RESUMO
The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
Assuntos
Complemento C3a/metabolismo , DNA/metabolismo , Interferon gama/metabolismo , Oligonucleotídeos/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Complemento C3a/imunologia , DNA/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Knockout , Oligonucleotídeos/imunologia , Ligação Proteica , Receptor para Produtos Finais de Glicação Avançada/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA(63), a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA(63) heptamer and for one residue (E126) on LF(N), the 30 kDa N-terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LF(N) was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time-resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488-labeled LF(N), whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LF(N) binds near the top of the ring-shaped (PA(63))(7) heptamer.
Assuntos
Antígenos de Bactérias , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Animais , Toxinas Bacterianas/genética , Sítios de Ligação , Cricetinae , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Hidrazinas/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Naftalenossulfonatos/química , Conformação ProteicaRESUMO
The structure-based design and synthesis of a series of novel biphenyl sulfonamide carboxylic acids as potent MMP-13 inhibitors with selectivity over MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-14, Aggrecanase 1, and TACE are described.