this enzyme in mammalian cells makes it a logical target for therapeutic agents. We designed three novel compounds, ACB41, ACD15, and ACD16, and investigated their effects on trypanosome alternative oxidase (TAO) enzymatic activity, parasite respiration, and parasite growth in vitro. All three compounds contain a 2-hydroxybenzoic acid moiety, analogous to that present in SHAM, and a prenyl side chain similar to that found in ubiquinol. ACD15 and ACD16 are further differentiated by the presence of a solubility-enhancing carbohydrate moiety. Kinetic studies with purified TAO show that all three compounds competitively inhibit TAO, and two compounds, ACB41 and ACD15, have inhibition constants five- and three-fold more potent than SHAM, respectively. All three compounds inhibited the respiration and growth of continuously cultured T. b. brucei bloodstream cells in a dose-dependent manner. None of the compounds interfered with respiration of rat liver mitochondria, nor did they inhibit the growth of a continuously cultured mammalian cell line. Collectively, the results suggest we have identified a new class of compounds that are inhibitors of TAO, have trypanocidal properties in vitro, and warrant further investigation in vivo.

Trypanosome alternative oxidase: from molecule to function

Trypanosome alternative oxidase (TAO) is the cytochrome-independent terminal oxidase of the mitochondrial electron transport chain. TAO is a diiron protein that transfers electrons from ubiquinol to oxygen, reducing the oxygen to water. The mammalian bloodstream forms of Trypanosoma brucei depend solely on TAO for respiration. The inhibition of TAO by salicylhydroxamic acid (SHAM) or ascofuranone is trypanocidal. TAO is present at a reduced level in the procyclic form of T. brucei, where it is engaged in respiration and is also needed for developmental processes. Alternative oxidases similar to TAO have been found in a wide variety of organisms but not in mammals, thus rendering TAO an important chemotherapeutic target for African trypanosomiasis.