JNK mediates differentiation, cell polarity and apoptosis during amphioxus development by regulating actin cytoskeleton dynamics and ERK signalling

c-Jun terminal kinase (JNK) is a multi-functional protein involved in a diverse array of context-dependent processes, including apoptosis, cell cycle regulation, adhesion and differentiation. It is integral to a number of signalling cascades, notably downstream of non-canonical Wnt and MAPK signalling pathways. As such, it is a key regulator of cellular behaviour and patterning during embryonic development across the animal kingdom. The cephalochordate amphioxus is an invertebrate chordate model system straddling the invertebrate to vertebrate transition and is thus ideally suited for comparative studies of morphogenesis. However, next to nothing is known about JNK signalling or cellular processes in this lineage. Pharmacological inhibition of JNK signalling using SP600125 during embryonic development arrests gastrula invagination and causes convergence extension-like defects in axial elongation, particularly of the notochord. Pharynx formation and anterior oral mesoderm derivatives like the preoral pit are also affected. This is accompanied by tissue-specific transcriptional changes, including reduced expression of six3/6 and wnt2 in the notochord, and ectopic wnt11 in neurulating embryos treated at late gastrula stages. Cellular delamination results in accumulation of cells in the gut cavity and a dorsal fin-like protrusion, followed by secondary Caspase3-mediated apoptosis of polarity-deficient cells, a phenotype only partly rescued by co-culture with the pan-caspase inhibitor Z-VAD-fmk. Ectopic activation of ERK signalling in the neighbours of extruded notochord and neural cells, possibly due to altered adhesive and tensile properties

The genotype-phenotype relationship in multicellular pattern-generating models - the neglected role of pattern descriptors

Background: A deep understanding of what causes the phenotypic variation arising from biological patterning processes, cannot be claimed before we are able to recreate this variation by mathematical models capable of generating genotype-phenotype maps in a causally cohesive way. However, the concept of pattern in a multicellular context implies that what matters is not the state of every single cell, but certain emergent qualities of the total cell aggregate. Thus, in order to set up a genotype-phenotype map in such a spatiotemporal pattern setting one is actually forced to establish new pattern descriptors and derive their relations to parameters of the original model. A pattern descriptor is a variable that describes and quantifies a certain qualitative feature of the pattern, for example the degree to which certain macroscopic structures are present. There is today no general procedure for how to relate a set of patterns and their characteristic features to the functional relationships, parameter values and initial values of an original pattern-generating model. Here we present a new, generic approach for explorative analysis of complex patterning models which focuses on the essential pattern features and their relations to the model parameters. The approach is illustrated on an existing model for Delta-Notch lateral inhibition over a two-dimensional lattice. Results: By combining computer simulations according to a succession of statistical experimental designs, computer graphics, automatic image analysis, human sensory descriptive analysis and multivariate data modelling, we derive a pattern descriptor model of those macroscopic, emergent aspects of the patterns that we consider of interest. The pattern descriptor model relates the values of the new, dedicated pattern descriptors to the parameter values of the original model, for example by predicting the parameter values leading to particular patterns, and provides insights that would have been hard to obtain by traditional methods. Conclusion: The results suggest that our approach may qualify as a general procedure for how to discover and relate relevant features and characteristics of emergent patterns to the functional relationships, parameter values and initial values of an underlying pattern-generating mathematical model

Understanding signaling cascades in melanoma

Understanding regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now appreciated that melanoma is the result of complex changes in multiple signaling pathways that affect growth control, metabolism, motility and the ability to escape cell death programs. Here we review the major signaling pathways currently known to be deregulated in melanoma with an implication to its development and progression. Among these pathways are Ras, B-Raf, MEK, PTEN, phosphatidylinositol-3 kinase (PI3Ks) and Akt which are constitutively activated in a significant number of melanoma tumors, in most cases due to genomic change. Other pathways discussed in this review include the [Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-beta pathways which are also activated in melanoma, although the underlying mechanism is not yet clear. As a paradigm for remodeled signaling pathways, melanoma also offers a unique opportunity for targeted drug development.Fil: Lopez Bergami, Pablo Roberto. Sanford-burnham Medical Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Fitchmann, B. Sanford-burnham Medical Research Institute; Estados UnidosFil: Ronai, Ze´ev. Sanford-burnham Medical Research Institute; Estados Unido

Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain

<p>Abstract</p> <p>Background</p> <p>The non-pathogenic bacterium <it>Mycobacterium smegmatis </it>is widely used as a near-native expression host for the purification of <it>Mycobacterium tuberculosis </it>proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1.</p> <p>Results</p> <p>In order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1ΔC. GroEL1ΔC, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1ΔC is no longer present in protein samples purified from the <it>groEL1ΔC </it>expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain.</p> <p>Conclusions</p> <p>This novel <it>Mycobacterium smegmatis </it>expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification.</p

Notch Signaling Regulates Bile Duct Morphogenesis in Mice

BACKGROUND: Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene. CONCLUSIONS/SIGNIFICANCE: Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification

Evasion of anti-growth signaling: a key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds

The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally-occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally-occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting

Modelling the Spatio-Temporal Cell Dynamics Reveals Novel Insights on Cell Differentiation and Proliferation in the Small Intestinal Crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4–5 days with 10–12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

Supplemental information: Document S1. Figures S1–S5, Tables S1–S3, and Methods S1 available at: https://www.cell.com/cms/10.1016/j.chembiol.2021.02.006/attachment/3e31e265-54fc-4337-ac40-861ecb85706e/mmc1.pdf (1.44 MB)Copyright © 2021 The Authors. BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.Wellcome Trust Translational Award ( 080083/Z/06/Z ); Seeding Drug Discovery Award ( 091058/Z/09/Z ); Medical Research Council (MRC) Program grants MC_UU_12022/1 and MC_UU_12022/8; Astex Pharmaceuticals

GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18–24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing

Notch Ankyrin Repeat Domain Variation Influences Leukemogenesis and Myc Transactivation

, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival., a direct Notch target that has an important role in Notch-associated T-ALL.We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis