The single mitochondrion of the kinetoplastid parasite Crithidia fasciculata is a dynamic network.

Mitochondria are central organelles in cellular metabolism. Their structure is highly dynamic, allowing them to adapt to different energy requirements, to be partitioned during cell division, and to maintain functionality. Mitochondrial dynamics, including membrane fusion and fission reactions, are well studied in yeast and mammals but it is not known if these processes are conserved throughout eukaryotic evolution. Kinetoplastid parasites are some of the earliest-diverging eukaryotes to retain a mitochondrion. Each cell has only a single mitochondrial organelle, making them an interesting model for the role of dynamics in controlling mitochondrial architecture. We have investigated the mitochondrial division cycle in the kinetoplastid Crithidia fasciculata. The majority of mitochondrial biogenesis occurs during the G1 phase of the cell cycle, and the mitochondrion is divided symmetrically in a process coincident with cytokinesis. Live cell imaging revealed that the mitochondrion is highly dynamic, with frequent changes in the topology of the branched network. These remodeling reactions include tubule fission, fusion, and sliding, as well as new tubule formation. We hypothesize that the function of this dynamic remodeling is to homogenize mitochondrial contents and to facilitate rapid transport of mitochondria-encoded gene products from the area containing the mitochondrial nucleoid to other parts of the organelle.

The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists.

BACKGROUND: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.

Orientation of DNA Minicircles Balances Density and Topological Complexity in Kinetoplast DNA.

Kinetoplast DNA (kDNA), a unique mitochondrial structure common to trypanosomatid parasites, contains thousands of DNA minicircles that are densely packed and can be topologically linked into a chain mail-like network. Experimental data indicate that every minicircle in the network is, on average, singly linked to three other minicircles (i.e., has mean valence 3) before replication and to six minicircles in the late stages of replication. The biophysical factors that determine the topology of the network and its changes during the cell cycle remain unknown. Using a mathematical modeling approach, we previously showed that volume confinement alone can drive the formation of the network and that it induces a linear relationship between mean valence and minicircle density. Our modeling also predicted a minicircle valence two orders of magnitude greater than that observed in kDNA. To determine the factors that contribute to this discrepancy we systematically analyzed the relationship between the topological properties of the network (i.e., minicircle density and mean valence) and its biophysical properties such as DNA bending, electrostatic repulsion, and minicircle relative position and orientation. Significantly, our results showed that most of the discrepancy between the theoretical and experimental observations can be accounted for by the orientation of the minicircles with volume exclusion due to electrostatic interactions and DNA bending playing smaller roles. Our results are in agreement with the three dimensional kDNA organization model, initially proposed by Delain and Riou, in which minicircles are oriented almost perpendicular to the horizontal plane of the kDNA disk. We suggest that while minicircle confinement drives the formation of kDNA networks, it is minicircle orientation that regulates the topological complexity of the network.

An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

The effect of tunicamycin on the glucose uptake, growth, and cellular adhesion in the protozoan parasite Crithidia fasciculata.

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.

Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine.

Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.

Probing why trypanosomes assemble atypical cytochrome c with an AxxCH haem-binding motif instead of CxxCH.

Mitochondrial cytochromes c and c1 are core components of the respiratory chain of all oxygen-respiring eukaryotes. These proteins contain haem, covalently bound to the polypeptide in a catalysed post-translational modification. In all eukaryotes, except members of the protist phylum Euglenozoa, haem attachment is to the cysteine residues of a CxxCH haem-binding motif. In the Euglenozoa, which include medically relevant trypanosomatid parasites, haem attachment is to a single cysteine residue in an AxxCH haem-binding motif. Moreover, genes encoding known c-type cytochrome biogenesis machineries are all absent from trypanosomatid genomes, indicating the presence of a novel biosynthetic apparatus. In the present study, we investigate expression and maturation of cytochrome c with a typical CxxCH haem-binding motif in the trypanosomatids Crithidia fasciculata and Trypanosoma brucei. Haem became attached to both cysteine residues of the haem-binding motif, indicating that, in contrast with previous hypotheses, nothing prevents formation of a CxxCH cytochrome c in euglenozoan mitochondria. The cytochrome variant was also able to replace the function of wild-type cytochrome c in T. brucei. However, the haem attachment to protein was not via the stereospecifically conserved linkage universally observed in natural c-type cytochromes, suggesting that the trypanosome cytochrome c biogenesis machinery recognized and processed only the wild-type single-cysteine haem-binding motif. Moreover, the presence of the CxxCH cytochrome c resulted in a fitness cost in respiration. The level of cytochrome c biogenesis in trypanosomatids was also found to be limited, with the cells operating at close to maximum capacity.

Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability.

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.

Role of transmembrane domain 4 in ligand permeation by Crithidia fasciculata equilibrative nucleoside transporter 2 (CfNT2).

Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.

Evidence for an ATP-sensitive K+ channel in mitoplasts isolated from Trypanosoma cruzi and Crithidia fasciculata.

Mammalian mitochondria, as well as rat, plant and Caenorhabditis elegans mitochondria, possess an ATP-sensitive K+ channel (mitoK(ATP)) that has been pharmacologically characterised. Opening of mitoK(ATP) and the subsequent K+ entry into the matrix was shown to have three effects on mitochondria physiology: (i) an increase in matrix volume (swelling), (ii) an acceleration of respiration, and (iii) an increase in reactive oxygen species (ROS) production. These effects on mitochondria bioenergetics have been shown to be part of distinct intracellular signalling pathways, to protect against cell death and to modulate gene transcription. To date, such a channel or its activity has not been described in trypanosomatids. In the present study, we show pharmacological evidence for the presence of a mitoK(ATP) in trypanosomatids. Cells were incubated in a hypotonic medium followed by mild detergent exposure to isolate mitoplasts from Trypanosoma cruzi and Crithidia fasciculata. Mitoplasts swelled when incubated in KCl medium due to respiration-driven K+ entry into the matrix. Swelling was sensitive to the presence of ATP when the mitoplast suspension was incubated in K+ -containing, but not in K+ -free, medium. The ATP inhibition of swelling was reversed by the mitoK(ATP) agonist diazoxide and the diazoxide-induced swelling was inhibited by the mitoK(ATP) blockers 5-hydroxydecanoate (5HD) or glibenclamide. Similar to mammalian and rat mitochondria, trypanosomatid mitoK(ATP) activity was modulated by the general protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) and antagonist chelerythrine. As expected, the potassium ionophore valinomycin could also reverse the ATP-inhibited state but this reversal was not sensitive to 5HD or glibenclamide. Dose response curves for ATP, diazoxide and 5HD are presented. These results provide strong evidence for the presence of an ATP-sensitive K+ in trypanosomatid mitochondria.

Evaluation of the topoisomerase II-inactive bisdioxopiperazine ICRF-161 as a protectant against doxorubicin-induced cardiomyopathy.

Anthracycline-induced cardiomyopathy is a major problem in anti-cancer therapy. The only approved agent for alleviating this serious dose limiting side effect is ICRF-187 (dexrazoxane). The current thinking is that the ring-opened hydrolysis product of this agent, ADR-925, which is formed inside cardiomyocytes, removes iron from its complexes with anthracyclines, hereby reducing the concentration of highly toxic iron-anthracycline complexes that damage cardiomyocytes by semiquinone redox recycling and the production of free radicals. However, the 2 carbon linker ICRF-187 is also is a catalytic inhibitor of topoisomerase II, resulting in the risk of additional myelosuppression in patients receiving ICRF-187 as a cardioprotectant in combination with doxorubicin. The development of a topoisomerase II-inactive iron chelating compound thus appeared attractive. In the present paper we evaluate the topoisomerase II-inactive 3 carbon linker bisdioxopiperazine analog ICRF-161 as a cardioprotectant. We demonstrate that this compound does chelate iron and protects against doxorubicin-induced LDH release from primary rat cardiomyocytes in vitro, similarly to ICRF-187. The compound does not target topoisomerase II in vitro or in cells, it is well tolerated and shows similar exposure to ICRF-187 in rodents, and it does not induce myelosuppression when given at high doses to mice as opposed to ICRF-187. However, when tested in a model of chronic anthracycline-induced cardiomyopathy in spontaneously hypertensive rats, ICRF-161 was not capable of protecting against the cardiotoxic effects of doxorubicin. Modulation of the activity of the beta isoform of the topoisomerase II enzyme by ICRF-187 has recently been proposed as the mechanism behind its cardioprotection. This concept is thus supported by the present study in that iron chelation alone does not appear to be sufficient for protection against anthracycline-induced cardiomyopathy.

Regulation of UMSBP activities through redox-sensitive protein domains.

UMSBP is a CCHC-type zinc finger protein, which functions during replication initiation of kinetoplast DNA minicircles and the segregation of kinetoplast DNA networks. Interactions of UMSBP with origin sequences, as well as the protein oligomerization, are affected by its redox state. Reduction yields UMSBP monomers and activates its binding to DNA, while oxidation drives UMSBP oligomerization and impairs its DNA-binding activity. Kinetics analyses of UMSBP-DNA interactions revealed that redox affects the association of free UMSBP with the DNA, but has little effect on its dissociation from the nucleoprotein complex. A previously proposed model, suggesting that binding of DNA is regulated via the reversible interconversions of active UMSBP monomers and inactive oligomers, was challenged here, revealing that the two redox-driven processes are not interrelated. No correlation could be observed between DNA-binding inhibition and UMSBP oligomerization, upon oxidation of UMSBP. Moreover, while the presence of zinc ions was found to be essential for the interaction of UMSBP with DNA, UMSBP oligomerization occurred through zinc-depleted, unfolded zinc finger domains. Site directed mutagenesis analysis of UMSBP suggested that its unique methionine residue, which can be oxidized into methionine sulfoxide, is not involved in the redox-mediated regulation of UMSBP-DNA interactions.

Enzymatic mechanism controls redox-mediated protein-DNA interactions at the replication origin of kinetoplast DNA minicircles.

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.

The rotational dynamics of kinetoplast DNA replication.

Kinetoplast DNA (kDNA), from trypanosomatid mitochondria, is a network containing several thousand catenated minicircles that is condensed into a disk-shaped structure in vivo. kDNA synthesis involves release of individual minicircles from the network, replication of the free minicircles and reattachment of progeny at two sites on the network periphery approximately 180 degrees apart. In Crithidia fasciculata, rotation of the kDNA disk relative to the antipodal attachment sites results in distribution of progeny minicircles in a ring around the network periphery. In contrast, Trypanosoma brucei progeny minicircles accumulate on opposite ends of the kDNA disk, a pattern that did not suggest kinetoplast motion. Thus, there seemed to be two distinct replication mechanisms. Based on fluorescence microscopy of the kDNA network undergoing replication, we now report that the T. brucei kinetoplast does move relative to the antipodal sites. Whereas the C. fasciculata kinetoplast rotates, that from T. brucei oscillates. Kinetoplast motion of either type must facilitate orderly replication of this incredibly complex structure.

Binding of the universal minicircle sequence binding protein at the kinetoplast DNA replication origin.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.

Identification and characterization of purine nucleoside transporters from Crithidia fasciculata.

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.

Trypanothione synthesis in crithidia revisited.

In Crithidia fasciculata the biosynthesis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBanktrade mark accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg(2+)-ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K(m) values for GSH, Mg(2+)-ATP, and glutathionylspermidine were 407, 222, and 480 microm, respectively, and the k(cat) was 8.7 s(-1) for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.

Presence of a poly(A) binding protein and two proteins with cell cycle-dependent phosphorylation in Crithidia fasciculata mRNA cycling sequence binding protein II.

Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

Leishmania major elongation factor 1B complex has trypanothione S-transferase and peroxidase activity.

In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified alpha, beta, and gamma subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg(-1)). Kinetic studies suggest a ternary complex mechanism, with Km values of 140 mum for trypanothione and 7.4 mm for cumene hydroperoxide and kcat=25 s(-1). Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.