Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.     

Transcription factors do it together: the hows and whys of studying protein-protein interactions

Protein–protein interactions are intrinsic to virtually every cellular process. Recent breakthroughs in techniques to study protein-interaction and the availability of fully sequenced plant genomes have attracted many plant scientists to undertake the first steps in the field of protein interactions. High-throughput screening systems allow the discovery of protein functions. Even without performing laborious functional assays, in planta functional homologues and redundant proteins can be accurately predicted based on protein-interaction maps. Therefore, protein–protein-interaction screenings are an essential supplement to the current functional-genomics toolbox.     

The use of floral homeotic mutants as a novel way to obtain durable resistance to insect pests

We have developed a novel strategy for the introduction of durable insect resistance in crops. This strategy was based on intervention in the natural relationship between plants and insects. For many insects, including pests such as thrips (Frankliniella occidentalis), the flower is an important factor in their life cycle, serving either as a food source or as a place for mating. The insects are attracted to the flower by scent, which is mainly produced by the petals, and by the bright colour of these floral organs. We therefore anticipated that removal or changing the identity of the petals would significantly reduce the attractiveness of the flower to thrips. To test this hypothesis, we used cucumber as a model species because most modern varieties are parthenocarpic, in which the fruit develops without fertilization. The cucumber mutant green petals, in which the petals are homeotically transformed into green sepals, was particularly useful for this study. The susceptibility of the cucumber plants to damage by thrips was determined by recording thrip numbers and by measuring leaf damage. Large differences were observed when greenhouse compartments with either wild-type or green petal mutant plants were compared. The rate of population growth of the insects on the mutant plants was significantly reduced and the leaves were almost undamaged. These results demonstrate that alterations in the structure of flowers may interfere with the life cycle of insects, providing the means for a novel and natural strategy for obtaining insect resistance.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Ubc13 and COOH terminus of Hsp70-interacting protein (CHIP) are required for growth hormone receptor endocytosis

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(βTrCP) (Skp Cullin F-box). Despite a specific interaction between β-transducin repeat-containing protein (βTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(βTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to βTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.