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Mario M. Müller Esther Klaile Olga Vorontsova Bernhard B. Singer Bj?rn ?brink 《The Journal of cell biology》2009,187(4):569-581
Carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CAM1 [CEACAM1]) mediates homophilic cell adhesion and regulates signaling. Although there is evidence that CEACAM1 binds and activates SHP-1, SHP-2, and c-Src, knowledge about the mechanism of transmembrane signaling is lacking. To analyze the regulation of SHP-1/SHP-2/c-Src binding, we expressed various CFP/YFP-tagged CEACAM1 isoforms in epithelial cells. The supramolecular organization of CEACAM1 was examined by cross-linking, coclustering, coimmunoprecipitation, and fluorescence resonance energy transfer. SHP-1/SHP-2/c-Src binding was monitored by coimmunoprecipitation and phosphotyrosine-induced recruitment to CEACAM1-L in cellular monolayers. We find that trans-homophilic CEACAM1 binding induces cis-dimerization by an allosteric mechanism transmitted by the N-terminal immunoglobulin-like domain. The balance of SHP-2 and c-Src binding is dependent on the monomer/dimer equilibrium of CEACAM1-L and is regulated by trans-binding, whereas SHP-1 does not bind under physiological conditions. CEACAM1-L homodimer formation is reduced by coexpression of CEACAM1-S and modulated by antibody ligation. These data suggest that transmembrane signaling by CEACAM1 operates by alteration of the monomer/dimer equilibrium, which leads to changes in the SHP-2/c-Src–binding ratio. 相似文献
144.
Tamara Azarashvili Irina Odinokova Anush Bakunts Vadim Ternovsky Olga Krestinina Jaana Tyynelä Nils-Erik Leo Saris 《Cell calcium》2014
Phosphorylated and non-phosphorylated forms of the F0F1-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca2+ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca2+. Collectively, the data allowed us to suppose that subunit c F0F1-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status. 相似文献
145.
Ana Beloqui Taras Y. Nechitaylo Nieves López-Cortés Azam Ghazi María-Eugenia Guazzaroni Julio Polaina Axel W. Strittmatter Oleg Reva Agnes Waliczek Michail M. Yakimov Olga V. Golyshina Manuel Ferrer Peter N. Golyshin 《Applied and environmental microbiology》2010,76(17):5934-5946
The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).Microorganisms producing diverse glycosyl hydrolases (GHs) are widespread and typically thrive in environments where plant materials tend to accumulate and deteriorate (42, 73). The habitats of microorganisms with great GH diversity are the ruminant animal rumen, mouse bowel, and rabbit cecum (10, 24, 26, 28, 49, 74). Microorganisms associated with soil invertebrates in general and with soil earthworms in particular carry out metabolic processes that contribute to element cycling and are essential in sustaining processes which their hosts are unable to perform (20, 52, 72, 76). Although some species of earthworms produce cellulases (15, 55), they generally rely on microbes inhabiting their gastrointestinal (GI) tracts to perform cellulose utilization processes (31, 47, 77). Casts are of special interest in this respect. Considering that the overall numbers of cellulolytic microbes in earthworm casts are greater than those in soil (57), earthworm casts seem to play an important role in the decomposition of plant litter, serving as an inoculum for cellulosic substrates (9). It is important to note that microorganisms from preingested substratum (soil or plant litter) are predominant in the gut lumen (20); however, microbial populations in earthworm casts differ from those in soil in terms of diversity and the relative abundance of different taxa (29, 57, 63). It is anticipated that the enzymatic repertoire of such microbial communities must be especially broad toward diverse sugar-based polymeric, oligomeric, and monomeric substrates; however, among approximately 115 families of GHs with thousands of members known to date (12), none of the GHs have been derived from microorganisms of earthworm-associated microbial communities.The aim of the present work was therefore to examine the diversity of GHs in metagenome libraries derived from fresh casts of Aporrectodea caliginosa and Lumbricus terrestris earthworms via functional screening. Other important tasks of this work were to characterize individual enzymes and to gain insight into their structural-functional features. Finally, we performed sequence analysis of large contiguous DNA fragments of fosmids harboring the genes for GHs to associate them with the organism(s) that may produce them, which was complemented by conventional small-subunit (SSU) rRNA clone library sequencing analysis. 相似文献
146.
Krupnik OV Fadeeva NV Kvitko N Shepelev VA Nielsen PE Lazurkin YS 《Journal of biomolecular structure & dynamics》2004,21(4):503-512
Structurally isomeric complexes formed between homopyrimidine bis-PNAs (T(2)JT(2)JT(4)-linker-T(4)CT(2)CT(2)) and single- and double-stranded DNA targets were investigated. These complexes are triplexes designated S1, S2 and S3 in order of increased mobility by polyacrylamide gel electrophoresis. It is shown that the S3 isomer is formed only on double-stranded DNA and possesses highest stability. Isomers S2 and S1 are formed upon binding of bis-PNA to double-stranded as well as to single-stranded DNA. It was found that the stability of the isomer S1 increases dramatically in the presence of excess single-stranded oligonucleotide complementary to the bis-PNA. The structure of the stabilized S1 isomer is proposed to consist of two bis-PNA/DNA triplexes. The relationship between the yield of the isomer S1 formed on single-stranded DNA and the bis-PNA concentration was investigated and a kinetic model of the formation of S1 is presented. 相似文献
147.
Silencing of subfamily I of protein phosphatase 2A catalytic subunits results in activation of plant defense responses and localized cell death 总被引:10,自引:0,他引:10
He X Anderson JC del Pozo O Gu YQ Tang X Martin GB 《The Plant journal : for cell and molecular biology》2004,38(4):563-577
The central importance of protein phosphorylation in plant defense responses has been demonstrated by the isolation of several disease-resistance genes that encode protein kinases. In addition, there are many reports of changes in protein phosphorylation accompanying plant responses to pathogens. In contrast, little is known about the role of protein dephosphorylation in regulating plant defenses. We report that expression of the LePP2Ac1 gene, which encodes a catalytic subunit of the heterotrimeric protein phosphatase 2A (PP2Ac), is rapidly induced in resistant tomato leaves upon inoculation with an avirulent strain of Pseudomonas syringae pv. tomato. By analysis of PP2Ac gene sequences from several plant species, we found that PP2Ac genes cluster into two subfamilies, with LePP2Ac1 belonging to subfamily I. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana was used to suppress expression of genes from subfamily I and not from subfamily II. The PP2Ac-silenced plants had greatly decreased PP2A activity, constitutively expressed pathogenesis-related (PR) genes, and developed localized cell death in stems and leaves. In addition, the plants were more resistant to a virulent strain of P. syringae pv. tabaci and showed an accelerated hypersensitive response (HR) to effector proteins from both P. syringae and the fungal pathogen, Cladosporium fulvum. Thus, catalytic subunits of PP2Ac subfamily I act as negative regulators of plant defense responses likely by de-sensitizing protein phosphorylation cascades. 相似文献
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149.
Elena M. Glinka Alla S. Andryushchenko Alexander M. Sapozhnikov Olga V. Zatsepina 《Plasmid》2009,62(2):119-127
Tumor-targeted vectors encoding toxic protein genes are promising tools for treating malignant tumors. We used the pEGFP-N1 vector to construct a novel plasmid (pCMV-ETA-EGFP) for eukaryotic expression of a truncated Pseudomonas aeruginosa exotoxin A (ETA) that is known to inhibit protein synthesis, and subsequently induce cell death, by inactivation of elongation factor-2. ETA was linked to the enhanced green fluorescent protein (EGFP) gene, and ETA-EGFP gene expression was driven by the cytomegalovirus (CMV) promoter. The time-lapse effects of pCMV-ETA-EGFP expression were examined in transiently transfected HeLa cells. HeLa cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and рЕGFP-N1 showed lower fluorescence intensity than cells transfected with pEGFP-N1 alone. Analysis of the number of dead cells further confirmed the highly toxic effect of the ETA-EGFP fusion protein on cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and рЕGFP-N1. ETA-EGFP fusion protein induced apoptotic cell death through the caspase-3 activation. By using the antibody against a marker nucleolar antigen A3 [Grigoryev, A.A., Bulycheva, T.I., Sheval, E.V., Kalinina, I.A., Zatsepina, O.V., 2008. Cytological indicators of the overall suppression of protein synthesis revealed by staining with new monoclonal antibody. Cell Tissue Biol. 2, 191–199], the distribution of which changes when HeLa cells are treated with known translation inhibitors, we obtained evidence to support the idea that protein synthesis is inhibited in transfected cells in situ. ETA-EGFP fusion protein was identified in lysates of transfected cells using anti-GFP-BL antibodies. Collectively, our results indicate that HeLa cells transfected with pCMV-ETA-EGFP synthesize the ETA-EGFP fusion protein that efficiently inhibits protein synthesis, leading to massive cell death by an apoptosis-mediated pathway with a participation of caspase-3. The constructed vector can be used in suicidal gene therapy of cancer and may also be useful for investigating the general effects of translational downregulation in human cancer cells. We also suggest a novel approach for detecting the activity of new vectors in transfected cells, which is based on the redistribution of nucleolar proteins in transfected cells. 相似文献
150.