Expression of alternative oxidase inhibits programmed cell death-like phenomenon in bloodstream form of Trypanosoma brucei rhodesiense

Trypanosoma brucei rhodesiense is one of the causative agents of African Trypanosomiasis. Programmed cell death (PCD) is fundamental in the development, homeostasis and immune mechanisms of multicellular organisms. It has been shown that, other than occurring in multicellular organisms, the PCD phenomenon also takes place in unicellular organisms. In the present study, we have found that under high-density axenic culture conditions, bloodstream form of T. b. rhodesiense depicts a PCD-like phenomenon. We investigated the association of the PCD-like phenomenon with expression of trypanosome alternative oxidase (TAO) under low-temperature stress conditions. We observed that bloodstream form of T. b. rhodesiense did not show any PCD but had up-regulated expression of TAO. Inhibition of TAO by the addition of ascofranone caused the development of PCD in bloodstream T. b. rhodesiense under low-temperature stress, implying that expression of TAO may contribute to the inhibition of PCD.     

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.     

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 (IC₅₀ value: 1 nM) to eliminate trypanosomes in human infective strain cultures.     

Kinetic and regulatory aspects of the function of the alternative oxidase in plant respiration

The kinetic modelling of the respiratory network in plant mitochondria is discussed, with emphasis on the importance of the choice of boundary conditions, and of modelling of both quinol-oxidising and quinone-reducing pathways. This allows quantitative understanding of the interplay between the different pathways, and of the functioning of the plant respiratory network in terms of the kinetic properties of its component parts. The effects of activation of especially succinate dehydrogenase and the cyanide-insensitive alternative oxidase are discussed. Phenomena, such as respiratory control ratios depending on the substrate, shortcomings of the Bahr and Bonner model for electron distribution between the oxidases and reversed respiratory control, are explained. The relation to metabolic control analysis of the respiratory network is discussed in terms of top-down analysis.     

Regulation of plant alternative oxidase activity: A tale of two cysteines

Two Cys residues, Cys_I and Cys_II, are present in most plant alternative oxidases (AOXs). Cys_I inactivates AOX by forming a disulfide bond with the corresponding Cys_I residue on the adjacent subunit of the AOX homodimer. When reduced, Cys_I associates with α-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the Cys_I site. Cys_II may also be a site of AOX activity regulation, through interaction with the small α-keto acid, glyoxylate. Comparison of Arabidopsis AOX1a (AtAOX1a) mutants with single or double substitutions at Cys_I and Cys_II confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at Cys_I) was limited to Cys_I. A variety of Cys_II substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at Cys_I, charge repulsion is involved. Independent effects at each Cys were suggested by lack of Cys_II substitution interference with pyruvate stimulation at Cys_I, and close to additive activation at the two sites. However, results obtained using diamide treatment to covalently link the AtAOX1a subunits by the disulfide bond indicated that Cys_I must be in the reduced state for activation at Cys_II to occur.     

Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase

The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.     

Expression and signal regulation of the alternative oxidase genes under abiotic stresses

Plants in their natural environment frequently face various abiotic stresses, such as drought, high salinity, and chilling. Plant mitochondria contain an alternative oxidase （AOX）, which is encoded by a small family of nuclear genes. AOX genes have been shown to be highly responsive to abiotic stresses. Using transgenic plants with varying levels of AOX expression, it has been confirmed that AOX genes are im- portant for abiotic stress tolerance. Although the roles of AOX under abiotic stresses have been extensively studied and there are several excellent reviews on this topic, the differential expression patterns of the AOX gene family members and the signal regulation of AOX gene（s） under abiotic stresses have not been extensively summarized. Here, we review and discuss the current progress of these two important issues.     

Regulation of alternative oxidase gene expression in soybean

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 °C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.     

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His⁹⁹ and Cys¹³⁶), and an Asp (Asp¹³⁴) in the potential S₁ binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of intact mitochondria. The incubation of mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated with AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order: AMP = GMP > GDP > GTP > MP > IMP. The apparent K(m) values for AMP in reactivation of the alternative oxidase of submitochondrial particles or mitochondria treated with Triton X-100 and incubated at 25 degrees C were calculated. Physiological aspects of activation of the alternative oxidase are discussed in connection with the impairment of electron transfer through the cytochrome pathway.     

Genomic structure and expression of alternative oxidase genes in legumes

Mitochondria isolated from chickpea (Cicer arietinum) possess substantial alternative oxidase (AOX) activity, even in non‐stressed plants, and one or two AOX protein bands were detected immunologically, depending on the organ. Four different AOX isoforms were identified in the chickpea genome: CaAOX1 and CaAOX2A, B and D. CaAOX2A was the most highly expressed form and was strongly expressed in photosynthetic tissues, whereas CaAOX2D was found in all organs examined. These results are very similar to those of previous studies with soybean and siratro. Searches of available databases showed that this pattern of AOX genes and their expression was common to at least 16 different legume species. The evolution of the legume AOX gene family is discussed, as is the in vivo impact of an inherently high AOX capacity in legumes on growth and responses to environmental stresses.     

The alternative oxidase in roots of poa annua after transfer from high-light to low-light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.     

Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme’s apparent V_max.     

A reporter gene system used to study developmental expressionof alternative oxidase and isolate mitochondrial retrograde regulationmutants in Arabidopsis

Perturbation of mitochondrial function causes altered nuclear gene expression in plants. To study this response, called mitochondrial retrograde regulation, and developmental gene expression, a transgenic Arabidopsis thaliana (Col-0) line containing a firefly luciferase gene controlled by a promoter region of the Arabidopsis alternative oxidase 1a gene (AtAOX1a) was created. The transgene and the endogenous gene were developmentally induced in young cotyledons to a level higher than in older cotyledons and leaves. Analysis of transgene expression suggests that this is true for emerging leaves as well. Antimycin A (AA), a mitochondrial electron transport chain inhibitor, and monofluroacetate (MFA), a TCA cycle inhibitor, induced expression of the transgene and the endogenous gene in parallel. The following comparative responses of the transgene to inhibitors were observed: (a) the response in cotyledons to AA treatment differed greatly in magnitude from the response in leaves; (b) the induction kinetics in cotyledons following MFA treatment differed greatly from the kinetics in leaves; and (c) the induction kinetics following MFA treatment differed from the kinetics of AA in both leaves and cotyledons. The transgenic line was used in a genetic screen to isolate mutants with greatly decreased transgene and AtAOX1a induction in response to AA. Some of these mutant lines showed greatly decreased induction by MFA, but one did not. Taken altogether, the data provide genetic evidence that suggests that induction of the AtAOX1a gene by distinct mitochondrial perturbations are via distinct, but overlapping signaling pathways that are tissue specific.