Biomedical subjects

Hans V Westerhoff

Publications and source records attributed to Hans V Westerhoff.

At least 19 recordsLinked to original sources

PubMed2003

Why the phosphotransferase system of Escherichia coli escapes diffusion limitation.

We calculated the implications of diffusion for the phosphoenolpyruvate:glucose phosphotransferase system (glucose-PTS) of Escherichia coli in silicon cells of various magnitudes. For a cell of bacterial size, diffusion limitation of glucose influx was negligible. Nevertheless, a significant concentration gradient for one of the enzyme species, nonphosphorylated IIA(Glc), was found. This should have consequences because the phosphorylation state of IIA(Glc) is an important intracellular signal. For mammalian cell sizes we found significant diffusion limitation, as well as strong concentration gradients in many PTS components, and strong effects on glucose and energy signaling. We calculated that the PTS may sense both extracellular glucose and the intracellular free-energy state. We discuss i), that the effects of diffusion on cell function should prevent this highly effective bacterial system from functioning in eukaryotic cells, ii), that in the larger eukaryotic cell any similar chain of mobile group-transfer proteins can neither sustain the same volumetric flux as in bacteria nor transmit a signal far into the cell, and iii), that systems such as these may exhibit spatial differentiation in their sensitivity to different signals.

Cell Size↗

PubMed2002

Modular response analysis of cellular regulatory networks.

The sheer complexity of intracellular regulatory networks, which involve signal transducing, metabolic, and genetic circuits, hampers our ability to carry out a quantitative analysis of their functions. Here, we describe an approach that greatly simplifies this type of analysis by capitalizing on the modular organization of such networks. Steady-state responses of the network as a whole are accounted for in terms of intermodular interactions between the modules alone; processes operating solely within modules need not be considered when analysing signal transfer through the entire network. The intermodular interactions are quantified through (local) response coefficients which populate an interaction map (matrix). This matrix can be derived from a biochemical or molecular biological analysis of (macro) molecular interactions that constitute the regulatory network. The approach is illustrated by two examples: (i) mitogenic signalling through the mitogen-activated protein kinase cascade in the epidermal growth factor receptor network and (ii) regulation of ammonium assimilation in Escherichia coli.

Animals↗

PubMed2002

Selectivity in overlapping MAP kinase cascades.

Some protein kinases operate in more than one mitogen-activated protein-kinase (MAPK) cascade. We here address the question whether specificity of the cascades necessitates physical sequestration of these "promiscuous" kinases (e.g. by binding to scaffolds). A model is constructed, in which two MAPK cascades depend on a single MAP-kinase kinase that is not sequestered in two subpopulations. We show that in this model selective signal transduction is possible provided that there is an additional interaction at the MAP-kinase level, there is no simultaneous activation of more than one response by either signal. We discuss a number of additional interactions that can generate the selectivity, as well as some kinetic features by which this mechanism may be recognized experimentally.

Adaptation, Physiological↗

PubMed2002

Untangling the wires: a strategy to trace functional interactions in signaling and gene networks.

Emerging technologies have enabled the acquisition of large genomics and proteomics data sets. However, current methodologies for analysis do not permit interpretation of the data in ways that unravel cellular networking. We propose a quantitative method for determining functional interactions in cellular signaling and gene networks. It can be used to explore cell systems at a mechanistic level or applied within a "modular" framework, which dramatically decreases the number of variables to be assayed. This method is based on a mathematical derivation that demonstrates how the topology and strength of network connections can be retrieved from experimentally measured network responses to successive perturbations of all modules. Importantly, our analysis can reveal functional interactions even when the components of the system are not all known. Under these circumstances, some connections retrieved by the analysis will not be direct but correspond to the interaction routes through unidentified elements. The method is tested and illustrated by using computer-generated responses of a modeled mitogen-activated protein kinase cascade and gene network.

Animals↗

PubMed2002

Product dependence and bifunctionality compromise the ultrasensitivity of signal transduction cascades.

Covalent modification cycles are ubiquitous. Theoretical studies have suggested that they serve to increase sensitivity. However, this suggestion has not been corroborated experimentally in vivo. Here, we demonstrate that the assumptions of the theoretical studies, i.e., irreversibility and absence of product inhibition, were not trivial: when the conversion reactions are close to equilibrium or saturated by their product, "zero-order" ultrasensitivity disappears. For high sensitivities to arise, not only substrate saturation (zero-order) but also high equilibrium constants and low product saturation are required. Many covalent modification cycles are catalyzed by one bifunctional 'ambiguous' enzyme rather than by two independent proteins. This makes high substrate concentration and low product concentration for both reactions of the cycle inconsistent; such modification cycles cannot have high responses. Defining signal strength as ratios of modified (e.g., phosphorylated) over unmodified protein, signal-to-signal response sensitivity equals 1: signal strength should remain constant along a cascade of ambiguous modification cycles. We also show that the total concentration of a signalling effector protein cannot affect the signal emanating from a modification cycle catalyzed by an ambiguous enzyme if the ratio of the two forms of the effector protein is not altered. This finding may explain the experimental result that the pivotal signal transduction protein PII plus its paralogue GlnK do not control steady-state N-signal transduction in Escherichia coli. It also rationalizes the absence of strong phenotypes for many signal-transduction proteins. Emphasis on extent of modification of these proteins is perhaps more urgent than transcriptome analysis.

Enzymes↗

PubMed2002

Putting intentions into cell biochemistry: an artificial intelligence perspective.

The living cell exists by virtue of thousands of nonlinearly interacting processes. This complexity greatly impedes its understanding. The standard approach to the calculation of the behaviour of the living cell, or part thereof, integrates all the rate equations of the individual processes. If successful extremely intensive calculations often lead the calculation of coherent, apparently simple, cellular "decisions" taken in response to a signal: the complexity of the behavior of the cell is often smaller than it might have been. The "decisions" correspond to the activation of entire functional units of molecular processes, rather than individual ones. The limited complexity of signal and response suggests that there might be a simpler way to model at least some important aspects of cell function. In the field of Artificial Intelligence, such simpler modelling methods for complex systems have been developed. In this paper, it is shown how the Artificial Intelligence description method for deliberative agents functioning on the basis of beliefs, desires and intentions as known in Artificial Intelligence, can be used successfully to describe essential aspects of cellular regulation. This is demonstrated for catabolite repression and substrate induction phenomena in the bacterium Escherichia coli. The method becomes highly efficient when the computation is automated in a Prolog implementation. By defining in a qualitative way the food supply of the bacterium, the make-up of its catabolic pathways is readily calculated for cases that are sufficiently complex to make the traditional human reasoning tedious and error prone.

Animals↗

PubMed2002

Simplicity in complexity: the photosynthetic reaction center performs as a simple 0.2 V battery.

The photosynthetic reaction center is one of the most complicated molecular complexes. Transducing photon energy to a transmembrane electrochemical potential difference for protons, it is the direct or indirect energy source for virtually all life. We show here that it operates in a simple, battery-like manner, with a maximum potential of 0.20 V. Intriguingly this is only one fifth of the energy of the absorbed photon.

Electric Stimulation↗

PubMed2002

Control analysis for autonomously oscillating biochemical networks.

It has hitherto not been possible to analyze the control of oscillatory dynamic cellular processes in other than qualitative ways. The control coefficients, used in metabolic control analyses of steady states, cannot be applied directly to dynamic systems. We here illustrate a way out of this limitation that uses Fourier transforms to convert the time domain into the stationary frequency domain, and then analyses the control of limit cycle oscillations. In addition to the already known summation theorems for frequency and amplitude, we reveal summation theorems that apply to the control of average value, waveform, and phase differences of the oscillations. The approach is made fully operational in an analysis of yeast glycolytic oscillations. It follows an experimental approach, sampling from the model output and using discrete Fourier transforms of this data set. It quantifies the control of various aspects of the oscillations by the external glucose concentration and by various internal molecular processes. We show that the control of various oscillatory properties is distributed over the system enzymes in ways that differ among those properties. The models that are described in this paper can be accessed on http://jjj.biochem.sun.ac.za.

Biochemistry↗

PubMed2002

DNA supercoiling by gyrase is linked to nucleoid compaction.

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 microm cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.

DNA Gyrase↗

PubMed2002

Sensitivity analysis of metabolic cascades catalyzed by bifunctional enzymes.

Covalent modification/demodification cycles are common in metabolism. When the modification and demodification steps are carried out by two independent enzymes, the degree of modification can be ultrasensitive to the total concentration of either catalyst. We recently showed that the degree of modification of a target molecule cannot exhibit ultrasensitivity to the free concentrations of effectors that decide whether a bifunctional enzyme acts as modifier or demodifier. However, here we can now demonstrate that the degree of modification of a target molecule can display ultrasensitivity to the total, rather than free, concentrations of such effectors. Our results clarify some general aspects of ultrasensitive responses to effectors, including competitive inhibitors, in mono-cyclic cascades.

Catalysis↗

PubMed2002

Flux control of the bacterial phosphoenolpyruvate:glucose phosphotransferase system and the effect of diffusion.

We analyzed the role of diffusion and cell size on the flux control properties of the glucose-PTS of Escherichia coli, in silicon cells under various metabolic conditions. To our surprise, the influence of the concentration of phosphoryl-donor PEP on the distribution of control was small. We found for cells of bacterial size that PTS-flux control was mainly located in processes taking place in the membrane and that diffusion hardly controlled the flux (< 2.8 %). Enlargement of the cells shifted the control from membrane to cytoplasm and from process rates to diffusion rates, the latter now having a total control of about 38 %. In the presence of glucose, nearly all diffusion flux control resided in the component that links the cytoplasmic processes to those in the membrane.

Diffusion↗

PubMed2002

ECA: control in ecosystems.

Although metabolic control analysis (MCA) cannot be applied directly to microbial ecological systems because of mass conservation and stoichiometric constraints, we demonstrate here that Hierarchical Control Analysis (HCA) can be applied to such systems. We illustrate the approach for a particular ecosystem example of the biological synthesis of acetic acid from glucose, and uncover some surprising aspects to the control of this miniature ecosystem.

Acetic Acid↗

PubMed2002

The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth.

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.

Adenosine Diphosphate↗

PubMed2002

Loss of fermentative capacity in baker's yeast can partly be explained by reduced glucose uptake capacity.

Initial attempts to increase fermentative capacity of baker's yeast focussed on the overproduction of single enzymes, which proved to be insufficient. Nowadays many components of the system are monitored simultaneously in a search for a correlation with fermentative capacity. However, this strategy has not yet proven fruitful either. Here we investigate an element previously neglected, the glucose transporter, and find that a loss of glucose transport capacity correlates with a decrease of fermentative capacity during nutrient starvation. However the correlation is not unique, suggesting that the loss of fermentative capacity cannot be attributed to an inactivation of glucose transport alone. Our results suggest the necessity to use a detailed kinetic model as an underlying working hypothesis and to use Metabolic Control Analysis to examine the pathway's control properties.

Biological Transport↗

PubMed2002

Metabolic control in integrated biochemical systems.

Traditional analyses of the control and regulation of steady-state concentrations and fluxes assume the activities of the enzymes to be constant. In living cells, a hierarchical control structure connects metabolic pathways to signal-transduction and gene-expression. Consequently, enzyme activities are not generally constant. This would seem to compromise analyses of control and regulation at the metabolic level. Here, we investigate the concept of metabolic quasi-steady state kinetics as a means of applying metabolic control analysis to hierarchical biochemical systems. We discuss four methods that enable the experimental determination of metabolic control coefficients, and demonstrate these by computer simulations. The best method requires extra measurement of enzyme activities, two others are simpler but are less accurate and one method is bound only to work under special conditions. Our results may assist in evaluating the relative importance of transcriptomics and metabolomics for functional genomics.

Enzymes↗

PubMed2002

DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase.

DNA of prokaryotes is in a nonequilibrium structural state, characterized as 'active' DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell 34, 105-113]. We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem. 266, 865-877]. Of the two regulatory mechanisms via which homeostasis is conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).

Base Sequence↗

PubMed2002

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis.

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.

Base Sequence↗

PubMed2002

The glycolytic flux in Escherichia coli is controlled by the demand for ATP.

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F(1) part of the membrane-bound (F(1)F(0)) H(+)-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.

Adenosine Triphosphate↗