Coevolved mutations reveal distinct architectures for two core proteins in the bacterial flagellar motor

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.JK was supported by Medical Research Council grant U117581331. SK was supported by seed funds from Lahore University of Managment Sciences (LUMS) and the Molecular Biology Consortium

Imbalance of neurotrophin receptor isoforms TrkB-FL/TrkB-T1 induces neuronal death in excitotoxicity

A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies

Lunar Phase-Dependent Expression of Cryptochrome and a Photoperiodic Mechanism for Lunar Phase-Recognition in a Reef Fish, Goldlined Spinefoot

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlight's pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response

Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

International audienceBACKGROUND: Neurotrophin receptors were initially identified in neural cells. They were recently detected in some cancers in association with invasiveness, but the function of these tyrosine kinase receptors was not previously investigated in colorectal cancer (CRC) cells. METHODS AND FINDINGS: We report herein that human CRC cell lines synthesize the neural growth factor Brain-derived neurotrophic factor (BDNF) under stress conditions (serum starvation). In parallel, CRC cells expressed high- (TrkB) and low-affinity (p75(NTR)) receptors at the plasma membrane, whereas TrkA and TrkC, two other high affinity receptors for NGF and NT-3, respectively, were undetectable. We demonstrate that BDNF induced cell proliferation and had an anti-apoptotic effect mediated through TrkB, as assessed by K252a, a Trk pharmacologic inhibitor. It suppressed both cell proliferation and survival of CRC cells that do not express TrkA nor TrkC. In parallel to the increase of BDNF secretion, sortilin, a protein acting as a neurotrophin transporter as well as a co-receptor for p75(NTR), was increased in the cytoplasm of primary and metastatic CRC cells, which suggests that sortilin could regulate neurotrophin transport in these cells. However, pro-BDNF, also detected in CRC cells, was co-expressed with p75(NTR) at the cell membrane and co-localized with sortilin. In contrast to BDNF, exogenous pro-BDNF induced CRC apoptosis, which suggests that a counterbalance mechanism is involved in the control of CRC cell survival, through sortilin as the co-receptor for p75(NTR), the high affinity receptor for pro-neurotrophins. Likewise, we show that BDNF and TrkB transcripts (and not p75(NTR)) are overexpressed in the patients' tumors by comparison with their adjacent normal tissues, notably in advanced stages of CRC. CONCLUSION: Taken together, these results highlight that BDNF and TrkB are essential for CRC cell growth and survival in vitro and in tumors. This autocrine loop could be of major importance to define new targeted therapies

Predicting Cellular Growth from Gene Expression Signatures

Maintaining balanced growth in a changing environment is a fundamental systems-level challenge for cellular physiology, particularly in microorganisms. While the complete set of regulatory and functional pathways supporting growth and cellular proliferation are not yet known, portions of them are well understood. In particular, cellular proliferation is governed by mechanisms that are highly conserved from unicellular to multicellular organisms, and the disruption of these processes in metazoans is a major factor in the development of cancer. In this paper, we develop statistical methodology to identify quantitative aspects of the regulatory mechanisms underlying cellular proliferation in Saccharomyces cerevisiae. We find that the expression levels of a small set of genes can be exploited to predict the instantaneous growth rate of any cellular culture with high accuracy. The predictions obtained in this fashion are robust to changing biological conditions, experimental methods, and technological platforms. The proposed model is also effective in predicting growth rates for the related yeast Saccharomyces bayanus and the highly diverged yeast Schizosaccharomyces pombe, suggesting that the underlying regulatory signature is conserved across a wide range of unicellular evolution. We investigate the biological significance of the gene expression signature that the predictions are based upon from multiple perspectives: by perturbing the regulatory network through the Ras/PKA pathway, observing strong upregulation of growth rate even in the absence of appropriate nutrients, and discovering putative transcription factor binding sites, observing enrichment in growth-correlated genes. More broadly, the proposed methodology enables biological insights about growth at an instantaneous time scale, inaccessible by direct experimental methods. Data and tools enabling others to apply our methods are available at http://function.princeton.edu/growthrate

Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Flexible mapping of homology onto structure with Homolmapper

<p>Abstract</p> <p>Background</p> <p>Over the past decade, a number of tools have emerged for the examination of homology relationships among protein sequences in a structural context. Most recent software implementations for such analysis are tied to specific molecular viewing programs, which can be problematic for collaborations involving multiple viewing environments. Incorporation into larger packages also adds complications for users interested in adding their own scoring schemes or in analyzing proteins incorporating unusual amino acid residues such as selenocysteine.</p> <p>Results</p> <p>We describe homolmapper, a command-line application for mapping information from a multiple protein sequence alignment onto a protein structure for analysis in the viewing software of the user's choice. Homolmapper is small (under 250 K for the application itself) and is written in Python to ensure portability. It is released for non-commercial use under a modified University of California BSD license. Homolmapper permits facile import of additional scoring schemes and can incorporate arbitrary additional amino acids to allow handling of residues such as selenocysteine or pyrrolysine. Homolmapper also provides tools for defining and analyzing subfamilies relative to a larger alignment, for mutual information analysis, and for rapidly visualizing the locations of mutations and multi-residue motifs.</p> <p>Conclusion</p> <p>Homolmapper is a useful tool for analysis of homology relationships among proteins in a structural context. There is also extensive, example-driven documentation available. More information about homolmapper is available at <url>http://www.mcb.ucdavis.edu/faculty-labs/lagarias/homolmapper_home/homolmapper%20web%20page.htm</url>.</p