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91.
COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the β-, γ-, δ- and ζ-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the α-, β'- and ε-COP subunits. Here, we present the structure of the appendage domain of γ-COP and show that it has a similar overall fold as the α-appendage of AP2. Again, like the α-appendage the γ-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian γ-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of γ-COP, Sec21p, a second binding site is proposed to exist on the γ-COP appendage that interacts with the α,β',ε COPI subcomplex. 相似文献
92.
Trischitta F Denaro MG Faggio C 《Journal of experimental zoology. Part A, Comparative experimental biology》2004,301(1):49-62
Ion transport in the intestine of Gobius niger, a euryhaline teleost, was studied in both isotonic and hypotonic conditions. Isolated tissues, mounted in Ussing chambers and bilaterally perfused with isotonic Ringer solution, developed a serosa negative transepithelial voltage and a short circuit current indicating a net negative current in absorptive direction. Bilateral removal of Cl- and Na+ from the bathing solutions as well as the luminal removal of K+in the presence of Ba2+(10(-3) M) almost abolished both Vt and Isc. Similar results were obtained by adding bumetanide (10(-5)M) to the luminal bath while other inhibitors of Cl- transport mechanisms were ineffective. These observations suggest that salt absorption begins with a coupled entry of Na+, Cl-, and K+ across the apical membrane; a Ba2+inhibitable K+ conductance, demonstrated also by micropuncture experiments, recycles the ion into the lumen. Salt entry into the cell is driven by the operation of the basolateral Na+/K(+)-ATPase since serosal ouabain (10(-4)M) completely abolished both Vt and Isc; this pump also completes the Na(+) absorption. The inhibitory effect of both serosal bumetanide (10(-4)M) and SITS (5 x 10(-4)M) suggests that Cl- would leave the cell via the KCl cotransport, the Cl/HCO3- antiport and/or conductive pathways. Bilateral exposure of tissues to hypotonic media produced a reduction of both the transepithelial voltage and the short circuit current probably due to the activation of homeostatic ionic fluxes involved in cell volume regulation. The results of experiments with both isolated enterocytes and intestine exposed to hypotonic solution suggested that the recovery of cell volume, after the initial cell swelling, involves a parallel opening of K+ and Cl- channels to facilitate net solute and water effluxes from the cell. J. Exp. Zool. 301A:49-62, 2004. 相似文献
93.
Due to their involvement in many pathological conditions, matrix metalloproteinases (MMPs), are very attractive therapeutic targets. Our study focuses on one of them, MMP-2, which is involved in tumor progression and metastasis. Recently, the solution structure of the catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020) was published by Feng et al. Using the Hanessian group published binding affinity data and the structure published by Feng as a basis, we have built a binding affinity model by targeting the S(2)' pocket of the enzyme with a set of nine alpha-N-sulfonylamino hydroxamic acid derivatives. Two binding geometries of each ligand have been generated corresponding to two binding modes denoted A and B, respectively, of which the first one is targeting the S(2)' pocket and the second one the S(1) pocket. For the binding affinity model developed for mode A the computed activities show a rmsd of 0.583 kcal/mol as compared with the experimental data, and a correlation coefficient r(2) of 0.779, while in the case of the binding mode B a rmsd of 0.834 kcal/mol and correlation coefficient r(2) of 0.500, respectively, were obtained. In conclusion, our data suggest a higher probability for the Phe(76) gated S(2)' open form pocket to accommodate the substituent alpha versus the wide solvent exposed S(1) subsite, probability which some research groups could have overlooked due to extensive use in their calculations of non revealing S(2)' pocket open state crystallographic structures instead of NMR ones. 相似文献
94.
Tripodi SA Del Vecchio MT Supuran CT Scozzafava A Gabrielli MG Pastorekova S Rossie R Fasolis G Puccetti L 《Journal of enzyme inhibition and medicinal chemistry》2004,19(3):287-291
Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis (p < 0.05). Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression (p = 0.057). The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC. 相似文献
95.
Gabrielli MG 《Journal of enzyme inhibition and medicinal chemistry》2004,19(3):283-286
The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and HCO3(-)/Cl(-) anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development. 相似文献
96.
P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading 总被引:1,自引:0,他引:1
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Di Stefano P Cabodi S Boeri Erba E Margaria V Bergatto E Giuffrida MG Silengo L Tarone G Turco E Defilippi P 《Molecular biology of the cell》2004,15(2):787-800
Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling. 相似文献
97.
The domains of glycoprotein D required to block apoptosis induced by herpes simplex virus 1 are largely distinct from those involved in cell-cell fusion and binding to nectin1
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Glycoprotein D (gD) interacts with two alternative protein receptors, nectin1 and HveA, to mediate herpes simplex virus (HSV) entry into cells. Fusion of the envelope with the plasma membrane requires, in addition to gD, glycoproteins gB, gH, and gL. Coexpression of the four glycoproteins (gD, gB, gH, and gL) promotes cell-cell fusion. gD delivered in trans is also capable of blocking the apoptosis induced by gD deletion viruses grown either in noncomplementing cells (gD(-/-)) or in complementing cells (gD(-/+)). While ectopic expression of cation-independent mannose-6 phosphate receptor blocks apoptosis induced by both stocks, other requirements differ. Thus, apoptosis induced by gD(-/-) virus is blocked by full-length gD (or two gD fragments reconstituting a full-length molecule), whereas ectopic expression of the gD ectodomain is sufficient to block apoptosis induced by gD(-/+) virus. In this report we took advantage of a set of gD insertion-deletion mutants to map the domains of gD required to block apoptosis by gD(-/-) and gD(-/+) viruses and those involved in cell-cell fusion. The mutations that resulted in failure to block apoptosis were the same for gD(-/-) and gD(-/+) viruses and were located in three sites, one within the immunoglobulin-type core region (residues 125, 126, and 151), one in the upstream connector region (residues 34 and 43), and one in the C-terminal portion of the ectodomain (residue 277). A mutant that carried amino acid substitutions at the three glycosylation sites failed to block apoptosis but behaved like wild-type gD in all other assays. The mutations that inhibited polykaryocyte formation were located in the upstream connector region (residues 34 and 43), at the alpha1 helix (residue 77), in the immunoglobulin core and downstream regions (residue 151 and 187), and at the alpha3 helix (residues 243 and 246). Binding of soluble nectin1-Fc to cells expressing the mutant gDs was generally affected by the same mutations that affected fusion, with one notable exception (Delta277-310), which affected fusion without hampering nectin1 binding. This deletion likely identifies a region of gD involved in fusion activity at a post-nectin1-binding step. We conclude that whereas mutations that affected all functions (e.g., upstream connector region and residue 151) may be detrimental to overall gD structure, the mutations that affect specific activities identify domains of gD involved in the interactions with entry receptors and fusogenic glycoproteins and with cellular proteins required to block apoptosis. The evidence that glycosylation of gD is required for blocking apoptosis supports the conclusion that the interacting protein is the mannose-6 phosphate receptor. 相似文献
98.
Herpes simplex virus glycoprotein K,but not its syncytial allele,inhibits cell-cell fusion mediated by the four fusogenic glycoproteins,gD, gB,gH, and gL
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A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial (syn) allele of gK (syn-gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm. 相似文献
99.
Genetic rescue of chondrodysplasia and the perinatal lethal effect of cartilage link protein deficiency 总被引:1,自引:0,他引:1
Czipri M Otto JM Cs-Szabó G Kamath RV Vermes C Firneisz G Kolman KJ Watanabe H Li Y Roughley PJ Yamada Y Olsen BR Glant TT 《The Journal of biological chemistry》2003,278(40):39214-39223
100.
Malovrh P Viero G Serra MD Podlesek Z Lakey JH Macek P Menestrina G Anderluh G 《The Journal of biological chemistry》2003,278(25):22678-22685
Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation. 相似文献