The Ser-Thr kinase mammalian target of rapamycin (mTOR) controls cell growth and metabolism by stimulating glycolysis and synthesis of proteins and lipids. To further understand the central role of ...mTOR in cell physiology, we used quantitative phosphoproteomics to identify substrates or downstream effectors of the two mTOR complexes. mTOR controlled the phosphorylation of 335 proteins, including CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase). CAD catalyzes the first three steps in de novo pyrimidine synthesis. mTORC1 indirectly phosphorylated CAD-S1859 through S6 kinase (S6K). CAD-S1859 phosphorylation promoted CAD oligomerization and thereby stimulated de novo synthesis of pyrimidines and progression through S phase of the cell cycle in mammalian cells. Thus, mTORC1 also stimulates the synthesis of nucleotides to control cell proliferation.
Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. ...Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.
Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic ...compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, ¹³C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPH-dependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype.
To counteract oxidative stress and reactive oxygen species (ROS), bacteria evolved various mechanisms, primarily reducing ROS through antioxidant systems that utilize cofactor NADPH. Cells must ...stabilize NADPH levels by increasing flux through replenishing metabolic pathways like pentose phosphate (PP) pathway. Here, we investigate the mechanism enabling the rapid increase in NADPH supply by exposing Escherichia coli to hydrogen peroxide and quantifying the immediate metabolite dynamics. To systematically infer active regulatory interactions governing this response, we evaluated ensembles of kinetic models of glycolysis and PP pathway, each with different regulation mechanisms. Besides the known inactivation of glyceraldehyde 3-phosphate dehydrogenase by ROS, we reveal the important allosteric inhibition of the first PP pathway enzyme by NADPH. This NADPH feedback inhibition maintains a below maximum-capacity PP pathway flux under non-stress conditions. Relieving this inhibition instantly increases PP pathway flux upon oxidative stress. We demonstrate that reducing cells' capacity to rapidly reroute their flux through the PP pathway increases their oxidative stress sensitivity.
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•Characterization of the immediate metabolic response of E. coli to oxidative stress•Development of a high-performance ensemble modeling computational pipeline•Identification of NADPH feedback inhibition on G6PDH as key regulatory interaction•Cells without reserve flux capacity in PP pathway are sensitive to oxidative stress
Christodoulou et al. describe the immediate metabolic response of E. coli to oxidative stress and reveal the key interactions that implement it. Combining quantitative metabolite dynamics with a high-performing ensemble modeling computational pipeline, they reveal the importance of a small molecule–protein interaction in the rapid cellular response. This interaction acts as a valve, maintaining a reserve of flux, which can be used rapidly when cells are exposed to stress. Cells without such a reserve capacity are more sensitive to oxidative stress.
Summary
DmpR is the obligate transcriptional activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR belongs to the AAA+ class of mechano‐transcriptional regulators that ...employ ATP‐hydrolysis to engage and remodel σ54‐RNA polymerase to allow transcriptional initiation. Previous work has established that binding of phenolic effectors by DmpR is a prerequisite to relieve interdomain repression and allow ATP‐binding to trigger transition to its active multimeric conformation, and further that a structured interdomain linker between the effector‐ and ATP‐binding domains is involved in coupling these processes. Here, we present evidence from ATPase and in vivo and in vitro transcription assays that a tyrosine residue of the interdomain linker (Y233) serves as a gatekeeper to constrain ATP‐hydrolysis and aromatic effector‐responsive transcriptional activation by DmpR. An alanine substitution of Y233A results in both increased ATPase activity and enhanced sensitivity to aromatic effectors. We propose a model in which effector‐binding relocates Y233 to synchronize signal‐reception with multimerisation to provide physiologically appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand‐responsive mechano‐transcriptional regulators, the model is likely to be pertinent for numerous members of this family and has implications for development of enhanced sensitivity of biosensor used to detect pollutants.
Chemical probes are important tools for understanding biological systems. However, because of the huge combinatorial space of targets and potential compounds, traditional chemical screens cannot be ...applied systematically to find probes for all possible druggable targets. Here, we demonstrate a novel concept for overcoming this challenge by leveraging high‐throughput metabolomics and overexpression to predict drug–target interactions. The metabolome profiles of yeast treated with 1,280 compounds from a chemical library were collected and compared with those of inducible yeast membrane protein overexpression strains. By matching metabolome profiles, we predicted which small molecules targeted which signaling systems and recovered known interactions. Drug–target predictions were generated across the 86 genes studied, including for difficult to study membrane proteins. A subset of those predictions were tested and validated, including the novel targeting of GPR1 signaling by ibuprofen. These results demonstrate the feasibility of predicting drug–target relationships for eukaryotic proteins using high‐throughput metabolomics.
Synopsis
High‐throughput metabolome profiling identifies potential drug‐target relationships by comparing drug‐treated and induced overexpression S. cerevisiae strains. The approach retrieves known as well as new potential drug‐gene relationships.
High‐throughput metabolomics is used to profile the metabolomes of S. cerevisiae perturbed by induced overexpression of 80 genes, or treatment with 1,280 drugs.
Through the comparison of the metabolome profiles, known as well as new drug‐target relationships are detected.
A new predicted relationship is that between the sugar sensor GPR1 and the drug ibuprofen, providing insight into the effect of the drug on filamentous growth.
The presented approach is suitable for analyzing hard‐to‐study membrane proteins, and for performing genome‐scale studies.
High‐throughput metabolome profiling identifies potential drug‐target relationships by comparing drug‐treated and induced overexpression S. cerevisiae strains. The approach retrieves known as well as new potential drug‐gene relationships.
The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host ...nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.
•New adaptive approach for the EMF estimation.•The EMF is estimated by observing the voltage change after the current interruption.•The approach enables an accurate SoC and capacity ...determination.•Real-time capable algorithm.
The online estimation of battery states and parameters is one of the challenging tasks when battery is used as a part of the pure electric or hybrid energy system. For the determination of the available energy stored in the battery, the knowledge of the present state-of-charge (SOC) and capacity of the battery is required. For SOC and capacity determination often the estimation of the battery electromotive force (EMF) is employed. The electromotive force can be measured as an open circuit voltage (OCV) of the battery when a significant time has elapsed since the current interruption. This time may take up to some hours for lithium-ion batteries and is needed to eliminate the influence of the diffusion overvoltages. This paper proposes a new approach to estimate the EMF by considering the OCV relaxation process within only some first minutes after the current interruption. The approach is based on an online fitting of an OCV relaxation model to the measured OCV relaxation curve. This model is based on an equivalent circuit consisting of a voltage source (represents the EMF) in series with the parallel connection of the resistance and a constant phase element (CPE). Based on this fitting the model parameters are determined and the EMF is estimated. The application of this method is exemplarily demonstrated for the state-of-charge and capacity estimation of the lithium-ion battery in an electrical vehicle. In the presented example the battery capacity is determined with the maximal inaccuracy of 2% using the EMF estimated at two different levels of state-of-charge. The real-time capability of the proposed algorithm is proven by its implementation on a low-cost 16-bit microcontroller (Infineon XC2287).
DmpR is the obligate transcriptional activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR belongs to the AAA
class of mechano-transcriptional regulators that employ ...ATP-hydrolysis to engage and remodel σ
-RNA polymerase to allow transcriptional initiation. Previous work has established that binding of phenolic effectors by DmpR is a prerequisite to relieve interdomain repression and allow ATP-binding to trigger transition to its active multimeric conformation, and further that a structured interdomain linker between the effector- and ATP-binding domains is involved in coupling these processes. Here, we present evidence from ATPase and in vivo and in vitro transcription assays that a tyrosine residue of the interdomain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and aromatic effector-responsive transcriptional activation by DmpR. An alanine substitution of Y233A results in both increased ATPase activity and enhanced sensitivity to aromatic effectors. We propose a model in which effector-binding relocates Y233 to synchronize signal-reception with multimerisation to provide physiologically appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family and has implications for development of enhanced sensitivity of biosensor used to detect pollutants.
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