A logical model provides insights into T cell receptor signaling

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.     

Benno Parthier (1932–2019)

Plant Molecular Biology -     

Hidden-Markov methods for the analysis of single-molecule actomyosin displacement data: the variance-Hidden-Markov method

In single-molecule experiments on the interaction between myosin and actin, mechanical events are embedded in Brownian noise. Methods of detecting events have progressed from simple manual detection of shifts in the position record to threshold-based selection of intermittent periods of reduction in noise. However, none of these methods provides a "best fit" to the data. We have developed a Hidden-Markov algorithm that assumes a simple kinetic model for the actin-myosin interaction and provides automatic, threshold-free, maximum-likelihood detection of events. The method is developed for the case of a weakly trapped actin-bead dumbbell interacting with a stationary myosin molecule (Finer, J. T., R. M. Simmons, and J. A. Spudich. 1994. Nature. 368:113-119). The algorithm operates on the variance of bead position signals in a running window, and is tested using Monte Carlo simulations to formulate ways of determining the optimum window width. The working stroke is derived and corrected for actin-bead link compliance. With experimental data, we find that modulation of myosin binding by the helical structure of the actin filament complicates the determination of the working stroke; however, under conditions that produce a Gaussian distribution of bound levels (cf. Molloy, J. E., J. E. Burns, J. Kendrick-Jones, R. T. Tregear, and D. C. S. White. 1995. Nature. 378:209-212), four experiments gave working strokes in the range 5.4-6.3 nm for rabbit skeletal muscle myosin S1.     

The Cenomanian Gastropoda of the Kassenberg quarry in Mülheim (Germany,Late Cretaceous)

Forty-six gastropod species are described from the Cenomanian of the Kassenberg quarry in Mülheim a. d. Ruhr, Germany, twelve of them are new. They are assigned to 33 known genera and two new genera.Deikella n. gen. is introduced for threeTurbo-like species which have strong axial ribs in contrast to the smooth RecentTurbo. Frydatinus n. gen. is introduced for a loosely coiled archaeogastropod with a round aperture and spine-like extensions on its outer side. A new species ofDelpeya is introduced, and it is suggested that this genus derived from the JurassicNummocalcar. The high number of pleurotomariids indicates that these gastropods still lived on rocky shores during the Cenomanian, in contrast to modern times, where they are restricted to hardgrounds in deep-water. The new species are:Bathrotomaria harasewychi, Deikella spinicostata, Deikella ruhrensis, Deikella? muelheimensis, Trochus rauenorum, Margarella (Promargarita) spiraloides, Margarella (Promargarita) trochoides, Semisolarium boehmi, Onkospira perrilliatae, Delpeya hilperti, Neritopsis kasei, andOtostoma kassenbergensis.     

AraPerox. A database of putative Arabidopsis proteins from plant peroxisomes

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.     

Are NADP-dependent isocitrate dehydrogenases and ferredoxin-dependent glutamate synthase co-regulated by the same photoreceptors?

Activities of NADP-dependent isocitrate dehydrogenases (cytosolic and plastidic isoforms, ICDH1 and ICDH2; EC 1.1.1.42) and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in turions of Spirodela polyrhiza were all stimulated by light. Single or repeated red light (R) pulses induced the activity of the enzymes and this effect was reverted by subsequent far-red light (FR) pulses. The enzymes are, therefore, co-regulated by the low-fluence response of phytochrome. For ICDH, this is reported here for the first time. Neither an effect of the very low-fluence response nor of the FR-mediated high-irradiance response was detectable. Irradiance with continuous R resulted in enhanced enzyme activities and protein levels (Western analysis using polyclonal antibodies against ICDH1 and Fd-GOGAT). These additional effects of continuous R (called a non-induction effect) could be inhibited for ICDH1 and ICDH2 by the inhibitor of photosynthetic electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and are therefore related to the effect of photosynthesis. In contrast, the non-induction effect of Fd-GOGAT was resistant against this inhibitor. Moreover, hourly R pulses did not replace the effect of continuous R. The non-induction effect of light on the activity and protein level of Fd-GOGAT was therefore tentatively classified as an R-mediated high-irradiance response. The activity of Fd-GOGAT but not that of ICDHs was additionally regulated by a specific blue-light receptor. It can be concluded that the levels of ICDHs and Fd-GOGAT were coordinated by light but were not co-regulated by the same photoreceptors. Nitrate is necessary for the light regulation of both enzymes, contributing to the coordinated expression of the relevant genes.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - Fd-GOGAT Ferredoxin-dependent glutamate synthase - FR Far-red light - HIR High-irradiance response - ICDH NADP-dependent isocitrate dehydrogenase - ICDH1 Cytosolic ICDH - ICDH2 Chloroplastic ICDH - LFR Low-fluence response - R Red light - SDS–PAGE Denaturing polyacrylamide gel electrophoresis - VLFR Very low-fluence response