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Protection from complement-dependent cytotoxicity by NF-(B p65
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Abstracts / Molecular Immunology 47 (2010) 2198–2294
explore a full range of solution conditions. The common Y402H variant of FH displayed lower binding affinity for CRP, and this may provide a mechanism for the known association of this polymorphism with increased risks of age-related macular degeneration. The second CRP binding site on FH suggests that CRP binding may also be involved with atypical haemolytic uraemic syndrome. doi:10.1016/j.molimm.2010.05.197 27 Protection from complement-dependent cytotoxicity by NF-(B p65 Dana Gancz, Zvi Fishelson Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Complement-dependent cytotoxicity (CDC) is regulated by intracellular signaling events that are poorly characterized. The role of NF-(B in cell protection from the complement membrane attack complex C5b-9 was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 (RelA) subunit of NF-(B or the IKK( subunit of the NF-(B activator complex (mouse embryonic fibroblasts isolated from p65(/( or IKK((/( mice) and in cells treated with p65 siRNA. In contrast, reconstitution of p65 into p65(/( cells and over-expression of p65 in wildtype cells reduced their sensitivity to CDC. Pretreatment with the IKK inhibitor PS1145 enhanced CDC of wildtype but not of p65(/( cells. Deletion of the nuclear localization signal in p65 had no effect on its anti-CDC activity although it blocked its nuclear translocation. In agreement, cell treatment with C5b-9 neither activated nuclear translocation of p65 nor degradation of I(B, the Inhibitor of NF-(B. The effect of p65 on the c-Jun N-terminal kinase (JNK) death signaling pathway activated by C5b-9 was next examined. The JNK inhibitor SP600125 reduced CDC in p65 +/+ and p65 (/( cells but was much more effective in p65 (/( cells. On the other hand, a JNK phosphatase inhibitor, phenylarsine oxide, enhanced CDC in wildtype cells but not in p65(/( cells. It is suggested that NF-(B activates the JNK phosphatase suppressing CDC. In conclusion, NF-(B mediates cell resistance to CDC probably by inhibiting the pro-lytic JNK pathway. doi:10.1016/j.molimm.2010.05.198 270 Oligomeric solution structures of factor H in the presence of heparin fragments Sanaullah Khan, Ami Miller, Jayesh Gor, Barbara Mulloy, Stephen J. Perkins Department of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London, WC1E 6BT, UK Factor H (FH) is a major serum regulator of complement, and FH is genetically associated with the development of age-related macular degeneration in the elderly. Binding studies of FH to heparin are used as a model for FH binding to heparan sulphate which is present on host cell surfaces and in the extracellular matrix. Heparin binds to sites found on at least the SCR-6/8 and SCR-19/20 regions of FH. While the solution structure of FH has been determined using X-ray solution scattering and analytical ultracentrifugation, the effects of heparin binding to intact FH are not yet known. To assess this, FH was studied in the presence of a range of purified heparin fragments by X-ray solution scattering and analytical ultracentrifugation. X-ray solution scattering data for FH in the presence of the smallest heparin fragments showed little effect on the radius
of gyration RG values. Dramatic increases in the RG values were seen with the larger heparin sizes. The maximum lengths of FHheparin complexes indicated that a series of compact complexes were formed. By analytical ultracentrifugation, the larger heparin fragments were observed to promote strong FH oligomer formation. Molecular modelling of these FH-heparin complexes based on the X-ray and ultracentrifuge data accounted for the formation of these oligomers. Further analyses of the heparin complex with the SCR-6/8 fragment of FH reveal complementary oligomer formation in the presence of heparin. These conformational changes and selfaggregation results provide the first molecular picture of how FH interacts with host cell surfaces and are directly relevance for an understanding of the sub-retinal pigmental endothelial deposits that form in Bruch’s membrane during the development of agerelated macular degeneration. doi:10.1016/j.molimm.2010.05.199 271 Coordinate activation of the factor B scissile bond with the Mg(2+)-dependent c3b-binding site during assembly of the alternative pathway convertase Dennis E. Hourcade, Lynne M. Mitchell Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110-1093, USA The C3b:factor B complex (C3bB) is an obligatory intermediate in the assembly of the alternative pathway C3 convertase (C3bBb). It is stabilized by a single Mg(2+) cation at the C3b:factor B interface. Crystallographic studies of factor B and of the cobra venom factor/factor B complex, a structural homolog of C3bB, have shown that the peptide bond cleaved by factor D (between Arg234 and Lys235) in the conversion of C3bB to C3bBb, is sequestered by two salt bridges which connect the Arg234 side-chain to the factor B linker region and von Willebrand A (vWA) domain. These observations raise the question as to how the scissile bond is cleaved. By one model, association of factor D with C3bB induces conformational changes that expose the scissile bond. By a second model, supported by EM studies of C3bB, a second C3bB conformation, in which the scissile bond is exposed, occurs spontaneously and independently of factor D. We are examining this issue by site-directed mutagenesis of factor B. Using SPR methodology, we have found that mutations of Arg234 (which preclude both salt bridges) result in up to a 10-fold increase of Mg(2+)-dependent C3b-binding. Most of this effect is replicated by the single E446 substitution that precludes the vWA salt bridge and results in active factor B that is particularly sensitive to cleavage at or near the scissile bond by non-specific proteases. Combining the new substitutions with previously described substitutions (D254G and N260D) results in C3bB complexes with a half-life in excess of 10 h. These observations account for the complex dissociation kinetics previously reported for C3bB and lend strong support to a model in which C3bB cleavage is mediated by a factor D-sensitive form that is stabilized by coordinate activation of the Mg(2+)-dependent binding site. While this “activation” form of C3bB appears to occur spontaneously (model 2), association of the “loading” form of C3bB with factor D might also promote its occurrence. doi:10.1016/j.molimm.2010.05.200
Abstracts / Molecular Immunology 47 (2010) 2198–2294
explore a full range of solution conditions. The common Y402H variant of FH displayed lower binding affinity for CRP, and this may provide a mechanism for the known association of this polymorphism with increased risks of age-related macular degeneration. The second CRP binding site on FH suggests that CRP binding may also be involved with atypical haemolytic uraemic syndrome. doi:10.1016/j.molimm.2010.05.197 27 Protection from complement-dependent cytotoxicity by NF-(B p65 Dana Gancz, Zvi Fishelson Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Complement-dependent cytotoxicity (CDC) is regulated by intracellular signaling events that are poorly characterized. The role of NF-(B in cell protection from the complement membrane attack complex C5b-9 was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 (RelA) subunit of NF-(B or the IKK( subunit of the NF-(B activator complex (mouse embryonic fibroblasts isolated from p65(/( or IKK((/( mice) and in cells treated with p65 siRNA. In contrast, reconstitution of p65 into p65(/( cells and over-expression of p65 in wildtype cells reduced their sensitivity to CDC. Pretreatment with the IKK inhibitor PS1145 enhanced CDC of wildtype but not of p65(/( cells. Deletion of the nuclear localization signal in p65 had no effect on its anti-CDC activity although it blocked its nuclear translocation. In agreement, cell treatment with C5b-9 neither activated nuclear translocation of p65 nor degradation of I(B, the Inhibitor of NF-(B. The effect of p65 on the c-Jun N-terminal kinase (JNK) death signaling pathway activated by C5b-9 was next examined. The JNK inhibitor SP600125 reduced CDC in p65 +/+ and p65 (/( cells but was much more effective in p65 (/( cells. On the other hand, a JNK phosphatase inhibitor, phenylarsine oxide, enhanced CDC in wildtype cells but not in p65(/( cells. It is suggested that NF-(B activates the JNK phosphatase suppressing CDC. In conclusion, NF-(B mediates cell resistance to CDC probably by inhibiting the pro-lytic JNK pathway. doi:10.1016/j.molimm.2010.05.198 270 Oligomeric solution structures of factor H in the presence of heparin fragments Sanaullah Khan, Ami Miller, Jayesh Gor, Barbara Mulloy, Stephen J. Perkins Department of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London, WC1E 6BT, UK Factor H (FH) is a major serum regulator of complement, and FH is genetically associated with the development of age-related macular degeneration in the elderly. Binding studies of FH to heparin are used as a model for FH binding to heparan sulphate which is present on host cell surfaces and in the extracellular matrix. Heparin binds to sites found on at least the SCR-6/8 and SCR-19/20 regions of FH. While the solution structure of FH has been determined using X-ray solution scattering and analytical ultracentrifugation, the effects of heparin binding to intact FH are not yet known. To assess this, FH was studied in the presence of a range of purified heparin fragments by X-ray solution scattering and analytical ultracentrifugation. X-ray solution scattering data for FH in the presence of the smallest heparin fragments showed little effect on the radius
of gyration RG values. Dramatic increases in the RG values were seen with the larger heparin sizes. The maximum lengths of FHheparin complexes indicated that a series of compact complexes were formed. By analytical ultracentrifugation, the larger heparin fragments were observed to promote strong FH oligomer formation. Molecular modelling of these FH-heparin complexes based on the X-ray and ultracentrifuge data accounted for the formation of these oligomers. Further analyses of the heparin complex with the SCR-6/8 fragment of FH reveal complementary oligomer formation in the presence of heparin. These conformational changes and selfaggregation results provide the first molecular picture of how FH interacts with host cell surfaces and are directly relevance for an understanding of the sub-retinal pigmental endothelial deposits that form in Bruch’s membrane during the development of agerelated macular degeneration. doi:10.1016/j.molimm.2010.05.199 271 Coordinate activation of the factor B scissile bond with the Mg(2+)-dependent c3b-binding site during assembly of the alternative pathway convertase Dennis E. Hourcade, Lynne M. Mitchell Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110-1093, USA The C3b:factor B complex (C3bB) is an obligatory intermediate in the assembly of the alternative pathway C3 convertase (C3bBb). It is stabilized by a single Mg(2+) cation at the C3b:factor B interface. Crystallographic studies of factor B and of the cobra venom factor/factor B complex, a structural homolog of C3bB, have shown that the peptide bond cleaved by factor D (between Arg234 and Lys235) in the conversion of C3bB to C3bBb, is sequestered by two salt bridges which connect the Arg234 side-chain to the factor B linker region and von Willebrand A (vWA) domain. These observations raise the question as to how the scissile bond is cleaved. By one model, association of factor D with C3bB induces conformational changes that expose the scissile bond. By a second model, supported by EM studies of C3bB, a second C3bB conformation, in which the scissile bond is exposed, occurs spontaneously and independently of factor D. We are examining this issue by site-directed mutagenesis of factor B. Using SPR methodology, we have found that mutations of Arg234 (which preclude both salt bridges) result in up to a 10-fold increase of Mg(2+)-dependent C3b-binding. Most of this effect is replicated by the single E446 substitution that precludes the vWA salt bridge and results in active factor B that is particularly sensitive to cleavage at or near the scissile bond by non-specific proteases. Combining the new substitutions with previously described substitutions (D254G and N260D) results in C3bB complexes with a half-life in excess of 10 h. These observations account for the complex dissociation kinetics previously reported for C3bB and lend strong support to a model in which C3bB cleavage is mediated by a factor D-sensitive form that is stabilized by coordinate activation of the Mg(2+)-dependent binding site. While this “activation” form of C3bB appears to occur spontaneously (model 2), association of the “loading” form of C3bB with factor D might also promote its occurrence. doi:10.1016/j.molimm.2010.05.200