Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes

Cross-linking of 4-1BB, a member of the TNFR family, increased tyrosine phosphorylation of TCR-signaling molecules such as CD3epsilon, CD3zeta, Lck, the linker for activation of T cells, and SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76). In addition, incubation of activated CD8+ T cells with p815 cells expressing 4-1BBL led to redistribution of the lipid raft domains and Lck, protein kinase C-theta;, SLP-76, and phospholipase C-gamma1 (PLC-gamma1) on the T cell membranes to the areas of contact with the p815 cells and recruitment of 4-1BB, TNFR-associated factor 2, and phospho-tyrosine proteins to the raft domains. 4-1BB ligation also caused translocation of TNFR-associated factor 2, protein kinase C-theta;, PLC-gamma1, and SLP-76 to detergent-insoluble compartments in the CD8+ T cells, and cross-linking of 4-1BB increased intracellular Ca2+ levels apparently by activating PLC-gamma1. The redistribution of lipid rafts and Lck, as well as translocation of PLC-gamma1, and degradation of IkappaB-alpha in response to 4-1BB were inhibited by disrupting the formation of lipid rafts with methyl-beta-cyclodextrin. These findings demonstrate that 4-1BB is a T cell costimulatory receptor that activates TCR-signaling pathways in CD8+ T cells.     

Selective precipitation and recovery of xylanase using surfactant and organic solvent

The selective precipitation of xylanase from an aqueous phase containing mixtures of xylanase and cellulase was studied using an ionic surfactant as precipitating ligand and a polar organic solvent as recovery solvent. Of four ionic surfactants tested, sodium di-(2-ethylhexyl) sulfosuccinate (AOT) showed a complete removal of xylanase at pH 4.5. The recovery of xylanase from the xylanase-AOT complex was a strong function of the type and the volume of the polar solvent and of the concentration of sodium acetate buffer in the final aqueous solution used to solubilize the recovered xylanase. With ethanol as a recovery solvent, a recovery of xylanase activity of 78 +/- 10% was obtained. The cellulase activity in the recovered xylanase was below the detection limit. The results demonstrate that an ionic surfactant can recover enzymes from aqueous solutions without loss in their activity.     

Activation of big MAP kinase 1 (BMK1/ERK5) inhibits cardiac injury after myocardial ischemia and reperfusion

Big MAP kinase 1 (BMK1/ERK5) plays a critical role in pre-natal development of the cardiovascular system and post-natal eccentric hypertrophy of the heart. Of the two isoforms upstream of MAPK-kinase 5 (MEK5) known to exist, only the longer MEK5alpha isoform potently activates BMK1. We generated cardiac-specific constitutively active form of the MEK5alpha (CA-MEK5alpha transgenic (Tg) mice), and observed a 3 to 4-fold increase in endogenous BMK1 activation and hyperphosphorylation of connexin 43 in the ventricles of the Tg compared to wild-type mice. The CA-MEK5alpha-Tg-mice demonstrated a profoundly accelerated recovery of left ventricular developed pressure after ischemia/reperfusion. We propose a novel role for BMK1 in protecting the heart from ischemia/reperfusion-induced cardiac injury.     

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.     

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.     

Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone

The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and L-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (-0.3+/-0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.