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261.
Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis,modification, and transport 总被引:31,自引:0,他引:31 下载免费PDF全文
Mathews H Clendennen SK Caldwell CG Liu XL Connors K Matheis N Schuster DK Menasco DJ Wagoner W Lightner J Wagner DR 《The Plant cell》2003,15(8):1689-1703
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Gärdby E Wrammert J Schön K Ekman L Leanderson T Lycke N 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(1):55-63
In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved. 相似文献
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Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and-as shown by the use of cholesterol analogs-the underlying interactions involve the 3beta-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses. 相似文献
266.
Rohde HM Cheong FY Konrad G Paiha K Mayinger P Boehmelt G 《The Journal of biological chemistry》2003,278(52):52689-52699
The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif. 相似文献
267.
Hilpert K Wessner H Schneider-Mergener J Welfle K Misselwitz R Welfle H Hocke AC Hippenstiel S Höhne W 《The Journal of biological chemistry》2003,278(27):24986-24993
Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase. 相似文献
268.
Buerger C Nagel-Wolfrum K Kunz C Wittig I Butz K Hoppe-Seyler F Groner B 《The Journal of biological chemistry》2003,278(39):37610-37621
Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation. 相似文献
269.
Scheper W Thaminy S Kais S Stagljar I Römisch K 《The Journal of biological chemistry》2003,278(39):37998-38003
Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins. 相似文献
270.
Nienhaus K Deng P Olson JS Warren JJ Nienhaus GU 《The Journal of biological chemistry》2003,278(43):42532-42544
We have combined Fourier transform infrared/temperature derivative (FTIR-TDS) spectroscopy at cryogenic temperatures and flash photolysis at ambient temperature to examine the effects of polar and bulky amino acid replacements of the highly conserved distal valine 68 in sperm whale myoglobin. In FTIR-TDS experiments, the CO ligand can serve as an internal voltmeter that monitors the local electrostatic field not only at the active site but also at intermediate ligand docking sites. Mutations of residue 68 alter size, shape, and electric field of the distal pocket, especially in the vicinity of the primary docking site (state B). As a consequence, the infrared bands associated with the ligand at site B are shifted. The effect is most pronounced in mutants with large aromatic side chains. Polar side chains (threonine or serine) have only little effect on the peak frequencies. Ligands that migrate toward more remote sites C and D give rise to IR bands with altered frequencies. TDS experiments separate the photoproducts according to their recombination temperatures. The rates and extent of ligand migration among internal cavities at cryogenic temperatures can be used to interpret geminate and bimolecular O2 and CO recombination at room temperature. The kinetics of geminate recombination can be explained by steric arguments alone, whereas both the polarity and size of the position 68 side chain play major roles in regulating bimolecular ligand binding from the solvent. 相似文献