RESUMO
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Virais Reguladoras e Acessórias/química , Sítios de Ligação , Cristalografia por Raios X , Ligação Proteica , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Homologia de Sequência , Proteínas Virais Reguladoras e Acessórias/metabolismo , Dedos de ZincoRESUMO
Cytokines and chemokines play a crucial role in regulating the immune system. Understanding how these molecules are co-regulated is important to understand general immunology, and particularly their role in clinical applications such as development and evaluation of novel drug therapies. Cytokines are today widely used as therapeutic targets and as biomarkers to monitor effects of drug therapies and for prognosis and diagnosis of diseases. Therapies that target a specific cytokine are also likely to affect the production of other cytokines due to their cross-regulatory functions and because the cytokines are produced by common cell types. In this study, we have perturbated the production of 17 different cytokines in a preclinical rat model of autoimmune arthritis, using 55 commercially available immunomodulatory drugs and clinical candidates. The majority of the studied drugs was selected for their anti-inflammatory role and was confirmed to inhibit the production of IL-2 and IFN-γ in this model but was also found to increase the production of other cytokines compared to the untreated control. Correlation analysis identified 58 significant pairwise correlations between the cytokines. The strongest correlations found in this study were between IL-2 and IFN-γ (r=0.87) and between IL-18 and EPO (r=0.84). Cluster analysis identified two robust clusters: (1) IL-7, IL-18 and EPO, and (2) IL-2, IL-17 and IFN-γ. The results show that cytokines are highly co-regulated, which provide valuable information for how a therapeutic drug might affect clusters of cytokines. In addition, a cytokine that is used as a therapeutic biomarker could be combined with its related cytokines into a biomarker panel to improve diagnostic accuracy.
Assuntos
Artrite Experimental/sangue , Citocinas/sangue , Fatores Imunológicos/farmacologia , Animais , Feminino , RatosRESUMO
OBJECTIVE: To identify genetic factors driving pathogenic autoantibody formation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), in order to better understand the etiology of RA and identify possible new avenues for therapeutic intervention. METHODS: We performed a genome-wide analysis of quantitative trait loci controlling autoantibody to type II collagen (anti-CII), anti-citrullinated protein antibody (ACPA), and rheumatoid factor (RF). To identify loci controlling autoantibody production, we induced CIA in a heterogeneous stock-derived mouse cohort, with contribution of 8 inbred mouse strains backcrossed to C57BL/10.Q. Serum samples were collected from 1,640 mice before arthritis onset and at the peak of the disease. Antibody concentrations were measured by standard enzyme-linked immunosorbent assay, and linkage analysis was performed using a linear regression-based method. RESULTS: We identified loci controlling formation of anti-CII of different IgG isotypes (IgG1, IgG3), antibodies to major CII epitopes (C1, J1, U1), antibodies to a citrullinated CII peptide (citC1), and RF. The anti-CII, ACPA, and RF responses were all found to be controlled by distinct genes, one of the most important loci being the immunoglobulin heavy chain locus. CONCLUSION: This comprehensive genetic analysis of autoantibody formation in CIA demonstrates an association not only of anti-CII, but interestingly also of ACPA and RF, with arthritis development in mice. These results underscore the importance of non-major histocompatibility complex genes in controlling the formation of clinically relevant autoantibodies.
Assuntos
Artrite Experimental/genética , Artrite Experimental/imunologia , Autoanticorpos/genética , Autoanticorpos/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , Feminino , Estudo de Associação Genômica Ampla , Imunoglobulina G/sangue , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Peptídeos Cíclicos/imunologia , Locos de Características Quantitativas/imunologia , Fator Reumatoide/imunologia , Especificidade da EspécieRESUMO
A locus on mouse chromosome 16 was found to control experimental autoimmune encephalomyelitis (EAE) in studies using congenic mice. Genes within the congenic region control encephalomyelitis but not arthritis, indicating the presence of genes in this region involved in central nervous system (CNS) specific mechanisms. Flow cytometry analyses of expression of two candidate genes within the linked locus, Cd200 and Btla, demonstrated a significantly lower expression of CD200 on CD4+ T cells and higher expression of BTLA on B cells from the congenic mice. These results suggest that genes within this mouse chromosome 16 locus specifically control EAE development possibly through immune-regulatory cell-surface molecules.
Assuntos
Cromossomos/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Loci Gênicos/genética , Loci Gênicos/imunologia , Animais , Cromossomos/genética , Progressão da Doença , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BLRESUMO
Resolving the genetic basis of complex diseases like rheumatoid arthritis will require knowledge of the corresponding diseases in experimental animals to enable translational functional studies. Mapping of quantitative trait loci in mouse models of arthritis, such as collagen-induced arthritis (CIA), using F(2) crosses has been successful, but can resolve loci only to large chromosomal regions. Using an inbred-outbred cross design, we identified and fine-mapped CIA loci on a genome-wide scale. Heterogeneous stock mice were first intercrossed with an inbred strain, B10.Q, to introduce an arthritis permitting MHCII haplotype. Homozygous H2(q) mice were then selected to set up an F(3) generation with fixed major histocompatibility complex that was used for arthritis experiments. We identified 26 loci, 18 of which are novel, controlling arthritis traits such as incidence of disease, severity and time of onset and fine-mapped a number of previously mapped loci.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Modelos Animais de Doenças , Animais , Cruzamentos Genéticos , Feminino , Genótipo , Haplótipos , Complexo Principal de Histocompatibilidade/genética , Masculino , Camundongos , Locos de Características Quantitativas/genéticaRESUMO
INTRODUCTION: In a cross between two mouse strains, the susceptible B10.RIII (H-2(r)) and resistant RIIIS/J (H-2(r)) strains, a locus on mouse chromosome 5 (Eae39) was previously shown to control experimental autoimmune encephalomyelitis (EAE). Recently, quantitative trait loci (QTL), linked to disease indifferent experimental arthritis models, were mapped to this region. The aim of the present study was to investigate whether genes within Eae39, in addition to EAE, control development of collagen-induced arthritis (CIA). METHODS: CIA, induced by immunisation with bovine type II collagen, was studied in Eae39 congenic and sub-interval congenic mice. Antibody titres were investigated with ELISA.Gene-typing was performed by micro-satellite mapping and statistics was calculated by standard methods. RESULTS: Experiments of CIA in Eae39 congenic- and sub-interval congenic mice, carrying RIIIS/J genes on the B10.RIII genetic background, revealed three loci within Eae39 that control disease and anti-collagen antibody titres. Two of the loci promoted disease and the third locus was protected against CIA development. By further breeding of mice with small congenic fragments, we identified a 3.2 mega base pair (Mbp)interval that regulates disease. CONCLUSIONS: Disease-promoting and disease-protecting genes within the Eae39 locus on mouse chromosome 5 control susceptibility to CIA. A disease-protecting locus in the telomeric part of Eae39 results in lower anti-collagen antibody responses. The study shows the importance of breeding sub-congenic mouse strains to reveal genetic effects on complex diseases.
Assuntos
Artrite Experimental/genética , Cromossomos/genética , Colágeno Tipo II/administração & dosagem , Predisposição Genética para Doença , Animais , Formação de Anticorpos , Bovinos , Colágeno Tipo II/imunologia , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos C57BL/genéticaRESUMO
The Eae2 locus on mouse chromosome 15 controls the development of experimental autoimmune encephalomyelitis (EAE); however, in this study we show that it also controls collagen-induced arthritis (CIA). To find the smallest disease-controlling locus/loci within Eae2, we have studied development of CIA in 676 mice from a partially advanced intercross. Eae2 congenic mice were bred with mice congenic for the Eae3/Cia5 locus on chromosome 3, previously shown to interact with Eae2. To create a large number of genetic recombinations within the congenic fragments, the offspring were intercrossed, and the eight subsequent generations were analyzed for CIA. We found that Eae2 consists of four Cia subloci (Cia26, Cia30, Cia31, and Cia32), of which two interacted with each other, conferring severe CIA. Genes within the other two loci independently interacted with genes in Eae3/Cia5. Investigation of the CD4/CD8 T cell ratio in mice from the partially advanced intercross shows that this trait is linked to one of the Eae2 subloci through interactions with Eae3/Cia5. Furthermore, the expression of CD86 on stimulated macrophages is linked to Eae2.