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C2 Domain (c2 + domain)
Selected AbstractsHLA-DR-restricted T-cell responses to factor VIII epitopes in a mild haemophilia A family with missense substitution A2201PHAEMOPHILIA, Issue 102 2010R. A. ETTINGER Summary., An HLA-DRA-DRB1*0101-restricted T-cell epitope in the factor VIII (FVIII) C2 domain occurred in a mild haemophilia A patient with missense substitution FVIII-A2201P. His T cells responded to synthetic peptides FVIII2186,2205 and FVIII2194,2213 (J Thromb Haemost 2007; 5: 2399). T cells from family members with genotype FVIII-A2201P were analysed to determine if FVIII-specific T cells occur in individuals with a haemophilic mutation but no clinically significant inhibitor response. Fluorescent MHC class II tetramers corresponding to subjects'HLA-DRB1 types were loaded with 20-mer peptides and utilized to label antigen-specific CD4+ T cells. T-cell responses to peptides spanning the FVIII-C2 sequence were evaluated. T cells recognizing specific peptides were cloned, and antigen specificity was verified by proliferation assays. Plasma and/or purified IgG samples were tested for FVIII inhibitory activity. CD4+ T cells and T-cell clones from two brothers who shared the DRB1*0101 allele responded to FVIII2194,2213. A haemophilic cousin's HLA-DRA-DRB1*1104-restricted response to FVIII2202,2221 was detected only when CD4+CD25+ cells were depleted. A great uncle and two obligate carriers had no detectable FVIII-C2-specific T cells. Concentrated IgG from the brother without a clinical inhibitor response showed a low-titre FVIII inhibitor. FVIII-specific T cells and inhibitory IgG were found in a previously infused, haemophilic subject who had a sub-clinical FVIII inhibitor. CD4+CD25+ depleted T cells from a non-infused haemophilic cousin recognized an overlapping FVIII epitope, indicating a latent HLA-DRA-DRB1*1104-restricted T-cell response to FVIII. Specific T-cell responses to FVIII can occur without clinically significant inhibitors. [source] Role of estrogenic compounds (diethylstibestrol, 17,-estradiol, and bisphenol A) in the phosphorylation of substrate by protein kinase C,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2009Jeong-Hun Kang Abstract Estrogenic compounds can activate protein kinase C (PKC), which is a calcium and phospholipid-dependent serine/threonine kinase. In the present study, we investigated the role of 17,-estradiol (E2), diethylstibestrol (DES), and bisphenol A (BPA) in the phosphorylation of substrate by PKC, using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The level of phosphorylated peptide was low in the absence of phosphatidylserine (PS). Moreover, reduction of phosphorylation ratios was identified in the presence of diacylglycerol (DAG) and Ca2+ or PS and Ca2+ after adding E2, DES, and BPA. However, no change in phosphorylation ratios was found in the presence of DAG and PS. Addition of E2, DES, and BPA also had no influence on the phosphorylation reaction of substrate by cell or tissue lysate samples. Our study suggests that E2, DES, and BPA can bind to the C2 domain of PKC, but have no effects on the phosphorylation reaction of substrates in the presence of DAG and PS. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:318,323, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20294 [source] Lipid binding region (2303,2332) is involved in aggregation of recombinant human FVIII (rFVIII),JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2005Karthik Ramani Abstract Factor VIII (FVIII) is a multi-domain protein that is important in the clotting cascade. Its deficiency causes Hemophilia A, a bleeding disorder. The unfolding of protein domains can lead to physical instability such as aggregation, and hinder their use in replacement therapy. It has been shown that the aggregation of rFVIIII is initiated by small fluctuations in the protein's tertiary structure (Grillo et al., 2001, Biochemistry 40:586,595). We have investigated the domain(s) involved in the initiation of aggregation using circular dichroism (CD), size exclusion chromatography (SEC), fluorescence anisotropy, domain specific antibody binding, and clotting activity studies. The studies indicated that aggregation may be initiated as a result of conformational change in the C2 domain encompassing the lipid-binding region (2303,2332). The presence of O -phospho- L -Serine (OPLS), which binds to the lipid-binding region of FVIII, prevented aggregation of the protein. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1288,1299, 2005 [source] Human CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIIIJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2003M. T. Reding Summary., Approximately 25% of severe hemophilia A patients develop antibodies (Ab) that neutralize the procoagulant function of factor (F)VIII (inhibitors). Autoimmune FVIII inhibitors may develop in individuals without congenital FVIII deficiency and cause acquired hemophilia. Low titers of anti-FVIII Ab may be present in hemophilia A patients without inhibitors and in healthy blood donors. FVIII-specific CD4+ T-cells drive the synthesis of anti-FVIII Ab. We examined the epitope repertoire of CD4+ T-cells from 15 healthy subjects, 10 hemophilia A patients without inhibitors, 11 hemophilia A patients with inhibitors, and six acquired hemophilia patients. Blood CD4+ T-cells were challenged in proliferation assays with a panel 16 overlapping synthetic peptides, spanning the sequence of the FVIII C2 domain. The sequence region 2291,2330 contained the most frequently and strongly recognized peptides in each of the four subject groups. Crystallographic B factor data and the location of these peptides within the three-dimensional structure of the C2 domain confirm that this region has a high degree of solvent exposure and flexibility within the peptide backbone, which are structural features typical of immunodominant universal CD4+ epitopes. Furthermore, this sequence region overlaps inhibitor-binding sites, suggesting that CD4+ T-cells recognizing peptide sequences within this region might be involved in inhibitor synthesis. The sequence regions 2191,2210 (recognized strongly by each study group except hemophilia A patients with inhibitors) and 2241,2290 (recognized primarily by acquired hemophilia patients and healthy subjects) share the same structural features, and also overlap inhibitor-binding sites. Although similar, there appear to be important differences in the CD4+ epitope repertoires of congenital and acquired hemophilia patients. [source] Autoinhibitory regulation of soluble adenylyl cyclaseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006James A. Chaloupka Abstract Soluble adenylyl cyclase is an evolutionarily conserved bicarbonate sensor that plays a crucial role in cAMP dependent processes that occur during mammalian fertilization. sAC protein is expressed at the highest levels in male germ cells, and is found to occur as one of two known isoforms: a truncated protein (sACt) that consists almost exclusively of the two conserved catalytic domains (C1 and C2), and a full-length form (sACfl) that contains an additional noncatalytic C-terminal region. Several studies suggested sACt was more active than sACfl. We now demonstrate that the specific activity of sACt is at least 10-fold higher than the specific activity of sACfl. Using deletion analysis and a novel genetic screen to identify activating mutations, we uncovered an autoinhibitory region just C-terminal to the C2 domain. Kinetic analysis of purified recombinant sAC revealed this autoinhibitory domain functions to lower the enzyme's Vmax without altering its affinity for substrate or regulation by any of the known modulators of sAC activity. Our results identify an additional regulatory mechanism specific to the sACfl isoform. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Von Willebrand factor protects the Ca2+ -dependent structure of the factor VIII light chainBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2009Masahiro Takeyama Summary We have recently reported that cation-exchange iminodiacetate resin completely inactivated factor VIII (FVIII) by direct deprivation of metal ions, predominantly Ca2+, from its molecules, and that von Willebrand factor (VWF) protected FVIII antigen from resin-induced degradation. The present study was further developed to investigate this mechanism. Western blotting analysis and enzyme-linked immunosorbent assay showed that the antigenic structure of the FVIII light chain, especially the C2 domain, was completely impaired by the resin, whilst that of the heavy chain was little affected. However, the complex formation with VWF protected the C2 domain from the resin-induced degradation. The resin-treated C2 domain failed to interact with VWF and phospholipid. Furthermore, the addition of Ca2+ competitively blocked the resin-induced impairment of the C2 domain structure. These results demonstrate that VWF protects the Ca2+ -dependent conformational structure of the FVIII light chain, especially the C2 domain, and may indicate that the C2 domain contains the Ca2+ -binding site(s). [source] The Dirichlet problem for the minimal surface system in arbitrary dimensions and codimensionsCOMMUNICATIONS ON PURE & APPLIED MATHEMATICS, Issue 2 2004Mu-Tao Wang Let , be a bounded C2 domain in ,n and , ,, , ,m be a continuous map. The Dirichlet problem for the minimal surface system asks whether there exists a Lipschitz map f : , , ,m with f|,, = , and with the graph of f a minimal submanifold in ,n+m. For m = 1, the Dirichlet problem was solved more than 30 years ago by Jenkins and Serrin [12] for any mean convex domains and the solutions are all smooth. This paper considers the Dirichlet problem for convex domains in arbitrary codimension m. We prove that if , : ¯, , ,m satisfies 8n, sup, |D2,| + ,2 sup,, |D,| < 1, then the Dirichlet problem for ,|,, is solvable in smooth maps. Here , is the diameter of ,. Such a condition is necessary in view of an example of Lawson and Osserman [13]. In order to prove this result, we study the associated parabolic system and solve the Cauchy-Dirichlet problem with , as initial data. © 2003 Wiley Periodicals, Inc. [source] The Freud-1/CC2D1A family: Transcriptional regulators implicated in mental retardationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Anastasia Rogaeva Abstract The CC2D1A gene family consists of two homologous genes, Freud-1/CC2D1A and Freud-2/CC2D1B, that share conserved domains, including several DM14 domains that are specific to this protein family, a C-terminal helix-loop-helix domain, and a C2 calcium-dependent phospholipid binding domain. Although the function of Freud-2 is unknown, Freud-1 has been shown to function as a transcriptional repressor of the serotonin-1A receptor gene that binds to a novel DNA element (FRE, 5,-repressor element). The DNA binding and repressor activities of Freud-1 are inhibited by calcium-calmodulin-dependent protein kinase. Recently, a deletion in the CC2D1A gene has been linked to nonsyndromic mental retardation. This deletion results in the truncation of the helix-loop-helix DNA binding and the C2 domains, crucial for Freud-1 repressor activity, and hence is predicted to generate an inactive or weakly dominant negative protein. The possible mechanisms by which inactivation of Freud-1 could lead to abnormal cortical development and cognitive impairment and the potential roles of Freud-1 gene targets are discussed. © 2007 Wiley-Liss, Inc. [source] Purification, crystallization and X-ray diffraction analysis of human synaptotagmin 1 C2A-C2BACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2006Miguel Montes Synaptotagmin acts as the Ca2+ sensor for neuronal exocytosis. The cytosolic domain of human synaptotagmin 1 is composed of tandem C2 domains: C2A and C2B. These C2 domains modulate the interaction of synaptotagmin with the phospholipid bilayer of the presynaptic terminus and effector proteins such as the SNARE complex. Human synaptotagmin C2A-C2B has been expressed as a glutathione- S -transferase fusion protein in Escherichia coli. The purification, crystallization and preliminary X-ray analysis of this protein are reported here. The crystals diffract to 2.7,Å and belong to the orthorhombic space group P212121, with unit-cell parameters a = 82.37, b = 86.31, c = 140.2,Å. From self-rotation function analysis, there are two molecules in the asymmetric unit. The structure determination of the protein using this data is ongoing. [source] | ||||||||||||||||