Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

YchF, a universally conserved protein, hitherto thought to be a GTPase, was shown to be an ATPase based on structural and biochemical studies on hOLA1, a human ortholog of YchF. However, the cellular role of YchF is unclear. Based on the presence of a RNA binding domain in this protein and significant homology to ribosome binding Obg family GTPases, we examined its ability to associate with the ribosome. Here, we show that Escherichia coli YchF binds the 50S and 70S ribosomal particles in a nucleotide independent manner and it hydrolyzes ATP utilizing a potassium dependent mechanism. A potassium mediated acceleration of hydrolysis activity was thus far known for a few GTPases. Like these, YchF too conserves the structural features required for K⁺ coordination, making it a unique ribosome binding ATPase utilizing a similar mechanism. Furthermore, we show that Lys78 is an important determinant of the potassium dependent ATPase activity.     

Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.     

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.     

Comparative genomics of prokaryotic GTP-binding proteins (the Era, Obg, EngA, ThdF (TrmE), YchF and YihA families) and their relationship to eukaryotic GTP-binding proteins (the DRG, ARF, RAB, RAN, RAS and RHO families)

Several GTP-binding proteins with poorly defined functions were previously identified in Escherichia coli (i.e. Era, ThdF (TrmE)), Bacillus subtilis (i.e. Obg) and Neisseria gonorrhoeae (i.e. EngA). In these species, every individual protein is encoded by an essential gene. BLAST searches were used to detect orthologs in genomes of various organisms. Alignments of orthologous sequences allowed the construction of phylogenetic trees and the definition of protein families. The BLAST searches also resulted in the identification of two additional families, the YchF and YihA families, named after the ychF and yihA genes of E. coli. Most families are not present in archaeal genomes, but representatives of each family were also detected in eukaryotic genomes. Only representatives of the YchF family are present in every genome sequenced to date, suggesting that YchF-like proteins might be involved in a fundamental life process. The GTP1/DRG family consisting of eukaryotic and archaeal proteins is related to the YchF family of GTP-binding proteins. The relationship of the six prokaryotic families of GTP-binding proteins and the GTP1/DRG family to eukaryotic GTPase families was also investigated: With the exception of the ARF family, a clear separation of the six prokaryotic families and the GTP1/DRG family with respect to eukaryotic (RAB, RAN, RAS and RHO) GTPases was observed.     

A new,expressed multigene family containing a hot spot for insertion of retroelements is associated with polymorphic subtelomeric regions of Trypanosoma brucei

We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons. The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome. From cDNA clones and subsequently in the T. brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position. Here these genes are called the RHS family, for retrotransposon hot spot. Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site. The RHS proteins share between 13 and 96% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains. Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus. Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)-genes. Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T. brucei strain TREU927/4 GUTat10.1. RHS genes comprise the remaining sequence of the size-polymorphic “repetitive region” described for T. brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.     

The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation

Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3′-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing.     

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.     

Trypanosoma brucei: Reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic “pulse-chase” analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8 h of initiating transformation. GPI-PLCp is not lost precipitously from newly transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.     

An Ancient P-Loop GTPase in Rice Is Regulated by a Higher Plant-specific Regulatory Protein

YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein.     

Expression and purification of non-glycosylated Trypanosoma brucei transferrin receptor in insect cells

The transferrin receptor of the parasite Trypanosoma brucei is a heterodimeric protein complex encoded by the 2 expression site-associated genes (ESAGs) 6 and 7. ESAG6 is a heterogeneously glycosylated protein of 50-60 kDa modified by a glycosylphosphatidylinositol anchor at the C-terminus, while ESAG7 is a 40-42 kDa glycoprotein carrying an unmodified C-terminus. In order to determine whether glycosylation is necessary for dimer formation and ligand binding, the receptor was expressed in insect cells in the presence of tunicamycin. When insect cells were infected with recombinant ESAG6/ESAG7 double expressor baculovirus and grown in the presence of tunicamycin, non-glycosylated forms of ESAG6 and ESAG7 of 46 and 36 kDa, respectively, were synthesized. The non-glycosylated ESAG6 and ESAG7 were capable of forming a heterodimer and of binding transferrin. This results shows that glycosylation is not necessary for synthesis of a functional T. brucei transferrin receptor.     

Mitochondrial tRNA import in Trypanosoma brucei is independent of thiolation and the Rieske protein

Due to a complete lack of the tRNA genes in the mitochondrial genome of Trypanosoma brucei, all tRNAs needed for mitochondrial translation have to be imported into the organelle from the cytosol. A previous study showed that the modified nucleotide s²U could act as a negative determinant for mitochondrial tRNA import in another kinetoplastid, Leishmania tarentolae. We have investigated whether the same type of cytosolic control for tRNA retention exists in T. brucei. Based on Northern analysis with subcellular RNA fractions and in vitro import assays, we demonstrate that silencing of the cysteine desulfurase, TbNfs (TbIscS), the key enzyme in tRNA thiolation (s²U) and Fe-S cluster formation in vivo, has no effect on tRNA partitioning. This observation is especially surprising in light of a recent report suggesting that in L. tropica the Rieske Fe-S protein is an essential component of the RNA import complex (RIC). In line with the above observation, we also show that down-regulation of the Rieske protein by RNA interference, similar to the TbNfs knockdowns, has no effect on import. The data presented here supports the view that in T. brucei: (1) s²U is not a negative determinant for tRNA import; (2) the Rieske protein is not an essential component of the import machinery, and (3) since the Rieske protein is essential for respiration and maintenance of inner mitochondrial membrane potential, neither process plays a critical role in tRNA import. We therefore suggest that the T. brucei import machinery differs substantially from what has been described in Leishmania.     

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.     

Na(+)-ATPase and protein kinase C are targets to 1-O-hexadecylphosphocoline (miltefosine) in Trypanosoma cruzi

Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na⁺-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na⁺-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na⁺-ATPase activity with IC₅₀ = 18 ± 5 μg mL⁻¹. This effect was shown to be reversible, dependent on the pH and Ca²⁺. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na⁺-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na⁺-ATPase and PKC activities.     

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30 min of incubation in lysis buffer at 4 °C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30 min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS^E approach. ProteinLynx Global Server (version 2.5) with Expression^E analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.     

Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins.

We have identified an essential Caulobacter crescentus gene (cgtA) that encodes a member of a recently identified subfamily of GTPases (the Obg family) conserved from Bacteria to Archaea to humans. This evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins possesses a fundamental, yet unknown, cellular role. In this report, we describe the isolation and sequence of the cgtA gene. The predicted CgtA protein displays striking similarity to the Obg family of small, monomeric GTP-binding proteins, both in the conserved guanine nucleotide-binding domains and throughout the N-terminal glycine-rich domain that is found in many members of the Obg family. Disruption of the cgtA gene was lethal, demonstrating that this gene is essential for cell growth. Immunoblot analysis revealed that CgtA protein levels remained constant throughout the C. crescentus cell cycle.