Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.     

Age-dependent increase of etheno-DNA-adducts in liver and brain of ROS overproducing OXYS rats

Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) by immunoaffinity/32P-postlabelling. While epsilondA and epsilondC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for epsilondA by immunostaining of nuclei. Brains from old OXYS rats accumulated epsilondA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.     

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.     

cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

A mutation in dVps28 reveals a link between a subunit of the endosomal sorting complex required for transport-I complex and the actin cytoskeleton in Drosophila

Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 in mammals. Here, we characterize a mutation in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated.     

The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells

Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H2O2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H2O2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.     

Tumor promoter TPA stimulates MMP-9 secretion from human keratinocytes by activation of superoxide-producing NADPH oxidase

Matrix metalloproteinase-9 (MMP-9) is involved in physiological tissue remodelling processes as well as in tumor invasion and metastasis. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases MMP-9 secretion from normal human epidermal keratinocytes (NHEK) in vivo and in vitro. Here we show that the flavoprotein inhibitor diphenyleneiodinium (DPI) and the NADPH oxidase inhibitor apocynin block TPA-induced MMP-9 secretion of NHEK in vitro. Furthermore, N-acetyl-L-cysteine and L-cysteine lowered TPA-induced MMP-9 secretion, suggesting an involvement of reactive oxygen species(ROS). TPA exerts its effect on MMP-9 gene expression and secretion via the superoxide-producing enzyme NADPH oxidase: TPA rapidly stimulates generation of superoxide anion as well as gene expression of two cytosolic NADPH oxidase subunits (p47-phox and p67-phox) after 2 h, which is followed by induction of MMP-9 gene expression after 4 h. Taken together, the novel finding herein is the TPA-induced MMP-9 secretion from normal human epidermal keratinocytes through a NADPH oxidase dependent pathway.     

Comparison of different heterocyclic scaffolds as substrate analog PDE5 inhibitors

Several different heterocyclic systems were compared as PDE5 inhibitor scaffolds. In addition to the known 3H-imidazo[5,1-f][1,2,4]triazin-4-ones and pyrazolopyrimidinones, isomeric imidazo[1,5-a][1,3,5]triazin-4(3H)-ones were also shown to be potent and selective PDE inhibitor scaffolds with in vivo activity. SAR trends were elucidated for sulfonamide derivatives with generality across different scaffolds.     

The dynamics and energetics of water permeation and proton exclusion in aquaporins

Aquaporins and aquaglyceroporins are passive membrane channels that, in many species, facilitate highly efficient yet strictly selective permeation of water and small solutes across lipid bilayers. Their ability to block proton flux is particularly remarkable, because other aqueous pores and water efficiently conduct protons, via the so-called Grotthuss mechanism. How efficient water permeation is achieved and how it is reconciled with the seemingly contradictory task of strict proton exclusion have been long-standing puzzles. Because neither the dynamics of the water molecules nor the mobility of protons inside the aquaporin channel could be experimentally accessed so far, several groups addressed this challenge using a variety of atomistic computer simulation methods.     

Interdependent regulation of insulin receptor kinase activity by ADP and hydrogen peroxide

Insulin signaling requires autophosphorylation of the insulin receptor kinase (IRK) domain. Using purified recombinant IRK fragments and the isolated intact insulin receptor, we show here that autophosphorylation is inhibited by ADP and that this effect is essentially reversed by hydrogen peroxide. Autophosphorylation was inhibited by hydrogen peroxide (60 microM) in the absence of ADP but enhanced in the presence of inhibitory concentrations of ADP (67 microM). Enhancement by hydrogen peroxide required direct interaction of hydrogen peroxide with the kinase domain and was not seen in insulin receptor mutants C1245A and C1308A. A similar enhancement was obtained in intact cells in the absence of insulin upon treatment with 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea, indicating that IRK activity can be alternatively enhanced by a shift in the thiol/disulfide redox status. Molecular modeling of the IRK domain indicated that the ATP-binding site becomes distorted after releasing the nucleotide unless the IRK domain is oxidatively derivatized at Cys1245. Recent clinical studies suggest that these effects may play a role in obesity due to the fact that cytoplasmic creatine kinase in combination with phosphocreatine normally ensures rapid removal of ADP in muscle cells but not in fat cells.