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1.
Nucleus ; 15(1): 2310452, 2024 Dec.
Article in English | MEDLINE | ID: mdl-38605598

ABSTRACT

The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.


Subject(s)
Nuclear Envelope , Trypanosoma , Humans , Nuclear Envelope/metabolism , Nuclear Pore/metabolism , Saccharomyces cerevisiae/metabolism , Nuclear Pore Complex Proteins/metabolism , Trypanosoma/metabolism
2.
J Biol Chem ; 299(1): 102726, 2023 01.
Article in English | MEDLINE | ID: mdl-36410438

ABSTRACT

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.


Subject(s)
Proteins , Proteomics , Biotinylation , Nuclear Pore , Proteins/chemistry , Proteomics/methods , Streptavidin/chemistry
3.
Trends Parasitol ; 38(10): 854-867, 2022 10.
Article in English | MEDLINE | ID: mdl-36028415

ABSTRACT

Export of RNA from the nucleus is essential for all eukaryotic cells and has emerged as a major step in the control of gene expression. mRNA molecules are required to complete a complex series of processing events and pass a quality control system to protect the cytoplasm from the translation of aberrant proteins. Many of these events are highly conserved across eukaryotes, reflecting their ancient origin, but significant deviation from a canonical pathway as described from animals and fungi has emerged in the trypanosomatids. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may also reveal additional, previously unsuspected, mRNA export pathways.


Subject(s)
RNA , Trypanosoma , Active Transport, Cell Nucleus/genetics , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , Trypanosoma/genetics , Trypanosoma/metabolism
4.
Nucleus ; 8(4): 340-352, 2017 Jul 04.
Article in English | MEDLINE | ID: mdl-28463551

ABSTRACT

The core architecture of the eukaryotic cell was established well over one billion years ago, and is largely retained in all extant lineages. However, eukaryotic cells also possess lineage-specific features, frequently keyed to specific functional requirements. One quintessential core eukaryotic structure is the nuclear pore complex (NPC), responsible for regulating exchange of macromolecules between the nucleus and cytoplasm as well as acting as a nuclear organizational hub. NPC architecture has been best documented in one eukaryotic supergroup, the Opisthokonts (e.g. Saccharomyces cerevisiae and Homo sapiens), which although compositionally similar, have significant variations in certain NPC subcomplex structures. The variation of NPC structure across other taxa in the eukaryotic kingdom however, remains poorly understood. We explored trypanosomes, highly divergent organisms, and mapped and assigned their NPC proteins to specific substructures to reveal their NPC architecture. We showed that the NPC central structural scaffold is conserved, likely across all eukaryotes, but more peripheral elements can exhibit very significant lineage-specific losses, duplications or other alterations in their components. Amazingly, trypanosomes lack the major components of the mRNA export platform that are asymmetrically localized within yeast and vertebrate NPCs. Concomitant with this, the trypanosome NPC is ALMOST completely symmetric with the nuclear basket being the only major source of asymmetry. We suggest these features point toward a stepwise evolution of the NPC in which a coating scaffold first stabilized the pore after which selective gating emerged and expanded, leading to the addition of peripheral remodeling machineries on the nucleoplasmic and cytoplasmic sides of the pore.


Subject(s)
Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/metabolism , Opisthorchis/chemistry , Proteomics , Trypanosoma/metabolism , Animals , Evolution, Molecular , Humans , Models, Biological , Protein Folding , Trypanosoma/chemistry
6.
J Cell Sci ; 130(8): 1379-1392, 2017 04 15.
Article in English | MEDLINE | ID: mdl-28232524

ABSTRACT

Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites.


Subject(s)
Endocytosis , Trypanosoma brucei brucei , Trypanosomiasis/metabolism , Adaptor Protein Complex 2/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Biological Evolution , Clathrin/metabolism , Cytoskeletal Proteins/metabolism , Humans , Phylogeny , Protein Serine-Threonine Kinases/metabolism , Protozoan Proteins/metabolism , SNARE Proteins/metabolism , Transcription Factor AP-1/metabolism
7.
Nucleic Acids Res ; 44(22): 10554-10570, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27625397

ABSTRACT

The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component.


Subject(s)
Nuclear Lamina/physiology , Nuclear Pore Complex Proteins/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Chromosomes/genetics , Chromosomes/metabolism , DNA Damage , Gene Expression Regulation , Nuclear Lamina/ultrastructure , Nuclear Pore/metabolism , Nuclear Pore/ultrastructure , Nuclear Pore Complex Proteins/genetics , Protein Transport , Protozoan Proteins/genetics , Transcriptome , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/ultrastructure
8.
Dev Cell ; 38(5): 445-6, 2016 09 12.
Article in English | MEDLINE | ID: mdl-27623377

ABSTRACT

Nuclear pore proteins at the base of cilia were thought to regulate transport into cilia. In this issue of Developmental Cell, Del Viso et al. (2016) challenge this view, showing instead that pore proteins localize to ciliary basal bodies and that their perturbation leads to congenital heart disease.


Subject(s)
Cilia/metabolism , Heart Defects, Congenital/metabolism , Nuclear Pore Complex Proteins/metabolism , Cilia/genetics , Cilia/pathology , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Humans , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/genetics , Protein Transport/genetics
9.
Mol Biochem Parasitol ; 209(1-2): 104-113, 2016.
Article in English | MEDLINE | ID: mdl-27475118

ABSTRACT

The parasitic protozoa Trypanosoma brucei and Plasmodium falciparum are lethal human parasites that have developed elegant strategies of immune evasion by antigenic variation. Despite the vast evolutionary distance between the two taxa, both parasites employ strict monoallelic expression of their membrane proteins, variant surface glycoproteins in Trypanosomes and the var, rif and stevor genes in Plasmodium, in order to evade their host's immune system. Additionally, both telomeric location and epigenetic controls are prominent features of these membrane proteins. As such, telomeres, chromatin structure and nuclear organization all contribute to control of gene expression and immune evasion. Here, we discuss the importance of epigenetics and sub-nuclear context for the survival of these disease-causing parasites.


Subject(s)
Genes, Protozoan , Genomics , Nuclear Envelope/metabolism , Parasites/genetics , Parasites/metabolism , Animals , Antigenic Variation , Cell Nucleus/genetics , Cell Nucleus/metabolism , Gene Expression Regulation , Gene Order , Genome , Genomics/methods , Host-Parasite Interactions/immunology , Parasites/immunology , Parasites/pathogenicity
10.
Methods Mol Biol ; 1411: 67-80, 2016.
Article in English | MEDLINE | ID: mdl-27147034

ABSTRACT

Functional understanding of the nuclear envelope requires the identification of its component proteins and their interactions. Trypanosomes cause human and livestock diseases worldwide but are so divergent from animals and fungi that in silico searches for homologs of proteins are frequently of low value. Here we describe a strategy for the straightforward identification of nuclear envelope proteins from trypanosomes that classifies proteins and their interaction networks in the nuclear pore complex. Milling frozen whole cells into a powder and rapid screening of buffer conditions for optimization of complex isolation is described. The method is inexpensive and potentially applicable to many organisms, providing fast access to functional information.


Subject(s)
Membrane Proteins/isolation & purification , Multiprotein Complexes/isolation & purification , Nuclear Envelope/metabolism , Protozoan Proteins/isolation & purification , Trypanosoma/metabolism , Chromatography, Affinity/instrumentation , Chromatography, Affinity/methods , Electrophoresis, Polyacrylamide Gel
11.
PLoS Biol ; 14(2): e1002365, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26891179

ABSTRACT

The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes.


Subject(s)
Evolution, Molecular , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/genetics , Microscopy, Immunoelectron , Nuclear Pore/chemistry , Nuclear Pore/metabolism , Protein Structure, Quaternary , Trypanosoma brucei brucei
12.
Nucleus ; 5(4): 304-10, 2014.
Article in English | MEDLINE | ID: mdl-25482119

ABSTRACT

The nuclear pore complex (NPC) is the sole mediator of bidirectional nucleo-cytoplasmic transport and is also an important scaffold for chromatin organization and transcriptional regulation. Proteomic studies of numerous diverse eukaryotic species initially characterized the NPC as built with a number of remarkably similar structural features, suggesting its status as an ancient and conserved eukaryotic cell component. However, further detailed analyses now suggest that several key specific NPC features have a more convoluted evolutionary history than initially assumed. Recently we reported on TbNup92, a component in trypanosomes of one such conserved structural feature, a basket-like structure on the nuclear face of the NPC. We showed that TbNup92 has similar roles to nuclear basket proteins from yeasts and animals (Mlp and Tpr, respectively) in interacting with both the NPC and the mitotic spindle. However, comparative genomics suggests that TbNup92 and Mlp/Tpr may be products of distinct evolutionary histories, raising the possibility that these gene products are analogs rather than direct orthologs. Taken together with recent evidence for divergence in the nuclear lamina and kinetochores, it is apparent that the trypanosome nucleus functions by employing several novel or highly divergent protein complexes in parallel with conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of hierarchical nuclear organization.


Subject(s)
Nuclear Pore/metabolism , Animals , Cell Nucleus/metabolism , Mitosis/physiology , Nuclear Envelope/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Proteins/metabolism , Protozoan Proteins/metabolism , Spindle Apparatus/metabolism , Trypanosoma brucei brucei/metabolism
13.
Dev Cell ; 22(4): 693-4, 2012 Apr 17.
Article in English | MEDLINE | ID: mdl-22516195

ABSTRACT

Cilia and flagella are membrane-sheathed, microtubule-based protrusions that decorate the surface of many eukaryotic cells. At their base, they form a selective barrier that concentrates certain proteins within the cilia but excludes others. Kee et al. (2012) now propose that nuclear pore complex proteins form a fundamental part of this diffusion barrier.

14.
BMC Genomics ; 13: 29, 2012 Jan 18.
Article in English | MEDLINE | ID: mdl-22257693

ABSTRACT

BACKGROUND: African trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive. RESULTS: We used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5. CONCLUSIONS: Our results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.


Subject(s)
Centromere/genetics , Tandem Repeat Sequences/genetics , Trypanosoma brucei brucei/genetics , Genome, Protozoan , Oligonucleotide Array Sequence Analysis , Restriction Mapping
15.
Genome Res ; 21(6): 915-24, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21363968

ABSTRACT

African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000 integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and "hypothetical" genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides much needed genetic validation to identify new drug targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.


Subject(s)
Genome, Protozoan/genetics , Genomics/methods , High-Throughput Nucleotide Sequencing/methods , Phenotype , RNA Interference , Sequence Analysis, DNA/methods , Trypanosoma brucei brucei/genetics , Computational Biology , DNA Primers/genetics , Gene Library , Genetic Fitness/genetics , Plasmids/genetics , Trypanosoma brucei brucei/physiology
16.
Nucleic Acids Res ; 39(3): 1023-33, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20864447

ABSTRACT

Topoisomerase-II accumulates at centromeres during prometaphase, where it resolves the DNA catenations that represent the last link between sister chromatids. Previously, using approaches including etoposide-mediated topoisomerase-II cleavage, we mapped centromeric domains in trypanosomes, early branching eukaryotes in which chromosome segregation is poorly understood. Here, we show that in bloodstream form Trypanosoma brucei, RNAi-mediated depletion of topoisomerase-IIα, but not topoisomerase-IIß, results in the abolition of centromere-localized activity and is lethal. Both phenotypes can be rescued by expression of the corresponding enzyme from T. cruzi. Therefore, processes which govern centromere-specific topoisomerase-II accumulation/activation have been functionally conserved within trypanosomes, despite the long evolutionary separation of these species and differences in centromeric DNA organization. The variable carboxyl terminal region of topoisomerase-II has a major role in regulating biological function. We therefore generated T. brucei lines expressing T. cruzi topoisomerase-II truncated at the carboxyl terminus and examined activity at centromeres after the RNAi-mediated depletion of the endogenous enzyme. A region necessary for nuclear localization was delineated to six residues. In other organisms, sumoylation of topoisomerase-II has been shown to be necessary for regulated chromosome segregation. Evidence that we present here suggests that sumoylation of the T. brucei enzyme is not required for centromere-specific cleavage activity.


Subject(s)
Antigens, Neoplasm/metabolism , Centromere/enzymology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Antigens, Neoplasm/chemistry , DNA Cleavage , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Molecular Sequence Data , RNA Interference , Sumoylation , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & development , Trypanosoma cruzi/enzymology
17.
Genome Biol ; 8(3): R37, 2007.
Article in English | MEDLINE | ID: mdl-17352808

ABSTRACT

BACKGROUND: Trypanosomes are parasitic protozoa that diverged early from the main eukaryotic lineage. Their genomes display several unusual characteristics and, despite completion of the trypanosome genome projects, the location of centromeric DNA has not been identified. RESULTS: We report evidence on the location and nature of centromeric DNA in Trypanosoma cruzi and Trypanosoma brucei. In T. cruzi, we used telomere-associated chromosome fragmentation and found that GC-rich transcriptional 'strand-switch' domains composed predominantly of degenerate retrotranposons are a shared feature of regions that confer mitotic stability. Consistent with this, etoposide-mediated topoisomerase-II cleavage, a biochemical marker for active centromeres, is concentrated at these domains. In the 'megabase-sized' chromosomes of T. brucei, topoisomerase-II activity is also focused at single loci that encompass regions between directional gene clusters that contain transposable elements. Unlike T. cruzi, however, these loci also contain arrays of AT-rich repeats stretching over several kilobases. The sites of topoisomerase-II activity on T. brucei chromosome 1 and T. cruzi chromosome 3 are syntenic, suggesting that centromere location has been conserved for more than 200 million years. The T. brucei intermediate and minichromosomes, which lack housekeeping genes, do not exhibit site-specific accumulation of topoisomerase-II, suggesting that segregation of these atypical chromosomes might involve a centromere-independent mechanism. CONCLUSION: The localization of centromeric DNA in trypanosomes fills a major gap in our understanding of genome organization in these important human pathogens. These data are a significant step towards identifying and functionally characterizing other determinants of centromere function and provide a framework for dissecting the mechanisms of chromosome segregation.


Subject(s)
Centromere , DNA , Repetitive Sequences, Nucleic Acid , Trypanosoma brucei brucei/genetics , Trypanosoma cruzi/genetics , Animals , Base Composition , Base Sequence , Chromosome Segregation , Chromosomes , DNA Topoisomerases, Type II , Genome, Protozoan , Multigene Family
18.
Mol Biochem Parasitol ; 147(1): 126-36, 2006 May.
Article in English | MEDLINE | ID: mdl-16569451

ABSTRACT

The cell surface of the epimastigote form of Trypanosoma cruzi is covered by glycoconjugates rich in galactose. The parasite cannot take up galactose through its hexose transporter, suggesting that the epimerisation of UDP-glucose to UDP-galactose may be the parasite's only route to this sugar. The T. cruzi UDP-glucose 4'-epimerase is encoded by the TcGALE gene. We were unable to make a CL-Brener strain T. cruzi epimastigote TcGALE-/- null mutant, suggesting that the gene is essential. Two TcGALE+/- single-allele knockout clones displayed aberrant morphology and haploid deficiency with respect to galactose metabolism. The morphological phenotypes included shortened flagella, increased incidence of spheromastigotes, agglutination and a novel walnut-like appearance. The reduced supply of UDP-galactose was manifest in the two clones as a six- or nine-fold reduction in the expression of galactopyranose-containing cell surface mucins and negligible or two-fold reduction in the expression of galactofuranose-containing glycoinositolphospholipids. The major loss of mucins as opposed to glycoinositolphospholipids may indicate that the latter are more important for basic parasite survival in culture. The apparent haploid deficiency suggests that epimerase levels are close to limiting, at least in the epimastigote form, and might be exploited as a potential drug target.


Subject(s)
Galactose/metabolism , Gene Expression Regulation , Trypanosoma cruzi/metabolism , Trypanosoma cruzi/ultrastructure , UDPglucose 4-Epimerase , Animals , Cell Membrane/metabolism , Gene Deletion , Glycolipids/chemistry , Glycolipids/metabolism , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Mucins/chemistry , Mucins/metabolism , Phospholipids/chemistry , Phospholipids/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Trypanosoma cruzi/genetics , Trypanosoma cruzi/growth & development , UDPglucose 4-Epimerase/chemistry , UDPglucose 4-Epimerase/genetics , UDPglucose 4-Epimerase/metabolism
19.
Genome Res ; 15(1): 36-43, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15632088

ABSTRACT

Trypanosomatids are an ancient family that diverged from the main eukaryotic lineage early in evolution, which display several unique features of gene organization and expression. Although genome sequencing is now complete, the nature of centromeres in these and other parasitic protozoa has not been resolved. Here, we report the functional mapping of a centromere in the American trypanosome, Trypanosoma cruzi, a parasite with an unusual mechanism of genetic exchange that involves the generation of aneuploidy by nuclear hybridization. Using a telomere-associated chromosome fragmentation approach, we show that the region required for the mitotic stability of chromosome 3 encompasses a transcriptional "strand-switch" domain constituted by a 16-kb GC-rich island. The domain contains several degenerate retrotransposon-like insertions, but atypically, lacks the arrays of satellite repeats normally associated with centromeric regions. This unusual type of organization may represent a paradigm for centromeres in T. cruzi and other primitive eukaryotes.


Subject(s)
Centromere/genetics , Chromosomes/genetics , DNA Fragmentation/genetics , Physical Chromosome Mapping/methods , Trypanosoma cruzi/genetics , Animals , Chromosome Aberrations , Genetic Vectors/genetics , Genome, Protozoan , Mitosis/genetics , Molecular Sequence Data , Monosomy/genetics
20.
Proc Natl Acad Sci U S A ; 99(21): 13453-8, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12351682

ABSTRACT

In most aerobic organisms hemoperoxidases play a major role in H(2)O(2)-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H(2)O(2). To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.


Subject(s)
Peroxidases/metabolism , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Animals , Ascorbate Peroxidases , Ascorbic Acid/metabolism , Base Sequence , DNA, Protozoan/genetics , Endoplasmic Reticulum/enzymology , Genes, Protozoan , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Molecular Sequence Data , Oxidation-Reduction , Peroxidases/chemistry , Peroxidases/genetics , Plants/enzymology , Sequence Homology, Amino Acid , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/genetics
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