RESUMO
Factor H (FH)-related proteins are a group of partly characterized complement proteins thought to promote complement activation by competing with FH in binding to surface-bound C3b. Among them, FH-related protein 1 (FHR-1) is remarkable because of its association with atypical hemolytic uremic syndrome (aHUS) and other important diseases. Using a combination of biochemical, immunological, nuclear magnetic resonance, and computational approaches, we characterized a series of FHR-1 mutants (including 2 associated with aHUS) and unraveled the molecular bases of the so-called deregulation activity of FHR-1. In contrast with FH, FHR-1 lacks the capacity to bind sialic acids, which prevents C3b-binding competition between FH and FHR-1 in host-cell surfaces. aHUS-associated FHR-1 mutants are pathogenic because they have acquired the capacity to bind sialic acids, which increases FHR-1 avidity for surface-bound C3-activated fragments and results in C3b-binding competition with FH. FHR-1 binds to native C3, in addition to C3b, iC3b, and C3dg. This unexpected finding suggests that the mechanism by which surface-bound FHR-1 promotes complement activation is the attraction of native C3 to the cell surface. Although C3b-binding competition with FH is limited to aHUS-associated mutants, all surface-bound FHR-1 promotes complement activation, which is delimited by the FHR-1/FH activity ratio. Our data indicate that FHR-1 deregulation activity is important to sustain complement activation and C3 deposition at complement-activating surfaces. They also support that abnormally elevated FHR-1/FH activity ratios would perpetuate pathological complement dysregulation at complement-activating surfaces, which may explain the association of FHR-1 quantitative variations with diseases.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Proteínas Sanguíneas/química , Complemento C3/química , Mutação , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Ligação ProteicaRESUMO
Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.
Assuntos
Subpopulações de Linfócitos B/citologia , Linfócitos T CD4-Positivos/imunologia , Catepsina L/metabolismo , Diferenciação Celular , Ativação do Complemento/fisiologia , Complemento C3/metabolismo , Homeostase/fisiologia , Adulto , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Sobrevivência Celular/imunologia , Criança , Complemento C3/imunologia , Complemento C3a/metabolismo , Complemento C3b/metabolismo , Regulação da Expressão Gênica/imunologia , HumanosRESUMO
The complement factor H (FH) mutation R1210C, which was described in association with atypical hemolytic uremic syndrome (aHUS), also confers high risk of age-related macular degeneration (AMD) and associates with C3 glomerulopathy (C3G). To reveal the molecular basis of these associations and to provide insight into what determines the disease phenotype in FH-R1210C carriers, we identified FH-R1210C carriers in our aHUS, C3G, and AMD cohorts. Disease status, determined in patients and relatives, revealed an absence of AMD phenotypes in the aHUS cohort and, vice versa, a lack of renal disease in the AMD cohort. These findings were consistent with differences in the R1210C-independent overall risk for aHUS and AMD between mutation carriers developing one pathology or the other. R1210C is an unusual mutation that generates covalent complexes between FH and HSA. Using purified FH proteins and surface plasmon resonance analyses, we demonstrated that formation of these FH-HSA complexes impairs accessibility to all FH functional domains. These data suggest that R1210C is a unique C-terminal FH mutation that behaves as a partial FH deficiency, predisposing individuals to diverse pathologies with distinct underlying pathogenic mechanisms; the final disease outcome is then determined by R1210C-independent genetic risk factors.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C3 , Nefropatias/genética , Glomérulos Renais , Degeneração Macular/genética , Mutação , Fator H do Complemento/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Síndrome Hemolítico-Urêmica Atípica/terapia , Complemento C3b/metabolismo , Fator B do Complemento/metabolismo , Hemoglobinúria Paroxística/terapia , Miastenia Gravis Autoimune Experimental/terapia , Animais , Anticorpos Bloqueadores/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Síndrome Hemolítico-Urêmica Atípica/imunologia , Bovinos , Linhagem Celular , C3 Convertase da Via Alternativa do Complemento/metabolismo , Fator B do Complemento/genética , Fator B do Complemento/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Hemoglobinúria Paroxística/imunologia , Humanos , Camundongos , Camundongos Knockout , Miastenia Gravis Autoimune Experimental/imunologia , Ligação Proteica/efeitos dos fármacos , Coelhos , Ratos , Ratos Endogâmicos Lew , OvinosAssuntos
Linfócitos B/metabolismo , Complemento C3/metabolismo , Memória Imunológica , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Complemento C3/imunologia , Feminino , Humanos , Masculino , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
Thrombotic microangiopathy (TMA) has different etiological causes, and not all of them are well understood. In atypical hemolytic uremic syndrome (aHUS), the TMA is caused by the complement dysregulation associated with pathogenic mutations in complement components and its regulators. Here, we describe a pediatric patient with aHUS in whom the relatively benign course of the disease confused the initial diagnosis. A previously healthy 8-year-old boy developed jaundice, hematuria, hemolytic anemia, thrombopenia, and mild acute kidney injury (AKI) in the context of a diarrhea without hypertension nor oliguria. Spontaneous and complete recovery was observed from the third day of admission. Persistent low C3 plasma levels after recovery raised the suspicion for aHUS, which prompted clinicians to discard the initial diagnosis of Shigatoxin-associated HUS (STEC-HUS). A thorough genetic and molecular study of the complement revealed the presence of an isolated novel pathogenic C3 mutation. The relatively benign clinical course of the disease as well as the finding of a de novo pathogenic C3 mutation are remarkable aspects of this case. The data are discussed to illustrate the benefits of identifying the TMA etiological factor and the relevant contribution of the MCP aHUS risk polymorphism to the disease severity.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C3/genética , Proteína Cofatora de Membrana/genética , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Criança , Humanos , Masculino , Mutação , Linhagem , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Introduction: The majority of kidneys used for transplantation are retrieved from brain-dead organ donors. In brain death, the irreversible loss of brain functions results in hemodynamic instability, hormonal changes and immunological activation. Recently, brain death has been shown to cause activation of the complement system, which is adversely associated with renal allograft outcome in recipients. Modulation of the complement system in the brain-dead donor might be a promising strategy to improve organ quality before transplantation. This study investigated the effect of an inhibitory antibody against complement factor B on brain death-induced renal inflammation and injury. Method: Brain death was induced in male Fischer rats by inflating a balloon catheter in the epidural space. Anti-factor B (anti-FB) or saline was administered intravenously 20 min before the induction of brain death (n = 8/group). Sham-operated rats served as controls (n = 4). After 4 h of brain death, renal function, renal injury, and inflammation were assessed. Results: Pretreatment with anti-FB resulted in significantly less systemic and local complement activation than in saline-treated rats after brain death. Moreover, anti-FB treatment preserved renal function, reflected by significantly reduced serum creatinine levels compared to saline-treated rats after 4 h of brain death. Furthermore, anti-FB significantly attenuated histological injury, as seen by reduced tubular injury scores, lower renal gene expression levels (>75%) and renal deposition of kidney injury marker-1. In addition, anti-FB treatment significantly prevented renal macrophage influx and reduced systemic IL-6 levels compared to saline-treated rats after brain death. Lastly, renal gene expression of IL-6, MCP-1, and VCAM-1 were significantly reduced in rats treated with anti-FB. Conclusion: This study shows that donor pretreatment with anti-FB preserved renal function, reduced renal damage and inflammation prior to transplantation. Therefore, inhibition of factor B in organ donors might be a promising strategy to reduce brain death-induced renal injury and inflammation.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Morte Encefálica , Fator B do Complemento/antagonistas & inibidores , Nefropatias/tratamento farmacológico , Animais , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Nefropatias/genética , Nefropatias/imunologia , Transplante de Rim , Masculino , Ratos Endogâmicos F344RESUMO
The ubiquitous and highly abundant glycolytic enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pivotal for the energy and carbon metabolism of most organisms, including human pathogenic bacteria. For bacteria that depend mostly on glycolysis for survival, GAPDH is an attractive target for inhibitor discovery. The availability of high-resolution structures of GAPDH from various pathogenic bacteria is central to the discovery of new antibacterial compounds. We have determined the X-ray crystal structures of two new GAPDH enzymes from Gram-positive bacterial pathogens, Streptococcus pyogenes and Clostridium perfringens. These two structures, and the recent structure of Atopobium vaginae GAPDH, reveal details in the active site that can be exploited for the design of novel inhibitors based on naturally occurring molecules. Two such molecules, anacardic acid and curcumin, have been found to counter bacterial infection in clinical settings, although the cellular targets responsible for their antimicrobial properties remain unknown. We show that both anacardic acid and curcumin inhibit GAPDH from two bacterial pathogens through uncompetitive and non-competitive mechanisms, suggesting GAPDH as a relevant pharmaceutical target for antibacterial development. Inhibition of GAPDH by anacardic acid and curcumin seems to be unrelated to the immune evasion function of pathogenic bacterial GAPDH, since neither natural compound interfere with binding to the human C5a anaphylatoxin.
RESUMO
C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b.
Assuntos
Complemento C3b/química , Complemento C3b/metabolismo , Degeneração Macular/genética , Modelos Moleculares , Animais , Complemento C3b/genética , Predisposição Genética para Doença , Humanos , Microscopia Eletrônica , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Domínios Proteicos/fisiologia , Estabilidade Proteica , TermodinâmicaRESUMO
The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio-ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio-ester-containing domain (TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3-convertase of the alternative pathway (AP). In addition, the displacement of the TED and its interaction with the macroglobulin 1 (MG1) domain generates an extended surface in C3b where the complement regulators factor H (FH), decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1) can bind, mediating accelerated decay of the AP C3-convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED can display a collection of conformations around the MG ring, as well as a few specialized positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation by affecting the interaction with regulators.
Assuntos
Ativação do Complemento , Complemento C3/metabolismo , Complemento C3b/metabolismo , Modelos Moleculares , Complemento C3/química , Complemento C3b/química , Humanos , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , ProteóliseRESUMO
Oxidative stress, a risk factor in the pathophysiology of Alzheimer's disease, is intimately associated with aging. We previously reported that the X-XOD free radical generating system acts as a modulator of lipid metabolism and a mild inducer of apoptotic death. Using the same cell model, the present study examines the metabolism/processing of the amyloid precursor protein (APP). Prior to inducing cell death, X-XOD promoted the secretion of α-secretase-cleaved soluble APP (sAPPα) and increased the level of APP carboxy-terminal fragments produced by α and γ secretase (αCTF and γCTF/AICD). In contrast, it reduced the activity of ß-secretase and the level of secreted Aß. The present results indicate that mild oxidative stress maintained throughout culturing regulates APP metabolism/processing in SK-N-MC human neuroblastoma cells.