Proteins In Trypanosomatids Research Articles

Analysis of Spliceosomal Proteins in Trypanosomatids Reveals Novel Functions in mRNA Processing

In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions.

An Experimental Approach for the Identification of Conserved Secreted Proteins in Trypanosomatids

Extracellular factors produced by Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei are important in the host-parasite relationship. Here, we describe a genome-based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway. Potentially secreted proteins were identified by bioinformatic analysis of the T. cruzi genome. A subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence in L. infantum, L. major, and T. brucei were transfected into L. infantum. Tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits. Secretion was confirmed for two L. infantum orthologs proteins using the same experimental system. Infectivity studies of transgenic Leishmania parasites suggest that one of the secreted proteins increases parasite replication inside macrophages. This methodology can identify conserved secreted proteins involved in the classical secretory pathway, and they may represent potential virulence factors in trypanosomatids.

Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.

Identification of New Kinetoplast DNA Replication Proteins in Trypanosomatids Based on Predicted S-Phase Expression and Mitochondrial Targeting

Trypanosomatid parasites contain an unusual form of mitochondrial DNA (kinetoplast DNA [kDNA]) consisting of a catenated network of several thousand minicircles and a smaller number of maxicircles. Many of the proteins involved in the replication and division of kDNA are likely to have no counterparts in other organisms and would not be identified by similarity to known replication proteins in other organisms. A new kDNA replication protein conserved in kinetoplastids has been identified based on the presence of posttranscriptional regulatory sequences associated with S-phase gene expression and predicted mitochondrial targeting. The Leishmania major protein P105 (LmP105) and Trypanosoma brucei protein P93 (TbP93) localize to antipodal sites flanking the kDNA disk, where several other replication proteins and nascent minicircles have been localized. Like some of these kDNA replication proteins, the LmP105 protein is only present at the antipodal sites during S phase. RNA interference (RNAi) of TbP93 expression resulted in a cessation of cell growth and the loss of kDNA. Nicked/gapped forms of minicircles, the products of minicircle replication, were preferentially lost from the population of free minicircles during RNAi, suggesting involvement of TbP93 in minicircle replication. This approach should allow the identification of other novel proteins involved in the duplication of kDNA.

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

C-terminal proteolysis of prenylated proteins in trypanosomatids and RNA interference of enzymes required for the post-translational processing pathway of farnesylated proteins

The C-terminal “CaaX”-motif-containing proteins usually undergo three sequential post-translational processing steps: (1) attachment of a prenyl group to the cysteine residue; (2) proteolytic removal of the last three amino acids “aaX”; (3) methyl esterification of the exposed α-carboxyl group of the prenyl-cysteine residue. The Trypanosoma brucei and Leishmania major Ras converting enzyme 1 (RCE1) orthologs of 302 and 285 amino acids-proteins, respectively, have only 13–20% sequence identity to those from other species but contain the critical residues for the activity found in other orthologs. The Trypanosoma brucei a-factor converting enzyme 1 (AFC1) ortholog consists of 427 amino acids with 29–33% sequence identity to those of other species and contains the consensus HExxH zinc-binding motif. The trypanosomatid RCE1 and AFC1 orthologs contain predicted transmembrane regions like other species. Membranes from Sf9 cells expressing the RCE1 ortholog of T. brucei or L. major showed proteolytic activity against farnesylated RAS-CVIM, whereas membranes containing T. brucei AFC1 ortholog were inactive. The results suggest that RCE1 is responsible for proteolytic removal of the C-terminal aaX from prenyl-CaaX proteins in these parasites. All the three enzymatic post-translational processes are thought to be required for proper cellular functioning of CaaX-proteins in eukaryotic cells. We carried out RNA interference experiments in Trypanosoma brucei of the enzymes involved in farnesyl protein post-translational modification to evaluate their importance in cell proliferation. Knockdown of T. brucei PFT β subunit and RCE1 mRNAs resulted in >20-fold suppression of cell growth and dramatic morphologic changes. Knockdown of PPMT mRNA caused less dramatic effects on growth but induced noticeable changes in cell morphology.

Relationships between the major kinetoplastid paraflagellar rod proteins: a consolidating nomenclature.

The Kinetoplastida comprise a family of flagellated microbes that are defined by the presence of a network of concatenated mitochondrial DNA called the kinetoplast and a range of other unique features. One such is the paraflagellar rod (PFR), which has an essential role in cell motility [1–3], an intricate sub-structural arrangement [4,5] and an interesting phylogenetic distribution [6,7]. Although the complete composition of the PFR is still unknown, the major structural components have been described in several species of Kinetoplastida. The first biochemical description in kinetoplastids identified the two major PFR proteins, PFR1 and PFR2, in Crithidia fasciculata [8]. In that paper, PFR1 was defined as the protein with the slower migration in SDS–PAGE gels while the faster migrating band was called PFR2. Since then, other descriptions of major PFR proteins in trypanosomatids have been made, including those of Herpetomonas megaseliae [9], Trypanosoma species [10,11] and Leishmania species [12,13]. At the time of many of these publications the correlation between major PFR proteins of different species was unclear and nomenclatures developed that were peculiar to each species and did not reflect the homologies amongst the proteins (see Table 1). However, with the increasing availability of DNA and protein sequences, we have now been able to define the levels of homology between PFR proteins (Fig. 1) and this reveals that the disconnected nomenclature

Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins.

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.

A trypanosomatid protein specifically interacts with a mammalian iron-responsive element.

Intracellular iron homeostasis of vertebrates and invertebrates is mediated through the interaction of iron-regulatory proteins (IRPs) with mRNAs containing a bulged hairpin-loop structure termed the iron-responsive element (IRE). We detected a protein within extracts prepared from Leishmania tarentolae that specifically interacts with a mammalian IRE; mutations to the IRE that inhibit the interaction with the mammalian protein have a corresponding effect on the interaction with the L. tarentolae protein. The disassociation constant noted for the interaction of the mammalian IRE with the L. tarentolae protein was 0.7+/-0.3 microM, whereas that recorded for the interaction with the mammalian IRP under these conditions was 5+/-2 nM. The interacting L. tarentolae protein potentially places the RNA-binding site of the IRP near the root of the eukaryotic evolutionary tree. However, unlike that of the mammalian IRPs, the L. tarentolae IRE-binding activity was not induced by growth in iron-depleted media.

The High Resolution Crystal Structure of Recombinant Crithidia fasciculata Tryparedoxin-I

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-A resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A right-handed redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted alpha-helix (alpha1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H...O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.

Leptomonas seymouri, Trypanosoma brucei:A Method for Isolating Trypanosomatid Nuclear Factors Which BindT. bruceiSingle-Stranded g-Rich Telomere Sequence

Sequential expression of variant surface glycoproteins (VSG) inTrypanosoma bruceiis the basis of antigenic variation which is essential for parasite survival. Telomere distal copies of VSG genes, so-called basic copies, provide a repository of VSG sequence information for variability, but actively expressed copies are found only at subtelomeric regions of chromosomes. Of eight or so expression sites (ES) in theT. bruceigenome, only one is active at one time. Movement of a basic copy VSG gene to an ES requires a recombination event of unknown mechanism. The properties of telomeres have been speculated to be important for control of VSG expression or basic copy mobilization, prompting us to begin to investigate telomere-binding proteins in trypanosomatids. TheT. bruceitelomere sequence is known, facilitating design of synthetic telomeric DNAs. Here we describe a method for preparation of active trypanosomatid nuclear extracts. We show that inT. bruceiandLeptomonas seymouri,factors can be detected which bind a g-rich single-strand telomere sequence based on theT. bruceitelomere. TheL. seymouritelomere-binding factor, LST-1, dissociates in the presence of high salt to produce a core factor, LST-2, migrating similarly to theT. bruceitelomere-binding factor TBT-1. The affinity of LST-2 and TBT-1 for DNA under high salt conditions is characteristic of telomere proteins.

Effect of RNA secondary structure and modified bases on the inhibition of trypanosomatid protein synthesis in cellfree extracts by antisense oligodeoxynucleotides

Every messenger RNA from leishmanias and trypanosomes has at its 5' end a conserved region termed the mini-exon sequence which, however, varies from species to species. In a systematic study mRNAs from Trypanosoma brucei, Trypanosoma vivax, and Leishmania enriettii were translated in cell-free extracts in the presence of oligodeoxynucleotides complementary to part of the mini-exon sequence. The affinity of the same oligonucleotides for target and non-target mRNAs was determined by thermal elution of filter-bound complexes showing that the critical temperature of half-dissociation of the complexes was linearly related to log (l + x), where l is the length of the oligomer and x its G + C content. A few oligomers exhibited a lower Tc value than expected which was ascribed to the presence of modified RNA bases or to the existence of a hairpin structure in the L. enriettii mini-exon. In most cases the efficiency of translation inhibition by the oligonucleotides was clearly correlated to their affinity for the target RNA. The modified bases weakened the inhibition of protein synthesis by oligonucleotides complementary to these regions.

Possible artifacts in the radioiodination of surface proteins of trypanosomatids

Three different methods for the radioiodination of surface proteins in trypanosomatids have been comparatively analyze. Data interpretation in the lactoperoxidase-mediated method was impaired by autoiodination of the enzyme and its tight retention by the cells. In the enzymobeads method, iodinated enzymes were released from the beads upon solubilization of the preparation with SDS, thus mixing with iodinated proteins of the trypanosomatids. Therefore, upon separation by polyacrylamide gel electrophoresis of the cell lysates, both methods yielded autoradiographic patterns in which prominent bands did not represent labeled cell surface components. In contrast, the autoradiographic patterns ontained by use of the Iodo-Gen method were apparently free of artifacts.