Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ethanolamine phosphoglycerol attachment to eEF1A is not essential for normal growth of Trypanosoma brucei.

ABSTRACT: Eukaryotic elongation factor 1A (eEF1A) is the only protein modified by ethanolamine phosphoglycerol (EPG). In mammals and plants, EPG is attached to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote, Trypanosoma brucei, a single EPG moiety is attached to domain III. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but the role of this unique protein modification in cellular growth and eEF1A function has remained elusive. Here we report, for the first time in a eukaryotic cell, a model system to study potential roles of EPG. By down-regulation of EF1A expression and subsequent complementation of eEF1A function using conditionally expressed exogenous eEF1A (mutant) proteins, we show that eEF1A lacking EPG complements trypanosomes deficient in endogenous eEF1A, demonstrating that EPG attachment is not essential for normal growth of T. brucei in culture.

SUBMITTER: Greganova E

PROVIDER: S-EPMC3275919 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Ethanolamine phosphoglycerol attachment to eEF1A is not essential for normal growth of Trypanosoma brucei.

Greganova Eva E Bütikofer Peter P

Scientific reports 20120209

Eukaryotic elongation factor 1A (eEF1A) is the only protein modified by ethanolamine phosphoglycerol (EPG). In mammals and plants, EPG is attached to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote, Trypanosoma brucei, a single EPG moiety is attached to domain III. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but the role of this unique protein modification in cellular ...[more]

PMID: 22355766

Similar Datasets

A structural domain mediates attachment of ethanolamine phosphoglycerol to eukaryotic elongation factor 1A in Trypanosoma brucei.

Project description:Ethanolamine phosphoglycerol (EPG) represents a protein modification that so far has only been found in eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is covalently attached to two conserved glutamate residues located in domains II and III of eEF1A. In contrast, Trypanosoma brucei eEF1A contains a single EPG attached to Glu362 in domain III. The sequence and/or structural requirements for covalent linkage of EPG to eEF1A have not been determined for any organism. Using a combination of biosynthetic labelling of parasites with tritiated ethanolamine and mass spectrometry analyses, we demonstrate that replacement of Glu362 in T. brucei eEF1A by site-directed mutagenesis prevents EPG attachment, whereas single or multiple amino acid substitutions around the attachment site are not critical. In addition, by expressing a series of eEF1A deletion mutants in T. brucei procyclic forms, we demonstrate that a peptide consisting of 80 amino acids of domain III of eEF1A is sufficient for EPG attachment to occur. Furthermore, EPG addition also occurs if domain III of eEF1A is fused to a soluble reporter protein. To our knowledge, this is the first report addressing amino acid sequence, or structure, requirements for EPG modification of eEF1A in any organism. Using T. brucei as a model organism, we show that amino acid substitutions around the modification site are not critical for EPG attachment and that a truncated version of domain III of eEF1A is sufficient to mediate EPG addition.

| S-EPMC2830473 | biostudies-literature

The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei.

Project description:Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.

| S-EPMC2784872 | biostudies-literature

An essential farnesylated kinesin in Trypanosoma brucei.

Project description:Kinesins are a family of motor proteins conserved throughout eukaryotes. In our present study we characterize a novel kinesin, Kinesin(CaaX), orthologs of which are only found in the kinetoplastids and not other eukaryotes. Kinesin(CaaX) has the CVIM amino acids at the C-terminus, and CVIM was previously shown to be an ideal signal for protein farnesylation in T. brucei. In this study we show Kinesin(CaaX) is farnesylated using radiolabeling studies and that farnesylation is dependent on the CVIM motif. Using RNA interference, we show Kinesin(CaaX) is essential for T. brucei proliferation. Additionally RNAi Kinesin(CaaX) depleted T. brucei are 4 fold more sensitive to the protein farneysltransferase (PFT) inhibitor LN-59, suggesting that Kinesin(CaaX) is a target of PFT inhibitors' action to block proliferation of T. brucei. Using tetracycline-induced exogenous tagged Kinesin(CaaX) and Kinesin(CVIMdeletion) (non-farnesylated Kinesin) expression lines in T. brucei, we demonstrate Kinesin(CaaX) is farnesylated in T. brucei cells and this farnesylation has functional effects. In cells expressing a CaaX-deleted version of Kinesin, the localization is more diffuse which suggests correct localization depends on farnesylation. Through our investigation of cell cycle, nucleus and kinetoplast quantitation and immunofluorescence assays an important role is suggested for Kinesin(CaaX) in the separation of nuclei and kinetoplasts during and after they have been replicated. Taken together, our work suggests Kinesin(CaaX) is a target of PFT inhibition of T. brucei cell proliferation and Kinesin(CaaX) functions through both the motor and farnesyl groups.

| S-EPMC3206815 | biostudies-literature

Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector.

Project description:Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

| S-EPMC5289646 | biostudies-literature

Assembly and maintenance of the flagellum attachment zone filament in Trypanosoma brucei.

Project description:Adhesion of motile flagella to the cell body in Trypanosoma brucei requires a filamentous cytoskeletal structure termed the flagellum attachment zone (FAZ). Despite its essentiality, the complete molecular composition of the FAZ filament and its roles in FAZ filament assembly remain poorly understood. By localization-based screening, we here identified a new FAZ protein, which we called FAZ2. Knockdown of FAZ2 disrupted the FAZ filament, destabilized multiple FAZ filament proteins and caused a cytokinesis defect. We also showed that FAZ2 depletion destabilized another new FAZ filament protein and several flagellum and cytoskeleton proteins. Furthermore, we identified CC2D and KMP11 as FAZ2 partners through affinity purification, and showed that they are each required for maintaining a stable complex. Finally, we demonstrated that FAZ filament proteins are incorporated into the FAZ filament from the proximal region, in contrast to the flagellum components, which are incorporated from the distal tip. In summary, we identified three new FAZ filament proteins and a FAZ filament protein complex, and our results suggest that assembly of the FAZ filament occurs at the proximal region and is essential to maintain the stability of FAZ filament proteins.

| S-EPMC4524107 | biostudies-literature

Phosphatidylinositol synthesis is essential in bloodstream form Trypanosoma brucei.

Project description:PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.

| S-EPMC1462709 | biostudies-literature

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

Project description:For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

| S-EPMC5207785 | biostudies-literature

A mitochondrial DNA primase is essential for cell growth and kinetoplast DNA replication in Trypanosoma brucei.

Project description:Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3'-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.

| S-EPMC2832486 | biostudies-literature

A novel protein kinase is essential in bloodstream Trypanosoma brucei.

Project description:Human African trypanosomiasis a fatal disease for which no vaccines exist and treatment regimens are difficult. Here, we evaluate a Trypanosoma brucei protein kinase, AEK1, as a potential drug target. Conditional knockouts confirmed AEK1 essentiality in bloodstream forms. For chemical validation, we overcame the lack of AEK1 inhibitors by creating parasites expressing a single, functional analog-sensitive AEK1 allele. Analog treatment of mice infected with this strain delayed parasitemia and death, with one-third of animals showing no parasitemia. These studies validate AEK1 as a drug target and highlight the need for further understanding of its function.

| S-EPMC4925258 | biostudies-literature

An essential RNase III insertion editing endonuclease in Trypanosoma brucei.

Project description:RNA editing adds and deletes uridine nucleotides in many preedited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and approximately 20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonuclease 2).

| S-EPMC1283813 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data