Nils Blüthgen

Effects of sequestration on signal transduction cascades

The building blocks of most signal transduction pathways are pairs of enzymes, such as kinases and phosphatases, that control the activity of protein targets by covalent modification. It has previously been shown [Goldbeter A & Koshland DE (1981) Proc Natl Acad Sci USA78, 6840–6844] that these systems can be highly sensitive to changes in stimuli if their catalysing enzymes are saturated with their target protein substrates. This mechanism, termed zero‐order ultrasensitivity, may set thresholds that filter out subthreshold stimuli. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations. Under these conditions a large fraction of the target protein may be sequestrated by the enzymes. This causes a reduction in ultrasensitivity so that the proposed mechanism is unlikely to account for ultrasensitivity under the conditions present in most in vivo signalling cascades. Furthermore, we show that sequestration changes the dynamics of a covalent modification cycle and may account for signal termination and a sign‐sensitive delay. Finally, we analyse the effect of sequestration on the dynamics of a complex signal transduction cascade: the mitogen‐activated protein kinase (MAPK) cascade with negative feedback. We show that sequestration limits ultrasensitivity in this cascade and may thereby abolish the potential for oscillations induced by negative feedback.

Efficient translation initiation dictates codon usage at gene start.

The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5–10 codons of protein‐coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.

RORγt+ Innate Lymphoid Cells Acquire a Proinflammatory Program upon Engagement of the Activating Receptor NKp44

RORγt⁺ innate lymphoid cells (ILCs) are crucial players of innate immune responses and represent a major source of interleukin-22 (IL-22), which has an important role in mucosal homeostasis. The signals required by RORγt⁺ ILCs to express IL-22 and other cytokines have been elucidated only partially. Here we showed that RORγt⁺ ILCs can directly sense the environment by the engagement of the activating receptor NKp44. NKp44 triggering in RORγt⁺ ILCs selectively activated a coordinated proinflammatory program, including tumor necrosis factor (TNF), whereas cytokine stimulation preferentially induced IL-22 expression. However, combined engagement of NKp44 and cytokine receptors resulted in a strong synergistic effect. These data support the concept that NKp44⁺ RORγt⁺ ILCs can be activated without cytokines and are able to switch between IL-22 or TNF production, depending on the triggering stimulus.

How robust are switches in intracellular signaling cascades

Since all-or-none decisions of the cell are controlled by extracellular signals, cells have biochemical switches within their intracellular signaling networks. Central elements of these switches are multisite phosphorylation, enzymic saturation, and amplification by cascades. Moreover, positive feedback can contribute to switch-like behavior termed also ultrasensitivity. Here we analyse the robustness of these mechanisms exemplified by models of the three-molecule MAPK-cascade and the single-molecule Goldbeter-Koshland switch. We show that the ultrasensitivity in the MAPK-cascades is more robust against changes of the kinetic parameters than the Goldbeter-Koshland switch. If multiple parameters are changed randomly, the effects of parameter changes can compensate each other in the cascade leading to a remarkable robustness of the switch-like behavior. The different degrees of robustness can be traced back to the different mechanisms of generating ultrasensitivity. While in the Goldbeter-Koshland switch the saturation of the enzymes are crucial, in the MAPK-cascade the adjustment of working ranges determines the ultrasensitivity. Our results indicate that amplification of ultrasensitivity in cascades and multisite phosphorylation might be a design principle to achieve robust switches.

Inferring combinatorial regulation of transcription in silico

In this paper, we propose a functional view on the in silico prediction of transcriptional regulation. We present a method to predict biological functions regulated by a combinatorial interaction of transcription factors. Using a rigorous statistic, this approach intersects the presence of transcription factor binding sites in gene upstream sequences with Gene Ontology terms associated with these genes. We demonstrate that for the well-studied set of skeletal muscle-related transcription factors Myf-2, Mef and TEF, the correct functions are predicted. Furthermore, starting from the well-characterized promoter of a gene expressed upon lipopolysaccharide stimulation, we predict functional targets of this stimulus. These results are in excellent agreement with microarray data.

Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway dependent gene signatures in colorectal cancer cells.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

How scaffolds shape MAPK signaling: what we know and opportunities for systems approaches

Scaffolding proteins add a new layer of complexity to the dynamics of cell signaling. Above their basic function to bring several components of a signaling pathway together, recent experimental research has found that scaffolds influence signaling in a much more complex way: scaffolds can exert some catalytic function, influence signaling by allosteric mechanisms, are feedback-regulated, localize signaling activity to distinct regions of the cell or increase pathway fidelity. Here we review experimental and theoretical approaches that address the function of two MAPK scaffolds, Ste5, a scaffold of the yeast mating pathway and KSR1/2, a scaffold of the classical mammalian MAPK signaling pathway. For the yeast scaffold Ste5, detailed mechanistic models have been valuable for the understanding of its function. For scaffolds in mammalian signaling, however, models have been rather generic and sketchy. For example, these models predicted narrow optimal scaffold concentrations, but when revisiting these models by assuming typical concentrations, rather a range of scaffold levels optimally supports signaling. Thus, more realistic models are needed to understand the role of scaffolds in mammalian signal transduction, which opens a big opportunity for systems biology.

A systems biological approach suggests that transcriptional feedback regulation by dual‐specificity phosphatase 6 shapes extracellular signal‐related kinase activity in RAS‐transformed fibroblasts

Mitogen‐activated protein kinase (MAPK) signaling determines crucial cell fate decisions in most cell types, and mediates cellular transformation in many types of cancer. The activity of MAPK is controlled by reversible phosphorylation, and the quantitative characteristics of MAPK activation determine the cellular response. Many systems biological studies have analyzed the activation kinetics and the dose–response behavior of the MAPK signaling pathway. Here we investigate how the pathway activity is controlled by transcriptional feedback loops. Initially, we predict that MAPK signaling regulates phosphatases, by integrating promoter sequence data and ontology‐based classification of gene function. From this, we deduce that MAPK signaling might be controlled by transcriptional negative feedback regulation via dual‐specificity phosphatases (DUSPs), and implement a mathematical model to further test this hypothesis. Using time‐resolved measurements of pathway activity and gene expression, we employ a model selection approach, and select DUSP6 as a highly likely candidate for shaping the activity of the MAPK pathway during cellular transformation caused by oncogenic RAS. Two predictions from the model were confirmed: first, feedback regulation requires that DUSP6 mRNA and protein are unstable; and second, the activation kinetics of MAPK are ultrasensitive. Taken together, an integrated systems biological approach reveals that transcriptional negative feedback controls the kinetics and the extent of MAPK activation under both physiological and pathological conditions.

Ant nests in tank bromeliads - an example of non-specific interaction

Summary: Four species of epiphytic tank bromeliads on an island in the Orinoco river in Venezuelan Amazonia were inhabited by 13 ant species from four subfamilies. None of these ant species are known as specialised plant-ants. A Monte Carlo randomisation test showed that ants were randomly distributed among host plants: (1) there was no association between particular ant species and bromeliad species, and (2) there was no vertical stratification of the ant community between bromeliads sampled on the ground and at two height classes in trees. This contrasts with the few published data on the distribution of ants on terrestrial myrmecophytes and epiphytes, respectively, to which we applied the same analytical method.

Quantitative analysis of ultrasensitive responses.

Ultrasensitive responses are common in cellular information transfer because they allow cells to decode extracellular stimuli in an all‐or‐none manner. Biochemical responses are usually analyzed by fitting the Hill equation, and the estimated Hill coefficient is taken as a measure of sensitivity. However, this approach is not appropriate if the response under consideration significantly deviates from the best‐fit Hill equation. In addition, Hill coefficients greater than unity do not necessarily imply ultrasensitive behaviour if basal activation is significant. In order to circumvent these problems we propose a general method for the quantitative analysis of sensitivity, the relative amplification plot, which is based on the response coefficient defined in metabolic control analysis. To quantify sensitivity globally (i.e. over the whole stimulus range) we introduce the integral‐based relative amplification coefficient. Our relative amplification approach can easily be extended to monotonically decreasing, bell‐shaped or nonsaturated responses.