A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes.

Understanding the dynamics of endogenous protein-protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical samples

Four plant defensins from an indigenous South African Brassicaceae species display divergent activities against two test pathogens despite high sequence similarity in the encoding genes

<p>Abstract</p> <p>Background</p> <p>Plant defensins are an important component of the innate defence system of plants where they form protective antimicrobial barriers between tissue types of plant organs as well as around seeds. These peptides also have other activities that are important for agricultural applications as well as the medical sector. Amongst the numerous plant peptides isolated from a variety of plant species, a significant number of promising defensins have been isolated from Brassicaceae species. Here we report on the isolation and characterization of four defensins from <it>Heliophila coronopifolia</it>, a native South African Brassicaceae species.</p> <p>Results</p> <p>Four defensin genes (<it>Hc-AFP1</it>-<it>4) </it>were isolated with a homology based PCR strategy. Analysis of the deduced amino acid sequences showed that the peptides were 72% similar and grouped closest to defensins isolated from other Brassicaceae species. The Hc-AFP1 and 3 peptides shared high homology (94%) and formed a unique grouping in the Brassicaceae defensins, whereas Hc-AFP2 and 4 formed a second homology grouping with defensins from <it>Arabidopsis </it>and <it>Raphanus</it>. Homology modelling showed that the few amino acids that differed between the four peptides had an effect on the surface properties of the defensins, specifically in the alpha-helix and the loop connecting the second and third beta-strands. These areas are implicated in determining differential activities of defensins. Comparing the activities after recombinant production of the peptides, Hc-AFP2 and 4 had IC₅₀values of 5-20 μg ml^-1against two test pathogens, whereas Hc-AFP1 and 3 were less active. The activity against <it>Botrytis cinerea </it>was associated with membrane permeabilization, hyper-branching, biomass reduction and even lytic activity. In contrast, only Hc-AFP2 and 4 caused membrane permeabilization and severe hyper-branching against the wilting pathogen <it>Fusarium solani</it>, while Hc-AFP1 and 3 had a mild morphogenetic effect on the fungus, without any indication of membrane activity. The peptides have a tissue-specific expression pattern since differential gene expression was observed in the native host. <it>Hc-AFP1 </it>and <it>3 </it>expressed in mature leaves, stems and flowers, whereas <it>Hc-AFP2 </it>and <it>4 </it>exclusively expressed in seedpods and seeds.</p> <p>Conclusions</p> <p>Two novel Brassicaceae defensin sequences were isolated amongst a group of four defensin encoding genes from the indigenous South African plant <it>H. coronopifolia</it>. All four peptides were active against two test pathogens, but displayed differential activities and modes of action. The expression patterns of the peptide encoding genes suggest a role in protecting either vegetative or reproductive structures in the native host against pathogen attack, or roles in unknown developmental and physiological processes in these tissues, as was shown with other defensins.</p

25th Annual Computational Neuroscience Meeting: CNS-2016

Abstracts of the 25th Annual Computational Neuroscience Meeting: CNS-2016 Seogwipo City, Jeju-do, South Korea. 2–7 July 201