NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.     

Methionine, folic acid and vitamin B12 in growing-finishing pigs: impact on growth performance and meat quality

Growth performance, metabolic variables, and meat quality were measured in 78 growing-finishing pigs using supplements of 0 (C), or 0.2% of DL-methionine (M), and three combinations of folic acid [mg/kg] and cyanocobalamin [microg/kg], respectively 0 and 0 (V0), 10 and 25 (V1), and 10 and 150 (V2) in a 2 x 3 factorial arrangement. Feed conversion was lower (p = 0.05) in M than in C pigs during the growing period (0-4 weeks). Both V1 and V2 treatments increased plasma vitamin B12 (p < 0.01) and decreased plasma homocysteine (p < 0.01). Plasma 5-methyl-tetrahydrofolates were the lowest, highest and intermediate in V0, V1 and V2 pigs (p < 0.04), respectively. In V2 meat, folates were 32% higher, vitamin B12, 55% higher and homocysteine, 28% lower than in V0 (p < 0.01). Oxidative stability of the fresh meat was similar among treatments during a storage period of 42 days. Therefore, methionine supplements improved growth performance during the growing period. Vitamin supplements interacted with the methionine cycle pathway, increased vitamin content of pork meat but did not improve oxidative stability of the fresh meat during storage.     

DeltaNp63 is essential for epidermal commitment of embryonic stem cells

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.     

Tyrosine phosphorylation of the UDP-glucose dehydrogenase of Escherichia coli is at the crossroads of colanic acid synthesis and polymyxin resistance

Background

In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides.

Methodology/Principal Findings

Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin.

Conclusions/Significance

Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology.     

Airway contribution to alveolar nitric oxide in healthy subjects and stable asthma patients

Alveolar nitric oxide (NO) concentration (Fa(NO)), increasingly considered in asthma, is currently interpreted as a reflection of NO production in the alveoli. Recent modeling studies showed that axial molecular diffusion brings NO molecules from the airways back into the alveolar compartment during exhalation (backdiffusion) and contributes to Fa(NO). Our objectives in this study were 1) to simulate the impact of backdiffusion on Fa(NO) and to estimate the alveolar concentration actually due to in situ production (Fa(NO,prod)); and 2) to determine actual alveolar production in stable asthma patients with a broad range of NO bronchial productions. A model incorporating convection and diffusion transport and NO sources was used to simulate Fa(NO) and exhaled NO concentration at 50 ml/s expired flow (Fe(NO)) for a range of alveolar and bronchial NO productions. Fa(NO) and Fe(NO) were measured in 10 healthy subjects (8 men; age 38 +/- 14 yr) and in 21 asthma patients with stable asthma [16 men; age 33 +/- 13 yr; forced expiratory volume during 1 s (FEV(1)) = 98.0 +/- 11.9%predicted]. The Asthma Control Questionnaire (Juniper EF, Buist AS, Cox FM, Ferrie PJ, King DR. Chest 115: 1265-1270, 1999) assessed asthma control. Simulations predict that, because of backdiffusion, Fa(NO) and Fe(NO) are linearly related. Experimental results confirm this relationship. Fa(NO,prod) may be derived by Fa(NO,prod) = (Fa(NO) - 0.08.Fe(NO))/0.92 (Eq. 1). Based on Eq. 1, Fa(NO,prod) is similar in asthma patients and in healthy subjects. In conclusion, the backdiffusion mechanism is an important determinant of NO alveolar concentration. In stable and unobstructed asthma patients, even with increased bronchial NO production, alveolar production is normal when appropriately corrected for backdiffusion.     

Biochemical and structural insights into bacterial organelle form and biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.