Jasmonates induce Arabidopsis bioactivities selectively inhibiting the growth of breast cancer cells through CDC6 and mTOR.

Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells. Here, direct incubation of leaf explants from the non-medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High-throughput LC-MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells. A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA-treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin-dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis-(+)-12-oxophytodienoic acid, in synergy, could act as anticancer compounds. Our work suggests a universal role for MeJA-treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti-cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments.

Aphids Pick Their Poison: Selective Sequestration of Plant Chemicals Affects Host Plant Use in a Specialist Herbivore.

In some plant-insect interactions, specialist herbivores exploit the chemical defenses of their food plant to their own advantage. Brassica plants produce glucosinolates that are broken down into defensive toxins when tissue is damaged, but the specialist aphid, Brevicoryne brassicae, uses these chemicals against its own natural enemies by becoming a "walking mustard-oil bomb". Analysis of glucosinolate concentrations in plant tissue and associated aphid colonies reveals that not only do aphids sequester glucosinolates, but they do so selectively. Aphids specifically accumulate sinigrin to high concentrations while preferentially excreting a structurally similar glucosinolate, progoitrin. Surveys of aphid infestation in wild populations of Brassica oleracea show that this pattern of sequestration and excretion maps onto host plant use. The probability of aphid infestation decreases with increasing concentrations of progoitrin in plants. Brassica brassicae, therefore, appear to select among food plants according to plant secondary metabolite profiles, and selectively store only some compounds that are used against their own enemies. The results demonstrate chemical and behavioral mechanisms that help to explain evidence of geographic patterns and evolutionary dynamics in Brassica-aphid interactions.

Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana.

The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.

Evidence for α-helices in the gas phase: a case study using Melittin from honey bee venom.

Gas phase methodologies are increasingly used to study the structure of proteins and peptides. A challenge to the mass spectrometrist is to preserve the structure of the system of interest intact and unaltered from solution into the gas phase. Small peptides are very flexible and can present a number of conformations in solution. In this work we examine Melittin a 26 amino acid peptide that forms the active component of honey bee venom. Melittin is haemolytic and has been shown to form an α-helical tetrameric structure by X-ray crystallography [M. Gribskov et al., The RCSB Protein Data Bank, 1990] and to be helical in high concentrations of methanol. Here we use ion mobility mass spectrometry, molecular dynamics and gas-phase HDX to probe its structure in the gas phase and specifically interrogate whether the helical form can be preserved. All low energy calculated structures possess some helicity. In our experiments we examine the peptide following nano-ESI from solutions with varying methanol content. Ion mobility gives collision cross sections (CCS) that compare well with values found from molecular modelling and from other reported structures, but with inconclusive results regarding the effect of solvent. There is only a slight increase in CCS with charge, showing minimal coloumbically driven unfolding. HDX supports preservation of some helical content into the gas phase and again shows little difference in the exchange rates of species sprayed from different solvents. The [M + 3H](3+) species has two exchanging populations both of which exhibit faster exchange rates than observed for the [M + 2H](2+) species. One interpretation for these results is that the time spent being analysed is sufficient for this peptide to form a helix in the 'ultimate' hydrophobic environment of a vacuum.

A comparison of mass spectrometry based hydrogen deuterium exchange methods for probing the cyclophilin A cyclosporin complex.

Direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques provide an increasingly popular route to determine quantitative information on protein-protein and protein-ligand interactions. When combined with hydrogen deuterium exchange (HDX), details on protein stability and complex conformation can be obtained; however, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting gas phase results. Zhu et al. [1] and Powell and Fitzgerald [2] have outlined LC-MS based techniques to probe the solution phase properties of the protein-ligand system in question. We here have taken the well characterised soluble immunophilin protein cyclophilin A, and examined it in complex with its endogenous ligand cyclosporin A. This ligand is widely used as an immunosuppressant following organ transplant, and the complex provides a basis for drug discovery efforts. We have used direct infusion, coupled with HDX, gas phase HDX and also the LC-HDX techniques PLIMSTEX and SUPREX. Results from each of these four HDX methodologies are presented here and discussed critically. From our direct infusion we find that there are 2 observable hydrogen populations in the protein, a very fast exchanging population, and a slower group. The exchange rate of both is lowered in the presence of the ligand. For PLIMSTEX we find a K(d) for ligand binding of 321 ± 128 nM, which is within one order of magnitude of values previously reported. SUPREX under a variety of conditions provides a range of K(d) values, but when we average these for experimental error we obtain a K(d) of 7.11 ± 0.29 nM which agrees well with measurements from other studies including via SUPREX. Finally gas phase HDX of the native complex shows more than 3 distinct populations of exchangeable hydrogens, for both the apo- and the holo protein consistent with an unfolding and refolding of the protein in the gas phase. The different techniques are compared with respect to the advantages and disadvantages they bring to the study of this protein-ligand system.