The structural and functional determinants of the Axin and Dishevelled DIX domains.

BACKGROUND: The dishevelled and axin genes encode multi-domain proteins that play key roles in WNT signalling. Dishevelled prevents beta-catenin degradation by interfering with the interaction of beta-catenin with the degradation-mediating Axin-APC-GSK3beta complex. This interference leads to an accumulation of cytoplasmic beta-catenin, which enters the nucleus and interacts with transcription factors that induce expression of Wnt-target genes. Axin, as a component of the degradation-mediating complex, is a potent negative regulator of Wnt signalling, whereas Dishevelled is a potent activator. Both Dishevelled and Axin possess a DIX (Dishevelled/Axin) domain, which mediates protein-protein interactions, specifically homodimerization. RESULTS: An evolutionary trace analysis of DIX domains identified conserved residues which, when mapped onto the crystal structure of the Axin DIX domain and a comparative model of the Dishevelled DIX domain, allow their categorization as residues of either structural or functional importance. We identify residues that are structural and functional determinants of the DIX domain fold, as well as those that are specific to homodimerization of Axin and Dishevelled. CONCLUSION: This report provides the first explanation of the mutant phenotypes caused by non-synonymous substitutions in the Dishevelled and Axin DIX domain by correlating their presumed functional significance with molecular structure.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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

ATP half-sites in RadA and RAD51 recombinases bind nucleotides.

Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.We would like to thank Dr Timothy Sharpe for help with MALS analysis of the monomeric RadA protein and Dr Tara Pukala for the mass spectrometric analysis of the same protein. We would like to thank X-ray crystallographic and Biophysics facilities at the Department of Biochemistry for access to their instrumentation. We thank Diamond Light Source for access to beamline I04 (proposal MX315), European Synchrotron Radiation Facility for access to beamline ID23-1 (proposal MX-705 17 and MX-857) and Swiss Light Source for access to beamline PXIII that contributed to the results presented here. This work was funded by Translational Award from the Wellcome Trust (080083/Z/06/Z).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Wiley

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

Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment

Intracellular antibodies can inhibit disease-relevant protein interactions, but inefficient cellular uptake limits their utility. Using a RAS-targeting intracellular antibody as a screening tool, the authors here identify small molecules that inhibit RAS-effector interactions and readily penetrate cells

The PE/PPE multigene family codes for virulence factors and are a possible source of mycobacterial antigenic variation: perhaps more?

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function