Biology subjects

Westerhoff, H. V.

Publications and source records attributed to Westerhoff, H. V..

5 recordsLinked to original sources

bioRxiv2026

Computer experimentation on E. coli ammonium transport and assimilation reveals mechanisms for energy coupling, balanced futile cycling, and robust growth

Nitrogen is essential for all life forms, and microorganisms prefer ammonium as a nitrogen source. Due to the low affinity of glutamine synthetase (GS) for ammonium, E. coli must maintain high intracellular ammonium (NH4+) concentrations to sustain its rapid growth. Under ammonium limitation, E. coli imports ammonium through the transporter AmtB and incorporates it into glutamine by using GS. On the basis of structural and mutagenesis information, mechanisms have been proposed for the transport of ammonia (NH3) and protons by AmtB through spatially (partly) separate routes. These mechanisms do not explain the required coupling between proton and ammonia transports. How does the membrane potential push the ammonia inward so as to attain high concentrations near GS? We here compare six candidate kinetic models of E. coli ammonium transport and assimilation in terms of how they reproduce experimental data from the literature: three variants of the electro-binding model in which the membrane potential affects AmtB-NH4+ binding, and three variants of the electro-flipping model in which it influences the conformational flip of the transporter. The computer simulations decide that the electro-binding models are 28 times more plausible than the electro-flipping models and suggest that the transmembrane electric potential affects AmtB-NH4+ binding from the cytoplasmic side. The addition of kinetic and thermodynamic features to existing structural information plus our requirement of an explanation of the coupling, suggest a new spatiotemporal mechanism of coupling of ammonia and proton flows in AmtB. Further simulations show that GS and AmtB regulation is coordinated via both the uridylyltransferase/uridylyl-removing enzyme (UTase) and 2-oxoglutarate binding, allowing the cell to minimize futile cycling while maintaining rapid growth. The free energy cost of transport-related futile cycling exceeded that of the GS reaction itself. Moreover, AmtB enabled robust growth under varying ammonium concentrations and pH levels, albeit at a cost of futile cycling that became substantial at low ammonium. These findings highlight the crucial roles of GS and AmtB in E. colis adaptations and provide new insights into the trade-off mechanism between nutrient acquisition and energy efficiency.

systems biology↗

bioRxiv2020

4sUDRB-sequencing for genome-wide transcription bursting quantification in breast cancer cells

Epigenetics maintains cell-identity specific gene-expression patterns. However, within a population of isogenic cells of the same identity, a substantial variability in gene expression and responsiveness is still observed. Transcription bursting is a substantial source of this gene-expression variability or noise, contributing to phenotypic heterogeneity and potentially driving both physiological and pathological processes such as differentiation or tumorigenesis and drug resistance. Identification of transcription-bursting dynamics at a genome-wide scale has been restricted to inferring bursts in mRNA production computationally from the heterogeneity of mRNA levels in single cell transcriptomic data. Systematic characterisation of the genomic and epigenetic chromatin context of genes with defined transcription bursting behaviour has been incomplete. Here, we measured the bursting of transcription itself by genome-wide nascent RNA sequencing of breast cancer MCF-7 cells upon synchronisation of transcription with a transcription elongation inhibitor and by calibration using live cell imaging of nascent PP7-tagged GREB1 transcription. Comparing across the entire genome, we find transcription bursting to be ubiquitous, with burst sizes of up to 160 transcripts. Transcription bursting is strongly correlated with steady state gene expression between genes, whereas both burst frequency and nascent transcript degradation only correlate weakly. Individual genes deviate strongly from this trend and engage both in anomalous burst size and frequency. We find that the presence of the TATA box or Inr sequence within gene promoters are significantly associated with a larger burst size, as are promoter-associated YY1 and E2F1 transcription-factor binding motifs. Enrichment of the transcription start site with epigenetic marks such as H3K79me2 and H3K27ac is also strongly associated with the transcription burst size. Finally, we show that in these MCF-7 breast-cancer cells, genes with a larger transcription burst size exhibit a larger immediate transcriptional response following endocrine drug treatment. Our genome-wide transcription-bursting analysis method paves the way to elucidate the dynamic role of epigenetic regulation on dynamic transcription in pathophysiology.

molecular biology↗

bioRxiv2019

Nature and Nurture integrated through an adjustable Flux Balance Analysis: The impact of cell-nutrition changes on the Warburg effect in hepatocellular carcinoma

How cancer cells utilize nutrients to support their growth and proliferation in complex nutritional systems is still an open question. However, it is certainly determined by both genetics and an environmental-specific context. The interactions between them lead to profound metabolic specialization, such as consuming glucose and glutamine and producing lactate at prodigious rates. To investigate whether and how glucose and glutamine availability impact metabolic specialization, we integrated computational modeling on the genome-scale metabolic reconstruction with an experimental study on cell lines. We used the most comprehensive human metabolic network model to date, Recon3D, to build cell line-specific models. RNA-Seq data was used to specify the activity of genes in each cell line and the uptake rates were quantitatively constrained according to nutrient availability. To integrated both constraints we applied a novel method, named GENSI (Gene Expression and Nutrients Simultaneous Integration), that translates the relative importance of gene expression and nutrient availability data into the metabolic fluxes based on an observed experimental feature(s). We applied GENSI to study hepatocellular carcinoma addiction to glucose/glutamine. We were able to identify that proliferation, and lactate production is associated with the presence of glucose but does not necessarily increase with its concentration when the latter exceeds the physiological concentration. There was no such association with glutamine. We show that the integration of gene expression and nutrient availability data into genome-wide models improves the prediction of metabolic phenotypes.

systems biology↗

bioRxiv2019

Design principles of ROS dynamic networks relevant to precision therapies for age-related diseases

The eminently complex regulatory network protecting the cell against oxidative stress, surfaces in several disease maps, including that of Parkinsons disease (PD). How this molecular networking achieves its various functionalities and how processes operating at the seconds-minutes time scale cause a disease at a time scale of multiple decennia is enigmatic.\n\nBy computational analysis, we here disentangle the reactive oxygen species (ROS) regulatory network into a hierarchy of subnetworks that each correspond to a different functionality. The detailed dynamic model of ROS management obtained integrates these functionalities and fits in vitro data sets from two different laboratories.\n\nThe model shows effective ROS-management for a century, followed by a sudden systems collapse due to the loss of p62 protein. PD related conditions such as lack of DJ-1 protein or increased -synuclein accelerated the systems collapse. Various in-silico interventions (e.g. addition of antioxidants or caffeine) slowed down the collapse of the system in silico, suggesting the model may help discover new medicinal and nutritional therapies.

systems biology↗

bioRxiv2017

Rational cell culture optimization enhances experimental reproducibility in cancer cells.

Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data.\n\nBaseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce.\n\nWe then devised metabolically rationalized standard assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays.

cell biology↗