European journal of biochemistry最新文献

The tissue-specific transcription factors HNF1alpha and HNF1beta are closely related homeodomain proteins conserved in vertebrate evolution. Heterozygous mutations in human HNF1beta but not in HNF1alpha genes are associated with kidney malformations. Overexpression of HNF1beta in Xenopus embryos leads to defective pronephros development, while HNF1alpha has no effect. We have defined the regions responsible for this functional difference between HNF1beta and HNF1alpha in transfected HeLa cells as well as in injected Xenopus embryos. Using domain swapping experiments, we located a nuclear localization signal in the POUH domain of HNF1beta, and showed that the POUS and POUH domains of HNF1beta mediate a high transactivation potential in transfected cells. In injected Xenopus embryos three HNF1beta domains are involved in nephrogenesis. These include the dimerization domain, the 26 amino acid segment specific for splice variant A as well as the POUH domain. As HNF1beta together with Pax8 and lim1 constitute the earliest regulators in the pronephric anlage, it is possible that they cooperate during early nephrogenesis. We have shown here that HNF1beta can overcome the enlargement and the induction of an ectopic pronephros mediated by overexpression of Pax8 and lim1. However, the phenotype induced by Pax8 and lim1 overexpression and characterized by cyst-like structures and thickening of the pronephric tubules was not altered by HNF1beta overexpression. Taken together, HNF1beta acts antagonistically to Pax8 and lim1 in only some processes during nephrogenesis, and a simple antagonistic relationship does not completely describe the functions of these genes. We conclude that HNF1beta has some distinct morphogenetic properties during nephrogenesis.

RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export. RHA has been reported to bind directly to the transcriptional coactivator, CREB-binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex. Using yeast two-hybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor kappaB (NF-kappaB). The interaction between p65 and RHA was confirmed by glutathione-S transferase pull-down assay in vitro, and by co-immunoprecipitation assay in vivo. In transient transfection assays, RHA enhanced NF-kappaB dependent reporter gene expression induced by p65, tumor necrosis factor-alpha, or NF-kappaB inducing kinase. The mutant form of RHA lacking ATP-binding activity inhibited NF-kappaB dependent reporter gene expression induced by these activators. Moreover, depletion of RHA using short interfering RNA reduced the NF-kappaB dependent transactivation. These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF-kappaB.

Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.

A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligonucleotides have been utilized and designated as primers 1 and 2. Primer 1, with its 5- and 3'-termini fully complementary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3'-end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure was predicted to prevent the annealing of the 3'-end of primer 2 with the template DNA, which leads to no primer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the appearance of double-stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature.

The red cell intercellular adhesion molecule-4 (ICAM-4) binds to different members of the integrin receptor families. To better define the ICAM-4 integrin receptor specificity, cell transfectants individually expressing various integrins were used to demonstrate that alphaLbeta2, alphaMbeta2, and alphaIIbbeta3 (activated) bind specifically and dose dependently to the recombinant ICAM-4-Fc protein. We also show that cell surface ICAM-4 interacts with the cell surface alphaVbeta3 integrin. In addition, using a alpha4beta1 cell transfectant and beta2 integrin-deficient LAD cells, we show here that ICAM-4 failed to interact with alpha4beta1 even after alpha4beta1 activation by phorbol ester or with the monoclonal antibody TS2/16 (+ Mn2+). ICAM-4 amino acids that are critical for alphaIIbbeta3 and alphaVbeta3 interaction were identified by domain deletion analysis, site-directed mutagenesis and synthetic peptide inhibition. Our results provide evidence that the beta3 integrin binding sites encompass the first and second Ig-like domains of ICAM-4. However, while the alphaIIbbeta3 contact site comprises the ABED face of domain D1 with an extension in the C'-E loop of domain D2, the alphaVbeta3 contact site comprises residues on both faces of D1 and in the C'-E loop of D2. These data, together with our previous results, demonstrate that different integrins bind to different but partly overlapping sites on ICAM-4, and that ICAM-4 may accommodate multiple integrin receptors present on leukocytes, platelets and endothelial cells.

Phosphorylation at multiple sites within the N-terminus of p53 promotes its dissociation from hdm2/mdm2 and stimulates its transcriptional regulatory potential. The large phosphoinositide 3-kinase-like kinases ataxia telangiectasia mutated gene product and the ataxia telangectasia and RAD-3-related kinase promote phosphorylation of human p53 at Ser15 and Ser20, and are required for the activation of p53 following DNA damage. DNA-dependent protein kinase (DNA-PK) is another large phosphoinositide 3-kinase-like kinase with the potential to phosphorylate p53 at Ser15, and has been proposed to enhance phosphorylation of these sites in vivo. Moreover, recent studies support a role for DNA-PK in the regulation of p53-mediated apoptosis. We have shown previously that colocalization of p53 and DNA-PK to structured single-stranded DNA dramatically enhances the potential for p53 phosphorylation by DNA-PK. We report here the identification of p53 phosphorylation at two novel sites for DNA-PK, Thr18 and Ser9. Colocalization of p53 and DNA-PK on structured DNA was required for efficient phosphorylation of p53 at multiple sites, while specific recognition of Ser9 and Thr18 appeared to be dependent upon additional determinants of p53 beyond the N-terminal 65 amino acids. Our results suggest a role for DNA-PK in the modulation of p53 activity resultant from the convergence of p53 and DNA-PK on structured DNA.

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.

Antifreeze proteins (AFPs) designate a class of proteins that are able to bind to and inhibit the growth of macromolecular ice. These proteins have been characterized from a variety of organisms. Recently, the structures of AFPs from the spruce budworm (Choristoneura fumiferana) and the yellow mealworm (Tenebrio molitor) have been determined by NMR and X-ray crystallography. Despite nonhomologous sequences, both proteins were shown to consist of beta-helices. We review the structures and dynamics data of these two insect AFPs to bring insight into the structure-function relationship and explore their beta-helical architecture. For the spruce budworm protein, the fold is a left-handed beta-helix with 15 residues per coil. The Tenebrio molitor protein consists of a right-handed beta-helix with 12 residues per coil. Mutagenesis and structural studies show that the insect AFPs present a highly rigid array of threonine residues and bound water molecules that can effectively mimic the ice lattice. Comparisons of the newly determined ryegrass and carrot AFP sequences have led to models suggesting that they might also consist of beta-helices, and indicate that the beta-helix might be used as an AFP structural motif in nonfish organisms.