scholarly journals Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases

2008 ◽  
Vol 415 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Federica Gibellini ◽  
William N. Hunter ◽  
Terry K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
Biochemical Characterization ◽  
De Novo ◽  
Carbon Chain ◽  
Surface Proteins ◽  
Membrane Phospholipids ◽  
Ethanolamine Kinase ◽  
Kennedy Pathway ◽  
Catalytically Active ◽  
Membrane Attachment

Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycerophosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4±0.9 and 219±29 μM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

scholarly journals Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host

2021 ◽  
Vol 15 (9) ◽  
pp. e0009504
Author(s):  
Arunasalam Naguleswaran ◽  
Paula Fernandes ◽  
Shubha Bevkal ◽  
Ruth Rehmann ◽  
Pamela Nicholson ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Tricarboxylic Acid Cycle ◽  
Surface Proteins ◽  
Metabolic Reprogramming ◽  
Tsetse Flies ◽  
Nucleotide Polymorphisms ◽  
Sequencing Technologies ◽  
A Genome ◽  
Animal Trypanosomiasis

Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5’ and 3’ untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

The myo-inositol-1-phosphate synthase gene is essential in Trypanosoma brucei

2005 ◽  
Vol 33 (5) ◽  
pp. 983-985 ◽  
Author(s):  
K.L. Martin ◽  
T.K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Essential Gene ◽  
Recombinant Expression ◽  
Conditional Knockout ◽  
Cellular Functions ◽  
Genome Database ◽  
Inositol 1 ◽  
Catalytically Active ◽  
Phosphate Synthase

The de novo synthesis of myo-inositol occurs via a two-step process: first, glucose 6-phosphate is converted into inositol 1-phosphate by an INO1 (myo-inositol-1-phosphate synthase; EC 5.5.1.4); then, it is dephosphorylated by an inositol monophosphatase. The myo-inositol can then be incorporated into PI (phosphatidylinositol), which is utilized in a variety of cellular functions, including the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. A putative INO1 was identified in the Trypanosoma brucei genome database and, by recombinant expression in Escherichia coli, was shown to be a catalytically active INO1. To investigate the importance of INO1, we created a conditional knockout, which, under non-permissive conditions, showed that INO1 is an essential gene in bloodstream form T. brucei and that the de novo synthesized myo-inositol is used for the formation of PI and GPI anchors.

scholarly journals Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host

2021 ◽  
Author(s):  
Arunasalam Naguleswaran ◽  
Paula Fernandes ◽  
Shubha Bevkal ◽  
Ruth Rehmann ◽  
Pamela Nicholson ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Tricarboxylic Acid Cycle ◽  
Surface Proteins ◽  
Metabolic Reprogramming ◽  
Tsetse Flies ◽  
Nucleotide Polymorphisms ◽  
Sequencing Technologies ◽  
A Genome ◽  
Animal Trypanosomiasis

Trypanosoma brucei ssp , unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of  T. brucei  show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing.  T. brucei brucei  EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks.  We present a genome assembly of EATRO1125, including contigs for the intermediate and mini-chromosomes that serve as repositories of VSG genes. In addition,  de novo  transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes.  Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus.  Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified.  Finally, RNA-Seq data were used to create an optimised annotation file with 5’ and 3’ untranslated regions accurately mapped for 9302 genes.  We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

scholarly journals Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants

2020 ◽  
Vol 6 (2) ◽  
Author(s):  
B F Koel ◽  
R M Vigeveno ◽  
M Pater ◽  
S M Koekkoek ◽  
A X Han ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
De Novo ◽  
Diagnostic Testing ◽  
Influenza Viruses ◽  
Surface Proteins ◽  
H3n2 Virus ◽  
Virus Population ◽  
Sample Collection ◽  
Routine Surveillance ◽  
H3n2 Influenza

Abstract Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4–7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

scholarly journals Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sheena Dass ◽  
Serena Shunmugam ◽  
Laurence Berry ◽  
Christophe-Sebastien Arnold ◽  
Nicholas J. Katris ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Phosphatidic Acid ◽  
De Novo ◽  
Lipid Synthesis ◽  
Parasite Development ◽  
Membrane Biogenesis ◽  
Membrane Phospholipids ◽  
Phosphatidic Acid Phosphatase ◽  
Intracellular Parasites ◽  
Parasite Survival

AbstractApicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by ‘lipotoxicity’ through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.

Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active

2013 ◽  
Vol 450 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Sweta Maheshwari ◽  
Marina Lavigne ◽  
Alicia Contet ◽  
Blandine Alberge ◽  
Emilie Pihan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cellular Localization ◽  
Biochemical Characterization ◽  
De Novo ◽  
Membrane Lipids ◽  
Polyclonal Antibodies ◽  
Homology Modelling ◽  
Site Directed Mutagenesis ◽  
Recombinant Enzymes ◽  
Parasite Life Cycle

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis–Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.

Catalytically active holo Homo sapiens adenosine deaminase I adopts a closed conformation

2022 ◽  
Vol 78 (1) ◽  
Author(s):  
Minh Thu Ma ◽  
Maria Rain Jennings ◽  
John Blazeck ◽  
Raquel L. Lieberman
Keyword(s):  
Adenosine Deaminase ◽  
T Cell Activation ◽  
Cell Activation ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Severe Combined Immunodeficiency ◽  
Closed Conformation ◽  
Catalytically Active

Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

Invariant surface proteins in bloodstream forms of Trypanosoma brucei

1994 ◽  
Vol 10 (2) ◽  
pp. 53-58 ◽  
Author(s):  
P. Overath ◽  
M. Chaudhri ◽  
D. Steverding ◽  
K. Ziegelbauer
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Proteins ◽  
Invariant Surface

scholarly journals A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II

2019 ◽  
Vol 116 (33) ◽  
pp. 16631-16640 ◽  
Author(s):  
José G. García-Cerdán ◽  
Ariel L. Furst ◽  
Kent L. McDonald ◽  
Danja Schünemann ◽  
Matthew B. Francis ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Thylakoid Membrane ◽  
De Novo Assembly ◽  
Biochemical Characterization ◽  
De Novo ◽  
Midpoint Potential ◽  
Photosynthetic Organisms ◽  
Redox Active ◽  
Photooxidative Damage

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559. Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

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