RESUMO
Complement component 8gamma (C8γ), a member of the lipocalin protein family, is suggested to act as a carrier protein for various chemicals. Although C8γ has been identified in both humans and rodents for some time, our understanding of the species differences in its chemical binding properties remains limited. In the present study, with the aim to elucidate the potential role of C8γ as a carrier protein in both humans and mice, we conducted a radioligand binding assay to examine the chemical binding properties of human C8γ (hC8γ) and mouse C8γ (mC8γ). Scatchard analysis revealed that [14C]TPT bound to hC8γ with an equilibrium dissociation constant (Kd) of 64.2 ± 32.4 nM, comparable to that of [14C]TPT to mC8γ. Competitive ligand-binding assays demonstrated binding of TPT and TBT to hC8γ, while diphenyltin, dibutyltin, monophenyltin, monobutyltin, and tetrabutyltin did not exhibit binding. These results suggest that for effective binding to C8γ, chemicals must possess substituents of appropriate bulkiness. Further analyses with other group 14 compounds with triphenyl substituents revealed that a central metal atom, rather than a central non-metal or semi-metal atom, is crucial for specific binding to both hC8γ and mC8γ. Overall our findings imply that C8γ may play a role in the physiological or toxicological actions of group 14 metal compounds with tributyl or triphenyl substituents by binding to these chemicals in both humans and mice.
Assuntos
Ligação Proteica , Animais , Humanos , Camundongos , Complemento C8/metabolismo , Complemento C8/química , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Ligação CompetitivaRESUMO
CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming ß-hairpins of C8 to form an intermolecular ß-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 ß-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.
Assuntos
Complemento C9 , Complexo de Ataque à Membrana do Sistema Complemento , Humanos , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Complemento C9/metabolismo , Microscopia Crioeletrônica , Antígenos CD59/metabolismo , Complemento C8/metabolismo , Ativação do ComplementoRESUMO
The complement system plays a crucial role in cognitive impairment in obstructive sleep apnea (OSA). The present study aimed to investigate the connections between complement component 8 gamma (C8G) levels in astrocyte-derived exosomes (ADEs) and cognitive impairment in OSA patients without dementia. This cross-sectional cohort study recruited 274 participants without dementia, including 124 OSA patients with mild cognitive impairment (MCI), 100 OSA patients without MCI, and 50 healthy control subjects. Enrolled participants underwent polysomnography (PSG) evaluation, neuropsychological scale assessment, magnetic resonance imaging scanning, and collection of peripheral blood samples for quantification of complement proteins in ADEs. The findings showed higher C8G concentrations in ADEs from OSA patients with MCI than in the controls and OSA without MCI group. Logistic regression analysis suggested that C8G levels in ADEs were independently associated with MCI in OSA patients. Multivariable linear regression analysis demonstrated that C8G levels in ADEs were significantly correlated with global cognitive scores and all cognitive subdomain scores after adjusting for demographic factors (age, sex, education), vascular risk factors (Body mass index, history of hypertension, diabetes, dyslipidemia), depressive symptoms measures, and apnea-hypopnea index (AHI) values. The levels of C8G were linearly positively related to the white matter hyperintensity (WMH) volumes in Pearson's correlation analysis. Our research confirmed that C8G levels are significantly associated with cognitive impairment in OSA patients, which paves the way for novel therapeutic targets for neurocognitive dysfunction progression in OSA patients in the future.
Assuntos
Disfunção Cognitiva , Demência , Exossomos , Apneia Obstrutiva do Sono , Humanos , Complemento C8 , Estudos Transversais , Astrócitos/patologia , Exossomos/patologia , Apneia Obstrutiva do Sono/complicações , Demência/complicaçõesRESUMO
The cytolytic activity of the membrane attack complex (MAC) is pivotal in the complement-mediated elimination of pathogens. Terminal complement pathway (TCP) genes encode the proteins that form the MAC. Although the TCP genes are well conserved within most vertebrate species, the early evolution of the TCP genes is poorly understood. Based on the comparative genomic analysis of the early evolutionary history of the TCP homologs, we evaluated four possible scenarios that could have given rise to the vertebrate TCP. Currently available genomic data support a scheme of complex sequential protein domain gains that may be responsible for the birth of the vertebrate C6 gene. The subsequent duplication and divergence of this vertebrate C6 gene formed the C7, C8α, C8ß, and C9 genes. Compared to the widespread conservation of TCP components within vertebrates, we discovered that C9 has disintegrated in the genomes of galliform birds. Publicly available genome and transcriptome sequencing datasets of chicken from Illumina short read, PacBio long read, and Optical mapping technologies support the validity of the genome assembly at the C9 locus. In this study, we have generated a > 120X coverage whole-genome Chromium 10x linked-read sequencing dataset for the chicken and used it to verify the loss of the C9 gene in the chicken. We find multiple CR1 (chicken repeat 1) element insertions within and near the remnant exons of C9 in several galliform bird genomes. The reconstructed chronology of events shows that the CR1 insertions occurred after C9 gene loss in an early galliform ancestor. Loss of C9 in galliform birds, in contrast to conservation in other vertebrates, may have implications for host-pathogen interactions. Our study of C6 gene birth in an early vertebrate ancestor and C9 gene death in galliform birds provides insights into the evolution of the TCP.
Assuntos
Complemento C8 , Complemento C9 , Animais , Galinhas/genética , Complemento C6 , Complemento C7/genética , Complemento C8/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/genética , Proteínas do Sistema Complemento/genética , GenomaRESUMO
Diagnosis of primary complement deficiencies requires a high index of suspicion. Thus, susceptible patients are often underdiagnosed and untreated. Here, we present a multicenter experience with two novel inborn errors of the classical complement system. This is a retrospective multicenter analysis of computerized medical records of children (<18 years) admitted in the period between 2012 and 2018 at Shaare Zedek Medical Center in Jerusalem and Edmond and Lily Safra Children's Hospital, Tel-Hashomer Medical Center, in Ramat Gan, Israel. Patients were genetically diagnosed by a complementary immune workup. We identified 5 patients (3 males) from four different families harboring two novel mutations in the complement components C6-C8. Genetic mutations were identified by whole-exome sequencing or by sequencing of the coding exons of a single gene based on the findings in the immune workup. Clinical manifestations consisted of meningitis with or without meningococcemia. The immune workup demonstrated nearly absent levels of CH50, compatible with a complement pathway defect. Diagnosis delay ranged between 0 and 30 years. CONCLUSION: Awareness of risk factors for primary complement deficiencies, even at the first infectious episode, should facilitate prompt immune and genetic workup, commencing diagnosis and proper treatment for the patient and family. WHAT IS KNOWN: ⢠Deficiencies in the classical terminal complement components increase susceptibility to invasive meningococcal infections. ⢠Recurrent meningococcal infections mandate a diagnostic workup of the complement system. WHAT IS NEW: ⢠Genetic workup can be utilized for prompt diagnosis of complement deficiencies. ⢠High rates of consanguinity, even in the presence of a single meningococcal infection, should promote immune and genetic workups.
Assuntos
Meningite Meningocócica , Infecções Meningocócicas , Neisseria meningitidis , Criança , Complemento C6 , Complemento C8/genética , Proteínas do Sistema Complemento/genética , Feminino , Doenças da Deficiência Hereditária de Complemento , Humanos , Masculino , Estudos RetrospectivosRESUMO
Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane ß-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complemento C8/química , Complemento C8/metabolismo , Complemento C9/química , Complemento C9/imunologia , Microscopia Crioeletrônica , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
Complement deficient patients are susceptible to rare meningococcal serogroups. A 6-year-old girl presented with serogroup Z meningitis. This led to identification of a C8 deficiency. The MenB-4C vaccine induced cross-reactive antibodies to serogroup Z and increased in vitro opsonophagocytic killing and may thus protect complement deficient patients.
Assuntos
Complemento C8/deficiência , Proteção Cruzada/imunologia , Meningite/microbiologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Sorogrupo , Anticorpos Antibacterianos/imunologia , Criança , Reações Cruzadas/imunologia , Feminino , Doenças da Deficiência Hereditária de Complemento , Humanos , Meningite/diagnóstico , Meningite/imunologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Neisseria meningitidis/classificação , OpsonizaçãoRESUMO
The complement system is part of the innate immune system that comprises several small proteins activated by sequential cleavages. The majority of these complement components, such as components 3a (C3a) and C5a, are chemotactic and pro-inflammatory. However, in this study, we revealed an inhibitory role of complement component 8 gamma (C8G) in neuroinflammation. In patients with Alzheimer's disease, who exhibit strong neuroinflammation, we found higher C8G levels in brain tissue, CSF, and plasma. Our novel findings also showed that the expression level of C8G increases in the inflamed mouse brain, and that C8G is mainly localized to brain astrocytes. Experiments using recombinant C8G protein and shRNA-mediated knockdown showed that C8G inhibits glial hyperactivation, neuroinflammation, and cognitive decline in acute and chronic animal models of Alzheimer's disease. Additionally, we identified sphingosine-1-phosphate receptor 2 (S1PR2) as a novel interaction protein of C8G and demonstrated that astrocyte-derived C8G interacts with S1PR2 to antagonize the pro-inflammatory action of S1P in microglia. Taken together, our results reveal the previously unrecognized role of C8G as a neuroinflammation inhibitor. Our findings pave the way towards therapeutic containment of neuroinflammation in Alzheimer's disease and related neurological diseases.
Assuntos
Doença de Alzheimer/complicações , Complemento C8/imunologia , Encefalite/imunologia , Doença de Alzheimer/imunologia , Animais , Astrócitos/imunologia , Células Cultivadas , Complemento C8/líquido cefalorraquidiano , Masculino , Camundongos Endogâmicos C57BL , Microglia/imunologia , Subunidades Proteicas/imunologia , Receptores de Esfingosina-1-Fosfato/imunologiaRESUMO
Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.
Assuntos
Quadruplex G/efeitos dos fármacos , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Viroses/tratamento farmacológico , Aminoquinolinas/farmacologia , Complemento C8/genética , Complemento C8/farmacologia , Proteínas de Ligação a DNA/genética , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Genótipo , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 16/ultraestrutura , Papillomavirus Humano 18/efeitos dos fármacos , Papillomavirus Humano 18/ultraestrutura , Humanos , Ligantes , Terapia de Alvo Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Viroses/genética , Viroses/patologiaRESUMO
Complement C8, as a main component of the membrane attack complex, has only been identified in vertebrates. C8 comprises three subunits encoded by individual genes: C8a (alpha chain), C8b (beta chain), and C8g (gamma chain). However, in fish, there have been limited studies on the evolutionary history and systematic function of C8. In the present study, phylogenetic analysis indicated the complete divergence of C8 genes in different fish species. Codon usage bias analysis revealed the evolutionary complexity of C8 genes. Selective pressure analysis found that C8 genes have been affected by negative selection during evolution. Sequence alignment identified the sites that are under selective pressure. The systematic functions of C8 were revealed by gene co-expression and protein-protein interaction (PPI) network analyses. Notably, gene ontology enrichment analysis suggested that C8 proteins in zebrafish function mainly in the neuroendocrine system. Protein structural comparisons showed that putative functional residues and domains were conserved between the C8 subunits of human and grass carp. A preliminary study on the theoretical interaction between C8a and CD59 was performed according to the simulated protein stereo structure. The first functionally-related site was absent in the simulated conformation of the grass carp (Ctenopharyngodon idella) C8a-CD59 protein complex. We speculated that Tyr63 is involved in the functional loss of CD59 binding. The docking of CD59 to four potential sites (Met390, Ser391, Leu392, and Val405) in grass carp C8a was analyzed. The results of the present study provide a deeper understanding of the evolution and function of fish complement C8.
Assuntos
Antígenos CD59/metabolismo , Carpas/metabolismo , Complemento C8/metabolismo , Proteínas de Peixes/metabolismo , Animais , Galinhas , Expressão Gênica/fisiologia , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Filogenia , Especificidade da Espécie , Tartarugas , Xenopus , Peixe-ZebraRESUMO
Complement component 8 γ (C8γ) is a subunit of complement protein 8 (C8), which itself is a subunit of the complement cytolytic membrane attack complex. However, C8γ is also suggested to be a carrier protein for the general clearance of endogenous and exogenous compounds because it belongs to the lipocalin family of small secreted proteins that have the common ability to bind small hydrophobic ligands. Although retinoic acid, a metabolite of vitamin A, has been suggested as a potential ligand of C8γ, it remains unclear which other substances are able to bind to C8γ as ligands. Here, we evaluated the binding affinity of several organotin compounds that are ligands of a receptor of retinoic acid, retinoid X receptor, by using radioligand binding assays. The amount of [14C]triphenyltin (TPT), a tri-substituted organotin, that bound to purified recombinant C8γ was increased with increasing protein concentration, whereas that of [3H]all-trans retinoic acid and [3H]9-cis retinoic acid was unchanged. Scatchard analysis revealed that [14C]TPT bound to C8γ with an equilibrium dissociation constant (Kd) of 56.2 ± 16.2 nM. Non-radiolabeled tributyltin (TBT), another tri-substituted organotin, blocked the binding of [14C]TPT to C8γ in a competitive manner, but non-radiolabeled mono- or di-substituted organotin compounds did not. Together, our present observations indicate that TBT and TPT, but not retinoic acid or mono- or di-substituted organotin compounds, are potent ligands of C8γ, suggesting that C8γ may be involved in the toxicities of these organotin compounds.
Assuntos
Proteínas de Transporte , Complemento C8 , Ligantes , Compostos Orgânicos de Estanho/toxicidade , Compostos de Trialquitina/toxicidade , Ligação Competitiva , Complexo de Ataque à Membrana do Sistema Complemento/química , Ligação Proteica , Receptores X de Retinoides/metabolismo , TretinoínaRESUMO
C8α-γ deficiency was examined in four unrelated African Americans. Two individuals were compound heterozygotes for a previously reported point mutation in exon 9. mRNA from the remaining six C8A alleles contained a 10 nt insertion between nt 992 and 993 corresponding to the junction between exons 6 and 7. This suggested that C8α-γ deficiency in these individuals was caused by a splicing defect. Genomic sequencing revealed a GâA point mutation in intron 6, upstream of the exon 7 acceptor site. This mutation converts a GG to an AG, generates a consensus 3' splice site that shifts the reading frame, and creates a premature stop codon downstream. To verify that the point mutation caused a splicing defect, we tested wild-type and mutant mRNA substrates, containing 333 nt of the C8α intron 6/exon 7 boundary, in an in vitro splicing assay. This assay generated spliced RNA containing the 10 bp insertion observed in the C8α mRNA of affected patients. In addition, in mutant RNA substrates, the new 3' splice site was preferentially recognized compared with wild-type. Preferential selection of the mutant splice site likely reflects its positioning adjacent to a polypyrimidine tract that is stronger than that adjacent to the wild-type site. In summary, we have identified a GâA mutation in intron 6 of C8A as a predominant cause of C8α-γ deficiency in African Americans. This mutation creates a new and preferred 3' splice site, results in a 10 nt insertion in mRNA, shifts the reading frame, and produces a premature stop codon downstream.
Assuntos
Negro ou Afro-Americano , Complemento C8/genética , Síndromes de Imunodeficiência/genética , Mutação Puntual/genética , Processamento de Proteína/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Alelos , Estudos de Associação Genética , Heterozigoto , Humanos , Análise de Sequência de DNARESUMO
Proteins are the active players in performing essential molecular activities throughout biology, and their dynamics has been broadly demonstrated to relate to their mechanisms. The intrinsic fluctuations have often been used to represent their dynamics and then compared to the experimental B-factors. However, proteins do not move in a vacuum and their motions are modulated by solvent that can impose forces on the structure. In this paper, we introduce a new structural concept, which has been called the structural compliance, for the evaluation of the global and local deformability of the protein structure in response to intramolecular and solvent forces. Based on the application of pairwise pulling forces to a protein elastic network, this structural quantity has been computed and sometimes is even found to yield an improved correlation with the experimental B-factors, meaning that it may serve as a better metric for protein flexibility. The inverse of structural compliance, namely the structural stiffness, has also been defined, which shows a clear anticorrelation with the experimental data. Although the present applications are made to proteins, this approach can also be applied to other biomolecular structures such as RNA. This present study considers only elastic network models, but the approach could be applied further to conventional atomic molecular dynamics. Compliance is found to have a slightly better agreement with the experimental B-factors, perhaps reflecting its bias toward the effects of local perturbations, in contrast to mean square fluctuations. The code for calculating protein compliance and stiffness is freely accessible at https://jerniganlab.github.io/Software/PACKMAN/Tutorials/compliance.
Assuntos
Complemento C8/química , Proteínas Fúngicas/química , Lectinas/química , Redes Neurais de Computação , Software , Agaricales/química , Fenômenos Biomecânicos , Elasticidade , Humanos , Internet , Simulação de Dinâmica MolecularRESUMO
C8α and C9 mediate the membrane attack complex formation and bacterial lysis and are important components in the complement system. The cDNA sequences of the C8α and C9 genes were cloned from Takifugu rubripes. The full-length cDNA of Tr-C8α was 1893 bp and included a 5'-UTR of 69 bp and 3'-UTR of 83 bp. The full-length cDNA of Tr-C9 was 2083 bp and included a 5'-UTR of 72 bp and 3'-UTR of 250 bp. The expression of Tr-C8α and Tr-C9 was detected in newly fertilized eggs of T. rubripes. The expression of these two genes was at a higher level in the liver than in other tissues tested. After lipopolysaccharide (LPS) challenge, the gene expression of Tr-C8α and Tr-C9 increased more significantly in the liver. With these combined results, we further understood how Tr-C8α and Tr-C9 function in the innate immunity of pufferfish. Our findings could deepen the understanding of immune regulation in pufferfish.
Assuntos
Complemento C8/genética , Complemento C9/genética , Proteínas de Peixes/genética , Fígado/fisiologia , Tetraodontiformes/imunologia , Animais , Células Cultivadas , Clonagem Molecular , Ativação do Complemento , Complemento C8/metabolismo , Complemento C9/metabolismo , Proteínas de Peixes/metabolismo , Imunidade Inata , Lipopolissacarídeos/imunologia , Filogenia , Transcriptoma , Regulação para CimaRESUMO
Noninvasive biomarkers are clinically useful for evaluating liver fibrosis stage in patients with nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to compare plasma proteins in patients with early nonalcoholic steatohepatitis (NASH) (F0-F1) versus NASH with significant/advanced fibrosis (F2-F4) to determine whether candidate proteins could be used as potential noninvasive biomarkers. Nineteen biopsy-proven NAFLD patients including ten early NASH patients and nine NASH patients with significant/advanced fibrosis were enrolled in the present study. High-resolution proteomics screening of plasma was performed with the SCIEX TripleTOF 5600 System. Proteins were quantified using two different software platforms, Progenesis Qi and Scaffold Q+, respectively. Progenesis Qi analysis resulted in the discovery of 277 proteins compared with 235 proteins in Scaffold Q+. Five consensus proteins (i.e. Complement component C7; α-2-macroglobulin; Complement component C8 γ chain; Fibulin-1; α-1-antichymotrypsin) were identified. Complement component C7 was three-fold higher in the NASH group with significant/advanced fibrosis (F2-F4) compared with the early NASH (F0-F1) group (q-value = 3.6E-6). Complement component C7 and Fibulin-1 are positively correlated with liver stiffness (P=0.000, P=0.002, respectively); whereas, Complement component C8 γ chain is negatively correlated (P=0.009). High levels of Complement C7 are associated with NASH with significant/advanced fibrosis and Complement C7 is a perfect classifier of patients included in this pilot study. Further studies will be needed in a larger validation cohort to confirm the utility of complement proteins as biomarkers or mechanistic determinants of NASH with significant/advanced fibrosis.
Assuntos
Complemento C7/análise , Cirrose Hepática/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Proteoma , Proteômica , Adulto , Idoso , Biomarcadores/sangue , Proteínas de Ligação ao Cálcio/sangue , Complemento C8/análise , Feminino , Humanos , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Projetos Piloto , Valor Preditivo dos Testes , Serpinas/sangue , alfa-Macroglobulinas/análiseRESUMO
The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/"tick-over" mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.
Assuntos
Via Alternativa do Complemento , Proteínas do Sistema Complemento/fisiologia , Vesiculovirus/imunologia , Animais , Chlorocebus aethiops , Complemento C3/metabolismo , Complemento C3/fisiologia , Complemento C5/metabolismo , Complemento C5/fisiologia , Complemento C8/metabolismo , Complemento C8/fisiologia , Fator B do Complemento/metabolismo , Fator B do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Ácido Edético , Ácido Egtázico , Humanos , Testes de Neutralização , Soro/imunologia , Soro/virologia , Células Vero , Vesiculovirus/fisiologia , Replicação Viral/imunologiaRESUMO
OBJECTIVE: To identify molecular correlates of primary angiitis of the CNS (PACNS) through proteomic analysis of CSF from a biopsy-proven patient cohort. METHODS: Using mass spectrometry, we quantitatively compared the CSF proteome of patients with biopsy-proven PACNS (n = 8) to CSF from individuals with noninflammatory conditions (n = 11). Significantly enriched molecular pathways were identified with a gene ontology workflow, and high confidence hits within enriched pathways (fold change >1.5 and concordant Benjamini-Hochberg-adjusted p < 0.05 on DeSeq and t test) were identified as differentially regulated proteins. RESULTS: Compared to noninflammatory controls, 283 proteins were differentially expressed in the CSF of patients with PACNS, with significant enrichment of the complement cascade pathway (C4-binding protein, CD55, CD59, properdin, complement C5, complement C8, and complement C9) and neural cell adhesion molecules. A subset of clinically relevant findings were validated by Western blot and commercial ELISA. CONCLUSIONS: In this exploratory study, we found evidence of deregulation of the alternative complement cascade in CSF from biopsy-proven PACNS compared to noninflammatory controls. More specifically, several regulators of the C3 and C5 convertases and components of the terminal cascade were significantly altered. These preliminary findings shed light on a previously unappreciated similarity between PACNS and systemic vasculitides, especially anti-neutrophil cytoplasmic antibody-associated vasculitis. The therapeutic implications of this common biology and the diagnostic and therapeutic utility of individual proteomic findings warrant validation in larger cohorts.
Assuntos
Proteínas do Sistema Complemento/líquido cefalorraquidiano , Moléculas de Adesão de Célula Nervosa/líquido cefalorraquidiano , Proteômica , Vasculite do Sistema Nervoso Central/líquido cefalorraquidiano , Adolescente , Adulto , Biópsia , Encéfalo/patologia , Antígenos CD55/líquido cefalorraquidiano , Antígenos CD59/líquido cefalorraquidiano , Estudos de Casos e Controles , Estudos de Coortes , Proteína de Ligação ao Complemento C4b/líquido cefalorraquidiano , Complemento C5/líquido cefalorraquidiano , Complemento C8/líquido cefalorraquidiano , Complemento C9/líquido cefalorraquidiano , Via Alternativa do Complemento , Feminino , Ontologia Genética , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Properdina/líquido cefalorraquidiano , Vasculite do Sistema Nervoso Central/patologiaRESUMO
The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.
Assuntos
Antígenos CD59/metabolismo , Membrana Celular/ultraestrutura , Complemento C9/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Multimerização Proteica , Membrana Celular/metabolismo , Complemento C5/metabolismo , Complemento C8/metabolismo , Humanos , Cinética , Microscopia de Força Atômica/métodos , Imagem Individual de Molécula/métodosRESUMO
BACKGROUND: Primary complement deficiencies are rare diseases. OBJECTIVE: To retrospectively evaluate the clinical and laboratory findings and complications of patients to increase awareness of pediatricians about complement deficiencies, which are rarely encountered. METHODS: In this study, the clinical and immunological characteristics of 21 patients who consulted the Immunology Department of our hospital between 2003 and 2017 and were diagnosed with classical or alternative pathway complement deficiency were obtained from the file records. RESULTS: Ten patients with C1 inhibitor deficiency, four patients with factor I deficiency, three patients with properdin deficiency, two patients with C8 deficiency, one patient with C1q deficiency, and one patient with C4B deficiency were assessed. The mean age of the patients at diagnosis was 11.4±4.7 years, the age of onset of symptoms was 7.9±3.9 years, and the follow-up period was 6.7±3.9 years. Fourteen cases had a similar medical history in the family. All patients with C1q, factor I, properdin, C8, and C4B deficiencies presented with an infection, and vasculitic rash was present in two patients with factor I deficiency. In addition, immune complex glomerulonephritis was present in one patient with factor I deficiency. Meningococcal, Haemophilus influenzae type B, and pneumococcal vaccines were administered and prophylactic antibiotic treatment was initiated in all patients except patients with C1 inhibitor deficiency. CONCLUSIONS: Early diagnosis of complement deficiencies can facilitate prevention of life-threatening complications such as severe bacterial infections by considering prophylactic antibiotics and vaccines. In patients with C1 inhibitor deficiency, achieving an acurate early diagnosis will assist in the management and timely treatment of life-threatening attacks such as upper airway obstruction and improve outcomes.
Assuntos
Infecções Bacterianas/genética , Via Alternativa do Complemento/genética , Via Clássica do Complemento/genética , Síndromes de Imunodeficiência/genética , Adolescente , Antibioticoprofilaxia , Infecções Bacterianas/diagnóstico , Criança , Proteína Inibidora do Complemento C1/genética , Complemento C1q/genética , Complemento C8/genética , Diagnóstico Precoce , Feminino , Fibrinogênio/genética , Seguimentos , Humanos , Síndromes de Imunodeficiência/diagnóstico , Masculino , Properdina/genética , Estudos RetrospectivosRESUMO
The membrane attack complex (MAC) is one of the immune system's first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant ß-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ß-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.