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81.
Lu HA Sun TX Matsuzaki T Yi XH Eswara J Bouley R McKee M Brown D 《The Journal of biological chemistry》2007,282(39):28721-28732
The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking. 相似文献
82.
Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9A resolution. Although the FD1 monomer shares a common fold with the fibrinogen gamma fragment and tachylectin-5A, the Asp-282-Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-d-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282-Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self- and non-self discrimination mechanism by ficolins. 相似文献
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85.
Zeller R Haramis AG Zuniga A McGuigan C Dono R Davidson G Chabanis S Gibson T 《Cell and tissue research》1999,298(1):85-93
Formin was originally isolated as the gene affected by the murine limb deformity (ld) mutations, which disrupt the epithelial-mesenchymal interactions regulating patterning of the vertebrate limb autopod. More
recently, a rapidly growing number of genes with similarity to formin have been isolated from many different species including fungi and plants. Genetic and biochemical analysis shows that formin family members function in cellular processes regulating either cytokinesis and/or cell polarisation. Another common feature
among formin family members is their requirement in morphogenetic processes such as budding and conjugation of yeast, establishment of
Drosophila oocyte polarity and vertebrate limb pattern formation. Vertebrate formins are predominantly nuclear proteins which control polarising activity in limb buds through establishment of the SHH/FGF-4
feedback loop. Formin acts in the limb bud mesenchyme to induce apical ectodermal ridge (AER) differentiation and FGF-4 expression in the posterior
AER compartment. Finally, disruption of the epithelial-mesenchymal interactions controlling induction of metanephric kidneys
in ld mutant embryos indicates that formin might function more generally in transduction of morphogenetic signals during embryonic pattern formation.
Received: 24 September 1998 / Accepted: 30 September 1998 相似文献
86.
Haneda K Tagashira M Yoshino E Takeuchi M Inazu T Toma K Iijima H Isogai Y Hori M Takamatsu S Fujibayashi Y Kobayashi K Takeuchi M Yamamoto K 《Glycoconjugate journal》2004,21(6):377-386
Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation. 相似文献
87.
Miyazaki T Matsunaga T Miyazaki S Hokari S Komoda T 《Journal of cellular biochemistry》2004,93(3):503-512
We investigated time-course changes in the expression of receptor activator of nuclear factor-kappaB (RANK), its ligand (RANKL), osteoprotegerin (OPG), bone-type alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase (TRAP) in ovariectomized (OVX) rats. Samples of sera and coccyges were used for analysis of the enzyme activities and expression levels of proteins and mRNAs, and an immunohistochemical analysis was also performed. Serum BAP activity increased to 158.6% of the pre-operation value at 1 week after OVX, and then decreased to 38.7% at 8 weeks after OVX. On the other hand, the serum TRAP activity increased to 130.9% of the pre-operation level at 1 week after OVX, and was maintained at a high level, compared with the pre-operation level. The patterns of BAP and TRAP activity in the coccyges specimens were similar to those seen in the sera. The expression profiles of TRAP, RANK, and RANKL proteins in the coccyx specimens were similar to the pattern of serum TRAP activity, while the profiles of the BAP and OPG proteins were similar to the pattern of serum BAP activity in OVX rats. The changes in the mRNA expression levels of the osteogenic proteins were similar to those for protein expression. These biochemical changes in OVX rats were confirmed by immunohistochemical studies. Our results suggest that not only osteoclastogenesis accelerated but also osteoblastogenesis transiently increased during the early phase of osteoporosis. 相似文献
88.
Takiya T Futo S Tsuna M Namimatsu T Sakano T Kawai K Suzuki T 《Bioscience, biotechnology, and biochemistry》2004,68(2):360-368
Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57 degrees C and 45 degrees C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results. 相似文献
89.
Yokose T Katamoto K Park S Matsuura H Yoshihara T 《Bioscience, biotechnology, and biochemistry》2004,68(12):2640-2642
The Solanum abutiloides plant is highly resistant to soil-borne pathogens such as Fusarium oxysporum f. sp. melongenae, Verticillium dahliae, and Ralstonia solanacearum. This species is utilized as a mating source of resistant cultivars and is also used as a rootstock. The root exudate of Solanum abutiloides was extracted from a soil system composed of charcoal and vermiculite. Anti-fungal activity was found in the extract, and an active ingredient was isolated. The chemical structure of the active compound was determined to be 3-beta-acetoxysolavetivone, a new sesquiterpenoid. The anti-fungal activity of 3-beta-acetoxysolavetivone examined by the inhibition of spore germination of Fusarium oxysporum was close to that of lubimin, and higher than that of solavetivone. 相似文献
90.
Yamagishi K Kimura T Suzuki M Shinmoto H Yamaki KJ 《Bioscience, biotechnology, and biochemistry》2004,68(5):1017-1026
In many fungi, the heterotrimeric G protein alpha subunits, and/or small G protein (RAS) control intracellular cAMP levels. But it is not clear which types of G proteins modulate cAMP levels in homobasidiomycete (mushrooms). To explain the mechanism, we expressed dominant active RAS (a homolog of S. cerevisiae RAS1) in homobasidiomycete Schizophyllum commune and compared the cAMP levels in the transformed clones with those of clones expressing dominant active heterotrimeric G protein alpha subunits ScGP-A, B, and C. The results demonstrated that the dominant active ScGP-A and C elevated the intracellular cAMP levels. In contrast, the dominant active S. commune RAS gene did not affect the cAMP levels, even though colony growth and formation of fruiting bodies were apparently repressed. These data suggest that the heterotrimeric G protein alpha subunits are involved in the mechanism of cAMP regulation, and that RAS modulates another signal-transduction pathway regulating cell growth and differentiation. 相似文献