Chemical Proteomics versus Leishmaniasis

In this issue of Chemistry & Biology, Wright et al. (2015) describe an elegant approach to evaluating substrates and the drug target potential of Leishmania donovani N-myristoyltransferase (NMT) using a technically simple and straightforward chemical proteomics approach

Significance of ISO7637 Pulse 2a to Integrated Circuits (ICs)

Year after year, an increasing number of electronic devices is introduced into vehicle designs. In order to address potential problems of these devices due to electrical transient disturbances, the standard ISO 7637 has been issued [1]. It deals with the emission of transients, transient transmission via electrical wiring and the potential susceptibility of electronic components to electrical transients. One of these transients is called “pulse 2a” [2]. It is supposed to simulate the voltage response of the partial load dump of a wiring harness. A corresponding schematic is shown in Figure 1. In this schematic, UA represents the power supply, SI is the ignition switch, LW models the inductive wiring harness, UDUT denotes the voltage drop across the device-under-test (DUT), RL represents the load and SL the load switch triggering a 2a pulse. The voltage waveform of UDUT with its characteristic parameters is depicted for two subsequent 2a pulses in Figure 2. The test pulses described in the different parts of ISO 7637 are stated to be characteristic of typical pulses [1]. Still, a device shall be subjected only to those tests in the relevant part of ISO 7637 which apply to that device. Only the tests necessary to replicate the use and mounting location of the device-under-test (DUT) shall be included in the test plan. In this way, ISO 7637 fosters a fit-for-use rather than a fit-for-standard qualification of devices as described in the Handbook of Robustness Validation [3]. Consequently, ISO 7637 points out the importance to know the correlation between laboratory tests and (the given) vehicle [1] and emphasizes the vehicle manufacturer's responsibility to define the test pulses (and failure criteria) required for a specific DUT [2]. EN 62215-3 [4] refers to ISO 7637-2 [2] and requires in chapter 4 and Annex D to apply pulse 2a directly to ICs, notwithstanding the application notes given in ISO 7637 [1] [2]. The only concession made to IC manufacturers is the allowance to demand an external protection. At the same time, it should be noted that 16 different pulse generators investigated in a round robin test in 2014 were found to give hardly reproducible results [5]. The reason for the poor reproducibility was attributed by the authors to the pulse shaping and decoupling networks of the pulse generators used as well as to loose or missing regulations in ISO 7637. Therefore, this paper investigates the applicability, usefulness and meaningfulness of pulse 2a as specified in ISO 7637 [1] [2] and its application to ICs as specified in EN 62215-3 [4]. The experimental set-ups are summarized in section 2. Two pulse 2a simulation models, their response to resistive and capacitive loads and their energy dissipation are introduced in section 3 and conclusions are drawn in section 4

Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant–pathogen interface

- The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae. - Using activity-based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). - The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active β-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. - This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3

Protease activities triggered by Ralstonia solanacearum infection in susceptible and tolerant tomato lines

Altres ajuts: CERCA Programme/Generalitat de CatalunyaActivity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum

Selective inhibition of carotenoid cleavage dioxygenases : phenotypic effects on shoot branching

Members of the carotenoid cleavage dioxygenase family catalyse the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signalling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesised in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at sub-micromolar concentrations. Application of certain inhibitors at 100 microM to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage