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B Domain (b + domain)
Selected AbstractsIn vivo potentiation of human oestrogen receptor , by Cdk7-mediated phosphorylationGENES TO CELLS, Issue 10 2004Saya Ito Phosphorylation of the Ser118 residue in the N-terminal A/B domain of the human oestrogen receptor , (hER,) by mitogen-activated protein kinase (MAPK), stimulated via growth factor signalling pathways, is known to potentiate ER, ligand-induced transactivation function. Besides MAPK, cyclin dependent kinase 7 (Cdk7) in the TFIIH complex has also been found to potentiate hER, transactivation in vitro through Ser118 phosphorylation. To investigate an impact of Cdk7 on hER, transactivation in vivo, we assessed activity of hER, in a wild-type and cdk7 inactive mutant Drosophila that ectopically expressed hER, in the eye disc. Ectopic expression of the wild-type or mutant receptors, together with a green fluorescent protein (GFP) reporter gene, allowed us to demonstrate that hER, expressed in the fly tissues was transcriptionally functional and adequately responded to hER, ligands in the patterns similar to those observed in mammalian cells. Replacement of Ser118 with alanine in hER, (S118A mutant) significantly reduced the ligand-induced hER, transactivation function. Importantly, while in cdk7 inactive mutant Drosophila the wild-type hER, exhibited reduced response to the ligand; levels of transactivation by the hER, S118A mutant were not affected in these inactive cdk7 mutant flies. Furthermore, phosphorylation of hER, at Ser118 has been observed in vitro by both human and Drosophila Cdk7. Our findings demonstrate that Cdk7 is involved in regulation of the ligand-induced transactivation function of hER,in vivo via Ser118 phosphorylation. [source] Molecular mechanism of a cross-talk between oestrogen and growth factor signalling pathwaysGENES TO CELLS, Issue 8 2000Shigeaki Kato Oestrogen (E2) plays significant roles in variety of biological events such as the development and maintenance of female reproductive organs, bone and lipid metabolisms. More recently, from study of knock-out mice deficient in oestrogen receptor (ER) , and ER, it turned out that normal spermatogenesis requires the E2 actions. Furthermore, this female steroid hormone is also well known to be deeply involved in many pathophysiological events such as osteoporosis and cancer development in female reproductive organs. It is particularly well known that most breast cancer is dependent on E2 in its development. Such E2 actions are thought to be mediated through two subtypes of ERs. Growth factors have been shown to synergize in this E2 signalling pathway, although the actual molecular mechanism largely remains unknown. Recently, we found that the MAP kinase activated by growth factors phosphorylates the Ser118 residue of the human ER, A/B domain and this phosphorylation potentiates the N-terminal transactivation function (AF-1) of human ER,, indicating the possible molecular mechanism of a novel cross-talk between E2 and growth factor signalling pathways. More recently, we have identified a coactivator associating with the hER, AF-1 in a MAPK-mediated phosphorylation-dependent manner. In this review, the molecular mechanism of this cross-talk is discussed in terms of the transactivation function of ERs, and their coactivators. [source] Functional roles of the factor VIII B domainHAEMOPHILIA, Issue 6 2009S. W. PIPE Summary., Unravelling the structure, function and molecular interactions of factor VIII (FVIII) throughout its life cycle from biosynthesis to clearance has advanced our understanding of the molecular mechanisms of haemophilia and the development of effective treatment strategies including recombinant replacement therapy. These insights are now influencing bioengineering strategies toward novel therapeutics. Whereas available molecular models and crystal structures have helped elucidate the structure and function of the A and C domains of FVIII, these models have not included detailed structural information of the B domain. Therefore, insights into the role of the FVIII B domain have come primarily from expression studies in heterologous systems, biochemical studies on bioengineered FVIII variants and clinical studies with B domain-deleted FVIII. This manuscript reviews the available data on the potential functional roles of the FVIII B domain. A detailed literature search was performed, and the data extracted were qualitatively summarized. Intriguing emerging evidence suggests that the FVIII B domain is involved in intracellular interactions that regulate quality control and secretion, as well as potential regulatory roles within plasma during activation, platelet binding, inactivation and clearance. [source] IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27,MHz quartz-crystal microbalanceJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2007Hideyuki Mitomo Abstract Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x,=,1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27,MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (,mmax), association constants (Ka), association and dissociation rate constants (k1 and k,1, respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains. Copyright © 2006 John Wiley & Sons, Ltd. [source] Antibody variable region interactions with Protein A: Implications for the development of generic purification processesBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2005Sanchayita Ghose Abstract In this paper, a wide range of antibodies from various subclasses and subfamilies are employed to evaluate the creation of generic separation processes using Protein A chromatography. The reasons for elution pH differences amongst several IgG1s, IgG2s, antibody fragments, and Fc-fusion proteins during Protein A chromatography are investigated using several complimentary techniques. The results indicate that variable region interactions play a major role in determining elution pH for VH3 subfamily antibodies while using traditional protein A chromatographic materials. On the other hand, experiments with a resin which employs a ligand consisting solely of B domain of Protein A indicate that variable region interactions can be mitigated, enabling the use of a single elution pH for a range of antibodies. Finally, the moderation of elution conditions associated with this engineered ligand are shown to minimize problems associated with low pH induced aggregation. It is expected that the findings reported in this paper will facilitate faster process development cycle times for this important class of human therapeutics. © 2005 Wiley Periodicals, Inc. [source] IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27,MHz quartz-crystal microbalanceJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2007Hideyuki Mitomo Abstract Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x,=,1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27,MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (,mmax), association constants (Ka), association and dissociation rate constants (k1 and k,1, respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains. Copyright © 2006 John Wiley & Sons, Ltd. [source] Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM familyMOLECULAR MICROBIOLOGY, Issue 6 2003Sreedhar R. Nallapareddy Summary A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen. [source] Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzaeMOLECULAR MICROBIOLOGY, Issue 4 2000Mumtaz Virji Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non-typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI-Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs. [source] | ||||||||||