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111.
Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells 总被引:7,自引:0,他引:7 下载免费PDF全文
The lysosomal protease cathepsin B has been implicated in a variety of pathologies including pancreatitis, tumor angiogenesis, and neuronal diseases. We used a tube formation assay to investigate the role of cathepsin B in angiogenesis. When cultured between two layers of collagen I, primary endothelial cells formed tubes in response to exogenously added VEGF. Overexpressing cathepsin B reduced the VEGF-dependent tube response, whereas pharmacologically or molecularly suppressing cathepsin B eliminated the dependence on exogenous VEGF. However, tube formation still required VEGF receptor activity, which suggested that endothelial cells generated VEGF. Indeed, VEGF mRNA and protein was detectable in cells treated with cathepsin B inhibitor, which correlated with a rise in the level of HIF-1alpha. In addition to boosting the level of proangiogenic factors, blocking cathepsin B activity reduced the amount of the antiangiogenic protein endostatin. Thus endothelial cells have the intrinsic capacity to generate pro- and antiangiogenic agents. These observations complement and expand our appreciation of how endothelial cell-derived proteases regulate angiogenesis. 相似文献
112.
Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1. 相似文献
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114.
Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation. 相似文献
115.
Lee SH Ahn SJ Im YJ Cho K Chung GC Cho BH Han O 《Biochemical and biophysical research communications》2005,330(4):1194-1198
Previous studies show that low temperature strongly induces suberin layers in the roots of chilling-sensitive cucumber plants, while in contrast, low temperature produces a much weaker induction of suberin layers in the roots of the chilling-tolerant figleaf gourd [S.H. Lee, G.C. Chung, S. Steudle, Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and -tolerant figleaf gourd, J. Exp. Bot. 56 (2005) 985-995; S.H. Lee, G.C. Chung, E. Steudle, Low temperature and mechanical stresses differently gate aquaporins of root cortical cells of chilling-sensitive cucumber and figleaf gourd, Plant Cell Environ. (2005) in press; S.J. Ahn, Y.J. Im, G.C. Chung, B.H. Cho, S.R. Suh, Physiological responses of grafted-cucumber leaves and rootstock roots affected by low root temperature, Scientia Hort. 81 (1999) 397-408]. Here, the effect of low temperature on fatty acid unsaturation and lipoxygenase activity was examined in cucumber and figleaf gourd. The double bond index demonstrated that membrane lipid unsaturation shows hyperbolic saturation curve in figleaf gourd roots while a biphasic response in cucumber roots to low temperature. In figleaf gourd, the hyperbolic response in the double bond index was primarily due to accumulation of linolenic acid. Chilling stress also significantly induced lipoxygenase activity in figleaf gourd roots. These results suggest that the degree of unsaturation of root plasma membrane lipids correlates positively with chilling-tolerance. Therefore, studies that compare the effects of chilling on cucumber and figleaf gourd may provide broad insight into stress response mechanisms in chilling-sensitive and chilling-tolerant plants. Furthermore, these studies may provide important information regarding the relationship between lipid unsaturation and lipoxygenase function/activity, and between lipoxygenase activity and water channeling during the response to chilling stress. The possible roles of these processes in chilling tolerance are discussed. 相似文献
116.
Xie GH Rah SY Kim SJ Nam TS Ha KC Chae SW Im MJ Kim UH 《Biochemical and biophysical research communications》2005,330(4):1290-1298
ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from beta-NAD(+). In this study, we examined the molecular basis of which beta-adrenergic receptor (betaAR) stimulation induces cADPR formation and characterized cardiac ADPR-cyclase. The results revealed that isoproterenol-mediated increase of [Ca(2+)](i) in rat cardiomyocytes was blocked by pretreatment with a cADPR antagonistic derivative 8-Br-cADPR, a PKA inhibitor H89 or high concentration of ryanodine. Moreover, incubation of ventricular lysates with isoproterenol, forskolin or cAMP resulted in activation of ADPR-cyclase that was inhibited by pretreatment with H89. Supporting the observations, the cADPR antagonist and H89 blocked 8-CPT-cAMP, a cell-permeant cAMP analog-induced increase in [Ca(2+)](i) but not cGMP-mediated increase. Characterization of partially purified cardiac ADPR-cyclase showed a molecular mass of approximately 42 kDa and no cross-activity with CD38 antibodies, and the enzyme activity was inhibited by Zn(2+) but not dithiothreitol. Microinjection of the enzyme into rat cardiomyocytes increased the level of [Ca(2+)](i) in a concentration-dependent manner. The enzyme-mediated increase of [Ca(2+)](i) was blocked by the cADPR antagonist. These findings suggest that betaAR-mediated regulation of [Ca(2+)](i) in rat cardiomyocytes is primed by activation of cardiac ADPR-cyclase via cAMP/PKA signaling and that cardiac ADPR-cyclase differs from CD38 in biochemical and immunological properties. 相似文献
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118.
Choe J Sun W Yoon SY Rhyu IJ Kim EH Kim H 《Biochemical and biophysical research communications》2005,331(1):43-49
The thymosin betas (Tbetas) are polypeptide regulators of actin dynamics that are critical for the growth and branching of neurites in developing neurons. We found that mRNAs for Tbeta4, Tbeta10, and Tbeta15 were highly expressed in the developing rat brain during neuritogenesis, supporting a role for the Tbetas in this process. Overexpression of the Tbetas increased the number of neurite branches per neuron in cultured hippocampal and cerebral cortex neurons, and Tbeta15 had the greatest effect. Actin binding activity appears to be essential for the branch-promoting activity of Tbetas because two mutants of Tbeta15 lacking monomeric actin binding activity failed to stimulate branch formation. We also found that transfection of siRNA against Tbeta15 reduced branching. Taken together, these data suggest that the three Tbetas, and especially Tbeta15, stimulate neurite branching during brain development. 相似文献
119.
To identify the binding site for bovine cytochrome b(5) (cyt b(5)) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with (2)H- and (15)N-enriched cyt c and unlabeled cyt b(5). In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b(5) concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A K(a) of (4 +/- 3) x 10(5) M(-)(1) was obtained with a lower limit of 855 s(-)(1) for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b(5) heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c-cyt b(5) complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein-protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces. 相似文献
120.
Im JS Yu KO Illarionov PA LeClair KP Storey JR Kennedy MW Besra GS Porcelli SA 《The Journal of biological chemistry》2004,279(1):299-310
CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function. 相似文献