SilicoTryp

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Aim: To provide quantitative data that will allow modeling of gene expression for all enzymes of redox metabolism and the pentose phosphate pathway. Modeling will be used to predict enzyme levels based on the integration of an RNA degradation model with translation and protein degradation rates.

Plan: The amounts of a protein in a cell can be determined by the rates of transcription, mRNA processing, translation, mRNA turnover and protein degradation. In trypanosomes analysis is simpler because ...

Aim. To provide critical quantitative parameter information and to model redox balance by determining the cellular concentration of all enzymes involved in the trypanothione-dependent hydroperoxide detoxification system of trypanosomes and by performing the kinetic characterization of the involved enzymes under pseudo-physiological conditions.

The aims of this investigation is to quantify metabolites associated with pathways involved in stress responses for parameterising models of oxidative stress metabolism; the measurement of metabolic fluxes of metabolites of interest with intracellular concentrations

Multiply perturbations of trypanosome redox metabolism, closing the feedback loop between experimentation and in silioc modelling, allowing model refinement or, where there are unexpected outcomes, re-evaluation. Providing a dynamic picture of cell physiology by examining programmed metabolic changes during the developmental life-cycle of these parasites as they adapt to very different external milieus, including distinct levels of oxidative stress and unique adaptations of their redox balance ...

Aim. Constructing a predictive, dynamic model of the redox metabolism of trypanosomes. Aided by this model we will quantify the impact of gene-expression and metabolic regulation on redox metabolism. The model will be constructed in an iterative cycle of experimentation – modelling – analysis – experimentation, such that it can be extended and refined based on new experimental insights.

Since over 40 enzymes will be investigated for their mRNA abundance, processing, and degradation kinetics, the less tedious and more accurate Next Generation Sequencing of the entire mRNA repertoire of the cell is employed. To optimise the proportion of useful sequence, while including RNA fragments that are products of of degradation, rRNA is depleted using the eukaryotic Ribominus kit (Ambion). Two biological replicates are treated with Sinefungin and Actinomycin D to inhibit RNA processing and ...

The enzyme Trypanothione Synthetase (TryS) is a complex enzyme that catalyses the two step reaction that forms trypanothione from 2 molecules of GSH and 1 molecule of Spd and the use of ATP

Flux will be measured using the metabolomics platforms based on absolute quantification method (isotope ratio based MS technique) by LC-MS, using heavy-isotope labelled precursors of the metabolites of interest. For example, 15N labelled cysteine, glycine and glutamate will be used to determine rates of synthesis of glutathione. 15N-labelled methionine to measure S-adenosyl methionine (and its decarboxylated form, as well as methionine cycle intermediates). 15N labelled arginine is used as precursor ...

In addition to the highly targeted quantification of metabolites already known to play major roles in oxidative stress, to provide data directly compatible with current models, we will also take an untargeted metabolomics approach. This will enable us to identify other areas of the metabolome influenced by, or influencing, oxidative stress and will allow us to compare changes in each of the stress-inducing stimuli. We have recently pioneered untargeted metabolite profiling of T. brucei using ...

We have already demonstrated that the key metabolites of polyamine biosynthesis (arginine, ornithine, putrescine and spermidine) can be identified using HILIC chromatography coupled to the Orbitrap mass spectrometer, as can glycine, glutamate and cysteine used in glutathione biosynthesis, glutathionyl spermidine and trypanothione itself. Furthermore the key metabolites of the methionine cycle (methionine, S-adenosyl methionine, decarboxylated S-adenosyl methionine, methylthioadenosine) can all ...

Parasites will be harvested at different growth phases and the total amount of the proteins will be followed by western blot. The absolut concentration will be obtained by comparison with a know amount of the recombinant untagged protein. The thiol redox state of the proteins will be followed by modification of the free cys with methoxy-ethyl-maleinimide poly(ethylenglycol) (Meo-PEG-mal).

The enzymes involved in the trypanothione metabolism will be studied in a uniform assay medium that mimics the intracellular milieu of the parasite.

Key enzymes of critical points in the pathways will be targeted for disruption by the generation of RNAi cell lines and lines which drive tetracycline-regulatable ectopic over expression of either wild type enzyme or, if appropriate, dominant-negative or mis-targeted mutants of these. In all cases perturbed lines will be analysed with respect to the mRNA, protein or enzymatic activities of other components of the subsystem, this being directed iteratively by the predictions from systems modelling. ...

The output of the initial model of redox metabolism will be compared to experimental flux and metabolite data. Deviations between model and experiment will be prioritized together with WP2. We will apply Metabolic Control Analysis (Fell 1992 PMID: 1530563) to diagnose which enzymes control the deviating metabolite concentrations and/or rates. When the agreement between model and experiment is sufficient we will first link it to the existing model of trypanosome glycolysis and repeat the same ...

Our current gene-expression model (Haanstra et al. 2008 PMID: 19008351) will be parameterized for the different genes of interest. The framework of this gene expression model has been used to include mRNA half life data into the model of glycolysis For the enzymes of redox metabolism we will use newly measured rates of transcription, RNA precursor degradation, mRNA degradation, concentrations of mature mRNAs and proteins, enzyme turnover, Vmax values and metabolic fluxes (WP3&5). Regulation ...

We are in the process of construct an ODE model of the trypanothione pathway. As input we will use newly determined and existing kinetic data and measured metabolite concentrations at the boundaries (from WP3&6). Recently the glycolysis model was extended with the pentose phosphate pathway. This pathway will yield the NAPDH that maintains trypanothione in a reduced state. For some complex enzymes (i.e trypanothione synthase) we are intensively discussing the kinetic data obtained on the ...

This assay is designed to measure the decay kinetics of mRNA in T. brucei blood forms. T. brucei lacks the canonical transcriptional regulation employed by other eukaryotes through transcription factors, and relies almost entirely on regulation of mRNA decay and further downstream steps in order to control gene expression. 3 replicates of 8 time points were taken to measure mRNA abundance in the cell using RNA-seq. The first time point was fot WT, untreated cells; the second was 5 min after the ...

This assay is designed to obtain the in vitro kinetic data of T. brucei recombinant trypanothione synthetase. The enzyme catalyzes the ATP-dependent ligation of spermidine (Spd) and GSH to generate glutathionylspermidine (Gsp) and also of Gsp and GSH to finally produce trypanothione (T(SH)2). The data was obtained in an spectrophotometric assay that links ADP production with NADH consumption through the piruvte kinase and lactate dehydrogenase.

This assay is for method development to quantify intra- and extra-cellular metabolites on T. brucei 427 bloodstream form using isotope ratio based MS technique with 13C-labelled E. coli extract

Intracellular metabolites in T. brucei at different stage of cell growth have been quantified absolutely by isotope ratio based MS technique using uniformly 13C-labelled E. coli extract. This is the case study for method development of absolute quantification for metabolic flux analysis.

Metabolite profiling on T. brucei exposed to methylene blue has been carried out using LC-MS to investigate metabolic changes caused by oxidative stress

26 intracellular metabolites (amino acids, polyamines, TCA intermediates) in T. brucei exposed to methylene blue have been absolutely quantified using isotope ratio based MS technique.

26 intracellular metabolites (amino acids, polyamines, TCA intermediates) in T. brucei under pH stress (pH8.7) have been absolutely quantified using isotope ratio based MS technique.

Extracellular metabolites in T. brucei at different stage of cell growth have been quantified absolutely by isotope ratio based MS technique using uniformly 13C-labelled E. coli extract. This is the case study for method development of absolute quantification for metabolic flux analysis.

Genes are transcribed in polysictronic messages (pre-mRNA) that are destined for either maturation into mRNAs, or degradation. Since transcription regulation is non-existent with few exceptions, the rate of pre-mRNA processing, together with mRNA decay and translation rates, are believed to control gene expression. In this assay, 2T1 blood form trypanosomes are subject to treatment by ActinomycinD for 5 minutes, inhibiting transcription. The cells are harvested, depleted for ribosomal RNA, and ...

We here create a kinetic model for a single enzyme within the T. brucei trypanothione synthesis pathway, the enzyme trypanothione synthetase based on the insights from the laboratory experiments

The RNAseq data on mRNA processing and mRNA decay were used to update a previously published model and to interrogate which process should be dependent on mRNA length

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of glutathione, and spermidine.

All datapoints that were measured are displayed together with the accompanying simulations by the computational model

This files contains the parameter values, life-times, half-lives and errors associated with modeling the decay of the transcriptome, based on 3 models described in Deneke et al. "Complex degradation processes lead to non-exponential decay patters and age-dependent decay rates of messenger RNA". PLoS One. 2013;8(2):e55442

The file contains the normalized relative read counts (RPM) of 2 mRNA decay experiments. Columns in blue correspond to experiment 1, columns in violet correspond to experiment 2. The time points are in column headers. The last 3 columns contain parameters and half lives calculated from an exponantial fit of all data points. Normalization was done in 2 steps :first by calculating RPM i.e. reads per million of aligned reads to unique ORFs, second by normalizing this to the total amount of mRNA ...

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of GSH, and Spd.

An extended model description of the TryS model

The file contains the initial rate measurements of TbTryS obtained under different substrate and product initial concentrations.

Untargeted and targeted metabolic analysis on T. b. brucei 427 grown under oxidative stress with methylene blue has been carried out. This work has been completed with 11 bio-reps and found significant metabolic changes as you can see in the IDEOM file attached. 'Comparison' tab in the data spread sheet shows heat maps and fold change analysis regarding different metabolite levels (T: T brucei, TMB: T. brucei exposed to methylene blue, numbers: time points, 0, 5, 60 & 120min). If you double ...

Metabolic changes of 26 intracellular metabolites extracted from T. b. brucei 427 wild type and arginine kinase knockout cells under high pH stress (pH8.7)

Intracellular concentrations of 50 metabolites measured by LC-MS using isotope ratio based mass spectrometry technique

Concentrations of 22 extracellular metabolites (major medium components) from T. b. brucei 427

Metabolic changes of 26 intracellular metabolites extracted from T. b. brucei 427 wild type and arginine kinase knockout cells exposed to methylene blue

Describes the assumptions made, how to integrate the new reactions to the rest of the glycolysis model, the parameters needed and the preliminary parameter values collected from the litterature.

Arginine kinase has been thought of as a potential stress marker (see 'Metabolic changes by oxidative stress in T. b. brucei 427'), the gene knockout cells have been constructed.

Mechanistical model of the catalytic cycle of Trypanothione Synthetase

SBML models without activity of the glycolytic enzymes in the cytosol:

Glycolysis_noActivityInCytosol_1a.xml Model 1a Glycolysis_noActivityInCytosol_1b.xml Model 1b Glycolysis_noActivityInCytosol_2.xml Model 2 Glycolysis_noActivityInCytosol_3.xml Model 3 Glycolysis_noActivityInCytosol_4.xml Model 4 Glycolysis_noActivityInCytosol_5.xml Model 5 Glycolysis_noActivityInCytosol_6.xml Model 6

SBML models with activity of the glycolytic enzymes in the cytosol:

Glycolysis_withActivityInCytosol_1a.xm Model ...

First darft of a model including glycolysis and the transcription and translation of the enzymes. See the datafile "Information on the darft transcription/translation model." for information.

Fixed parameter model, where the glycolysis model of bloodstream form T. brucei is extended with the pentose phosphate pathway and a ribokinase in the glycosome. Non-final version.

Fixed parameter model, where the glycolysis model of bloodstream form T. brucei is extended with the pentose phosphate pathway and an ATP:ADP antiporter over the glycosomal membrane. Non-final version.

SBML file supplementary material of the publication.

This method describes how to derivatize the N-glutathionylspermidine and trypanothione produced by T. brucei trypanothione synthetase under in vivo-like conditions

This is a protocol for determining the activity of the T. brucei Trypanothione synthetase under in vivo-like conditions.

This method describes the preparation of the in vivo-like buffer for the measurement of bloodstream T. brucei recombinant enzymes under pseudo-physiological conditions.

Sample preparation procedure for metabolic analysis on T. b. brucei 427 using LC/MS

Labelling and extraction procedure for uniformly 13C-labelled E. coli (MG1655) for absolute quantification using isotope dilution technique by LC-MS

Sample preparation procedure for metabolic footprint analysis on T. b. brucei 427 using LC/MS

For the study of mRNA decay rates, transcription was inhibited with ActinomycinD, and RNA splicing with Sinefungin, at different time points, in the Matthews lab. rRNA depleted RNA was extracted from each of the samples in the Clayton lab, and sent for deep sequencing at the BioQuant facility in Heidelberg

We routinely select specific RNAi gene targets (400–600 bp) and primers using the RNAit software http://trypanofan.path.cam.ac.uk/software/RNAit.html. A single pair of PCR primers are designed that incorporate four selected restriction sites (not present in the RNAi target fragment) such that a single PCR product can be differentially digested and sequentially cloned. For example, using MCS1/2, the following primers could be used to clone antisense followed by sense fragments: Primer 1, XbaI–BamHI-5′ ...

Poster presented during the ICSB 2011 conference in Mannheim

Oral presentation given at ICSB 2011 in Mannheim/Heidelberg

Poster presented during the ICSB 2011 conference in Mannheim