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Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.
Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.
Copyright © 2016 by the Genetics Society of America.
The non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway plays a crucial role in embryonic development. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed Wnt/PCP signalling as a therapeutic target. Here we show that the archetypal Wnt/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumour growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2-p62/SQSTM1-JNK pathway. This proliferative signalling cascade is upregulated in breast cancer patients with shorter survival and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2-p62/SQSTM1 interaction. VANGL2-JNK signalling is thus a potential target for breast cancer therapy.
Batrachochytrium dendrobatidis is a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, but B. dendrobatidis releases factors that inhibit in vitro and in vivo lymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Wounded cells such as Xenopus oocytes respond to damage by assembly and closure of an array of actin filaments and myosin-2 controlled by Rho GTPases, including Rho and Cdc42. Rho and Cdc42 are patterned around wounds in a characteristic manner, with active Rho concentrating in a ring-like zone inside a larger, ring-like zone of active Cdc42. How this patterning is achieved is unknown, but Rho and Cdc42 at wounds are subject to regulation by other proteins, including the protein kinases C. Specifically, Cdc42 and Rho activity are enhanced by PKCβ and inhibited by PKCη. We adapt a mathematical model of Simon and coworkers to probe the possible roles of these kinases. We show that PKCβ likely affects the magnitude of positive Rho-Abr feedback, whereas PKCη acts on Cdc42 inactivation. The model explains both qualitative and some overall quantitative features of PKC-Rho GTPase regulation. It also accounts for the previous, peculiar observation that ∼ 20% of cells overexpressing PKCη display zone inversions--that is, displacement of active Rho to the outside of the active Cdc42.
© 2015 Holmes, Liao, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Billions of base pairs of DNA must be replicated trillions of times in a human lifetime. Complete and accurate replication once and only once per cell division cycle is essential to maintain genome integrity and prevent disease. Impediments to replication fork progression including difficult to replicate DNA sequences, conflicts with transcription, and DNA damage further add to the genome maintenance challenge. These obstacles frequently cause fork stalling, but only rarely cause a failure to complete replication. Robust mechanisms ensure that stalled forks remain stable and capable of either resuming DNA synthesis or being rescued by converging forks. However, when failures do happen the fork collapses leading to genome rearrangements, cell death and disease. Despite intense interest, the mechanisms to repair damaged replication forks, stabilize them, and ensure successful replication remain only partly understood. Different models of fork collapse have been proposed with varying descriptions of what happens to the DNA and replisome. Here, I will define fork collapse and describe what is known about how the replication checkpoint prevents it to maintain genome stability.
Copyright © 2015 Elsevier B.V. All rights reserved.
Screens for small-molecule modulators of biological pathways typically utilize cultured cell lines, purified proteins, or, recently, model organisms (e.g., zebrafish, Drosophila, C. elegans). Herein, we describe a method for using Xenopus laevis egg extract, a biologically active and highly tractable cell-free system that recapitulates a legion of complex chemical reactions found in intact cells. Specifically, we focus on the use of a luciferase-based fusion system to identify small-molecule modulators that affect protein turnover.
The principal Afrotropical malaria vector mosquito, Anopheles gambiae remains a significant threat to human health. In this anthropophagic species, females detect and respond to a range of human-derived volatile kairomones such as ammonia, lactic acid, and other carboxylic acids in their quest for blood meals. While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered. In this study, we have identified and characterized two candidate ammonium transporter genes, AgAmt and AgRh50 that are expressed in the mosquito antenna and may contribute to physiological and behavioral responses to ammonia, which is an important host kairomone for vector mosquitoes. AgAmt transcripts are highly enhanced in female antennae while a splice variant of AgRh50 appears to be antennal-specific. Functional expression of AgAmt in Xenopus laevis oocytes facilitates inward currents in response to both ammonium and methylammonium, while AgRh50 is able to partially complement a yeast ammonium transporter mutant strain, validating their conserved roles as ammonium transporters. We present evidence to suggest that both AgAmt and AgRh50 are in vivo ammonium transporters that are important for ammonia sensitivity in An. gambiae antennae, either by clearing ammonia from the sensillar lymph or by facilitating sensory neuron responses to environmental exposure. Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies.
To explore the structural basis for odorant specificity in odorant receptors of the human malaria vector mosquito, Anopheles gambiae, odorant-binding subunits (Agam\Ors) expressed in Xenopus oocytes in combination with Agam\Orco (coreceptor subunit) were assayed by 2-electrode voltage clamp against 25 structurally related odorants. Agam\Or13 and Agam\Or15 display 82% amino acid identity and had similar, but somewhat distinct odorant response profiles. The ratio of acetophenone to 4-methylphenol responses was used in a mutation-based analysis of Agam\Or15, interchanging 37 disparate residues between Agam\Or15 and Agam\Or13. Eleven mutations caused significant changes in odorant responsiveness. Mutation of alanine 195 resulted in the largest shift in response ratio from Agam\Or15 toward Agam\Or13. Concentration-response analysis for a series of mutations of residue 195 revealed a large effect on acetophenone sensitivity, with EC50 values varying by >1800-fold and correlating with residue side chain length. Similar results were obtained for propiophenone and benzaldehyde. But, for other odorants, such as 4-methylphenol, 4-methylbenzaldehyde, and 4-methylpropiophenone, the effect of mutation was much smaller (EC50 values varied by ≤16-fold). These results show that alanine 195, putatively located at the second extracellular loop/fourth transmembrane domain interface, plays a critical role in determining the odorant response specificity of Agam\Or15.
© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: email@example.com.
NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 μM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 μM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC50 value for PYD of 13 μM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC50. PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor.
Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.