RESUMO
Phospholipids are important membrane components involved in diverse biological activities ranging from cell signaling to infection by viral particles. A thorough understanding of protein-phospholipid interaction dynamics is thus crucial for deciphering basic cellular processes as well as for targeted drug discovery. For any specific phospholipid-protein binding experiment, various groups have reported different binding constants, which are strongly dependent on applied conditions of interactions. Here, we report a method for accurate determination of the binding affinity and specificity between proteins and phospholipids using a model interaction between PLC-δ1/PH and phosphoinositide phospholipid PtdIns(4,5)P2. We developed an accurate Force Distance Spectroscopy (FDS)-based assay and have attempted to resolve the problem of variation in the observed binding constant by directly measuring the bond force. We confirm the FDS findings of a high bond strength of â¼0.19 ± 0.04 nN by Surface Plasmon Resonance (SPR) data analysis, segregating non-specific interactions, which show a significantly lower K(D) suggesting tight binding.
Assuntos
Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Microscopia de Força Atômica/métodos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Cinética , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b-9. Elafin was also found to promote angiogenesis through activation of the extracellular signal-regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.
Assuntos
Ciclosporina/farmacologia , Sinergismo Farmacológico , Elafina/farmacologia , Rejeição de Enxerto/prevenção & controle , Microvasos/patologia , Transplante de Órgãos/efeitos adversos , Traqueia/transplante , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Complemento C3/metabolismo , Quimioterapia Combinada , Endotélio Vascular/efeitos dos fármacos , Feminino , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Terapia de Imunossupressão , Elastase de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microcirculação , Microvasos/efeitos dos fármacos , Perfusão , Inibidores de Proteases/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
A number of distinct beta-amyloid (Abeta) variants or multimers have been implicated in Alzheimer's disease (AD), and antibodies recognizing such peptides are in clinical trials. Humans have natural Abeta-specific antibodies, but their diversity, abundance, and function in the general population remain largely unknown. Here, we demonstrate with peptide microarrays the presence of natural antibodies against known toxic Abeta and amyloidogenic non-Abeta species in plasma samples and cerebrospinal fluid of AD patients and healthy controls aged 21-89 years. Antibody reactivity was most prominent against oligomeric assemblies of Abeta and pyroglutamate or oxidized residues, and IgGs specific for oligomeric preparations of Abeta1-42 in particular declined with age and advancing AD. Most individuals showed unexpected antibody reactivities against peptides unique to autosomal dominant forms of dementia (mutant Abeta, ABri, ADan) and IgGs isolated from plasma of AD patients or healthy controls protected primary neurons from Abeta toxicity. Aged vervets showed similar patterns of plasma IgG antibodies against amyloid peptides, and after immunization with Abeta the monkeys developed high titers not only against Abeta peptides but also against ABri and ADan peptides. Our findings support the concept of conformation-specific, cross-reactive antibodies that may protect against amyloidogenic toxic peptides. If a therapeutic benefit of Abeta antibodies can be confirmed in AD patients, stimulating the production of such neuroprotective antibodies or passively administering them to the elderly population may provide a preventive measure toward AD.