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1.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
2.
Nat Protoc ; 15(10): 3361-3379, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32908315

RESUMO

RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and skin removal are used to permeate tissues and preserve antigen epitopes, making this protocol applicable to both whole-mount embryos and larvae. Off-target hybridization and FISH background are reduced by using PCR-amplified DNA templates and stringent buffers. This protocol simultaneously detects multiple mRNAs and proteins with high sensitivity, and enables detection at single-cell resolution. The protocol can be completed within 6 days, overcoming the shortage of reliable antibodies available for zebrafish and exploiting the advantages of zebrafish for studying organ development and regeneration.


Assuntos
Embrião não Mamífero/diagnóstico por imagem , Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Animais , Anticorpos/metabolismo , Larva/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética
3.
Nat Commun ; 11(1): 4115, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807795

RESUMO

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.


Assuntos
Anticorpos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Anticorpos/genética , Western Blotting , Calorimetria , Cristalografia por Raios X , Citometria de Fluxo , Polarização de Fluorescência , Imunofluorescência , Humanos , Espectrometria de Massas , Ligação Proteica , Domínios Proteicos/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Biologia Sintética
4.
Medicine (Baltimore) ; 99(34): e21887, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32846849

RESUMO

INTRODUCTION: The incidence of hepatocellular carcinoma (HCC) ranks sixth in the world, but its mortality is the third highest due to the lack of early diagnostic markers. Nowadays, the increase of autoantibody levels has been found in many cancers, and many studies have begun to pay attention to the detection of anti-p53 antibodies in HCC. The purpose of this study is to quantitatively and comprehensively analyze the potential diagnostic value of anti-p53 autoantibodies in HCC METHODS:: English articles up to November 2019 were collected. The overall sensitivity and specificity were calculated. Besides, the positive likelihood ratio, negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and summary receiver operating characteristic curves of the overall diagnostic accuracy of anti-p53 antibody were calculated by STATA software. Finally, according to the heterogeneity of the results, the subgroup analysis, and the publication bias were performed. RESULTS: A total of 16 eligible studies were incorporated into this meta-analysis, including 1323 patients with HCC and 1896 control. The pooled sensitivity was 0.28(0.17-0.41) and specificity was 0.98 (0.95-0.99). The pooled DOR was 10.44 (6.31-17.29) and the pooled NLR was 0.74 (0.63-0.86). The area under ROC curve of symmetrical ROC was 0.840. CONCLUSIONS: The anti-p53 antibody has a high specificity for HCC, but the low sensitivity is not perfect and would limit the clinical application. The anti-p53 antibody would help rule out HCC but not help rule in HCC for early diagnosis. Whether combined as a diagnostic panel with other biomarkers or laboratory tests may prove useful requires further study.


Assuntos
Anticorpos/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/patologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Estudos de Casos e Controles , Estudos de Avaliação como Assunto , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/antagonistas & inibidores
5.
Proc Natl Acad Sci U S A ; 117(36): 22341-22350, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32855302

RESUMO

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.


Assuntos
Anticorpos , Diversidade de Anticorpos/genética , Autoantígenos , Rearranjo Gênico do Linfócito B/genética , Mutação/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Afinidade de Anticorpos/genética , Autoanticorpos/química , Autoanticorpos/genética , Autoanticorpos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Regiões Determinantes de Complementaridade/genética , Imunidade Humoral/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Hipermutação Somática de Imunoglobulina/genética
6.
J Vis Exp ; (160)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32628177

RESUMO

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.


Assuntos
Olho/citologia , Olho/diagnóstico por imagem , Citometria de Fluxo , Fagócitos/citologia , Animais , Anticorpos/metabolismo , Células Dendríticas/citologia , Feminino , Corantes Fluorescentes/metabolismo , Lasers , Macrófagos/citologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/citologia , Monócitos/citologia , Fagócitos/imunologia
7.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
8.
Nature ; 583(7816): 425-430, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612231

RESUMO

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Barreira Hematoencefálica/metabolismo , Transcitose , Fosfatase Alcalina/metabolismo , Animais , Anticorpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasma/metabolismo , Proteoma/administração & dosagem , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores da Transferrina/imunologia , Transcrição Genética , Transferrina/metabolismo
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 1029-1035, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701237

RESUMO

OBJECTIVE: To investigate the classification of idiopathic inflammatory myopathies (IIM) based on clinical manifestations and myositis- specific antibodies using cluster analysis. METHODS: We retrospectively analyzed the data of patients with IIM admitted in Nanfang Hospital in 2015-2019. The clinical data of the patients including serum creatine kinase (CK), interstitial lung disease (ILD), cancer, and myositis-specific antibodies were collected for two-step cluster analysis to identify the distinct clusters of patients, whose clinical characteristics were subsequently analysed. RESULTS: A total of 71 patients with IIM were included in this study, including 30 (42.3%) with polymyositis (PM), 20 (28.2%) with classic dermatomyositis (DM), 16 (22.5%) with amyopathic dermatomyositis (CADM), and 5 (7.0%) with immune-mediated necrotizing myopathy (IMNM). Two-step cluster analysis identified 3 distinctive subgroups: Cluster 1 of 15 (51.7%) patients characterized by rash, positive anti-MDA5 antibody and hypoproteinemia (P < 0.05) with normal or slightly elevated CK level, mainly corresponding to CADM; Cluster 2 of 4 (57.1%) patients with significantly elevated CK and positive anti-SRP antibody (P < 0.001) corresponding to IMNM; and Cluster 3 of 17 (48.6%) patients consisting primarily of patients with PM, characterized by positivity for anti- aminoacyl transfer RNA synthetases antibodies (P=0.022) corresponding to antisynthetase syndrome (ASS). CONCLUSIONS: Patients with IIM can be divided into 3 subgroups based on their clinical and serological characteristics (especially myositis-specific antibodies), and among them ASS may represent an independent IIM subgroup with unique clinical characteristics.


Assuntos
Miosite , Anticorpos/metabolismo , Humanos , Miosite/classificação , Miosite/fisiopatologia , Estudos Retrospectivos
10.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32630064

RESUMO

Vimentin is an intermediate filament protein that plays key roles in integration of cytoskeletal functions, and therefore in basic cellular processes such as cell division and migration. Consequently, vimentin has complex implications in pathophysiology. Vimentin is required for a proper immune response, but it can also act as an autoantigen in autoimmune diseases or as a damage signal. Although vimentin is a predominantly cytoplasmic protein, it can also appear at extracellular locations, either in a secreted form or at the surface of numerous cell types, often in relation to cell activation, inflammation, injury or senescence. Cell surface targeting of vimentin appears to associate with the occurrence of certain posttranslational modifications, such as phosphorylation and/or oxidative damage. At the cell surface, vimentin can act as a receptor for bacterial and viral pathogens. Indeed, vimentin has been shown to play important roles in virus attachment and entry of severe acute respiratory syndrome-related coronavirus (SARS-CoV), dengue and encephalitis viruses, among others. Moreover, the presence of vimentin in specific virus-targeted cells and its induction by proinflammatory cytokines and tissue damage contribute to its implication in viral infection. Here, we recapitulate some of the pathophysiological implications of vimentin, including the involvement of cell surface vimentin in interaction with pathogens, with a special focus on its role as a cellular receptor or co-receptor for viruses. In addition, we provide a perspective on approaches to target vimentin, including antibodies or chemical agents that could modulate these interactions to potentially interfere with viral pathogenesis, which could be useful when multi-target antiviral strategies are needed.


Assuntos
Vírus da SARS/fisiologia , Vimentina/metabolismo , Viroses/patologia , Anticorpos/imunologia , Anticorpos/metabolismo , Anticorpos/uso terapêutico , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/tratamento farmacológico , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Vimentina/química , Vimentina/imunologia , Viroses/tratamento farmacológico , Viroses/metabolismo , Replicação Viral/efeitos dos fármacos
11.
PLoS Biol ; 18(6): e3000288, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516310

RESUMO

Unc-51-like autophagy activating kinase 1 (ULK1)-autophagy-related 13 (ATG13) is the most upstream autophagy initiation complex that is phosphorylated by mammalian target-of-rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) to induce autophagy in asynchronous conditions. However, their phospho-regulation and functions in mitosis and cell cycle remain unknown. Here we show that ULK1-ATG13 complex is differentially regulated throughout the cell cycle, especially in mitosis, in which both ULK1 and ATG13 are highly phosphorylated by the key cell cycle machinery cyclin-dependent kinase 1 (CDK1)/cyclin B. Combining mass spectrometry and site-directed mutagenesis, we found that CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Moreover, double knockout (DKO) of ULK1 and ATG13 could block cell cycle progression and significantly decrease cancer cell proliferation in cell line and mouse models. Our results not only bridge the mutual regulation between the core machinery of autophagy and mitosis but also illustrate the positive function of ULK1-ATG13 and their phosphorylation by CDK1 in mitotic autophagy regulation.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteína Quinase CDC2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Animais , Anticorpos/metabolismo , Linhagem Celular , Ciclina B/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Especificidade por Substrato
12.
Ann N Y Acad Sci ; 1475(1): 34-42, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32594556

RESUMO

The cannabinoid CB1 receptor (CB1 R) is the most abundant G protein-coupled receptor in the central nervous system, consistent with the important role of endocannabinoids as neuromodulators. Cannabinoids also modulate the function of G protein-coupled receptor 55 (GPR55), which forms heteroreceptor complexes with the CB1 R in the striatum. The aim was to characterize cannabinoid CB1 R-GPR55 heteromers (CB1 R/GPR55Hets) in the basal ganglia input nuclei of nonhuman primates, Macaca fascicularis, both in projection neurons and interneurons, by the in situ proximity ligation assay. Striatal projecting neurons were identified by the retrograde neuroanatomical tracer, biotinylated dextran amine (BDA), injected into external or internal subdivisions of the globus pallidus. Triple immunofluorescent stains were carried out to visualize (1) BDA-labeled neurons, (2) CB1 R/GPR55Hets, and (3) striatal interneurons positive for choline acetyltransferase, parvalbumin, calretinin, or nitric oxide synthase. CB1 R/GPR55Hets were identified within both types of projection neurons as well as all interneurons except those that are cholinergic. Moreover, CB1 R/GPR55Hets were found specifically in the neuronal cell surface, and also in intracellular membranes. Further research efforts will be needed to confirm the intracellular occurrence of heteromers and their potential as therapeutic targets in diseases related to motor control imbalances, particularly within a parkinsonian context (with or without levodopa-induced dyskinesia).


Assuntos
Corpo Estriado/metabolismo , Neurônios/metabolismo , Multimerização Proteica , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Anticorpos/metabolismo , Biomarcadores/metabolismo , Interneurônios/metabolismo , Macaca fascicularis , Masculino
13.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1216-1222, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597071

RESUMO

A rapid and simple method to detect tumor markers in liver cancer was established by combining immunochromatography technique with fluorescent microsphere labeling. According to the principle of double antibody sandwich, the cytoskeleton-associated protein 4 (CKAP4) paired antibody was used as the labeled and coated antibody, and the goat anti-rabbit polyclonal antibody was used as the quality control line coated antibody in the preparation of the CKAP4 fluorescent immunochromatographic test strips. After the preparation, the test strips were evaluated on various performance indicators, such as linearity, precision and stability. The CKAP4 immunochromatographic strip prepared by time-resolved fluorescent microspheres had high sensitivity, and good specificity. Its precision was within 15%, recovery between 85% and 115%, and linear range between 25 and 1 000 pg/mL. The test strip could be kept stable at 37 °C for 20 days, and it correlated well with commercial ELISA kits. The CKAP4 fluorescence immunochromatography method can quantitatively detect the content of CKAP4 in serum. Furthermore, it is rapid, sensitive, simple, economical and single-person operation. This method has the potential of becoming a new method for the diagnosis and treatment of liver cancer.


Assuntos
Cromatografia de Afinidade , Neoplasias Hepáticas , Proteínas de Membrana , Técnicas de Diagnóstico Molecular , Animais , Anticorpos/metabolismo , Fluorescência , Humanos , Neoplasias Hepáticas/diagnóstico , Proteínas de Membrana/isolamento & purificação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e Especificidade
14.
Proc Natl Acad Sci U S A ; 117(24): 13509-13518, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32493749

RESUMO

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Anticorpos/imunologia , Agregados Proteicos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Especificidade de Anticorpos , Caenorhabditis elegans , Modelos Animais de Doenças , Epitopos , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único
15.
Proc Natl Acad Sci U S A ; 117(21): 11624-11635, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385154

RESUMO

Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, and cMyc/IgH translocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID-hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.


Assuntos
Anticorpos , Citidina Desaminase , Quebras de DNA , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Motivos de Ligação ao RNA/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Hipermutação Somática de Imunoglobulina/genética
16.
Cancer Immunol Immunother ; 69(9): 1833-1840, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32350593

RESUMO

BACKGROUND: Bladder cancer is diagnosed by the use of several biomarkers, including survivin. This protein has an important role in the cancer progression by controlling the rate of cell apoptosis. Findings show that there is no survivin in normal tissues, whereas the level of survivin expression increases in tumor cells. DESIGN: The purpose of this study was to specify the reactive antibodies to survivin protein as a biomarker to determine the bladder cancer stage with ELISA method and using GNPs conjugated with survivin antibody. The serum and urine samples of patients with bladder cancer were collected among those referred to Sina Hospital, Tehran, Iran. The survivin protein level was measured in the serum and urine by ELISA technique and in the urine by GNPs conjugated with survivin. RESULTS: Based on the results of ELISA, the serum and urinary levels of survivin increased significantly in T3 and T4 stages of the disease (high grades), compared with the healthy individuals. Also, using conjugated GNPs, survivin protein was detected in the urine specimens of patients at all grades (low and high grades). CONCLUSION: Our findings showed that using the ELISA technique, the increased level of survivin could be identified in high grades of bladder cancer, but using anti-survivin antibody-conjugated GNPs, bladder cancer can be detected in early stages. The applied method was found to be a rapid tool, dependent on visible color changes and colorimetric detection, without any need for reader devices.


Assuntos
Anticorpos/metabolismo , Ouro/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Survivina/metabolismo , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Masculino , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/métodos
17.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L71-L81, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32374670

RESUMO

SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 2 (SPOCK2) was previously associated with genetic susceptibility to bronchopulmonary dysplasia in a French population of very preterm neonates. Its expression increases during lung development and is increased after exposure of rat pups to hyperoxia compared with controls bred in room air. To further investigate the role of SPOCK2 during lung development, we designed two mouse models, one that uses a specific anti-Spock2 antibody and one that reproduces the hyperoxia-induced Spock2 expression with a transgenic mouse model resulting in a conditional and lung-targeted overexpression of Spock2. When mice were bred under hyperoxic conditions, treatment with anti-Spock2 antibodies significantly improved alveolarization. Lung overexpression of Spock2 altered alveolar development in pups bred in room air and worsened hyperoxia-induced lesions. Neither treatment with anti-Spock2 antibody nor overexpression of Spock2 was associated with abnormal activation of matrix metalloproteinase-2. These two models did not alter the expression of known players in alveolar development. This study brings strong arguments for the deleterious role of SPOCK2 on lung alveolar development especially after lung injury, suggesting its role in bronchopulmonary dysplasia susceptibility. These effects are not mediated by a deregulation in metalloproteases activity and in expression of factors essential to normal alveolarization. The balance between types 1 and 2 epithelial alveolar cells may be involved.


Assuntos
Hiperóxia/patologia , Proteoglicanas/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Animais , Anticorpos/metabolismo , Ativação Enzimática , Hiperóxia/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo
18.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G907-G911, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249590

RESUMO

The mammalian intestine is host to a vast number of microbial organisms. The immune system must balance tolerance with innate and adaptive defense mechanisms to maintain homeostasis with the microbial community. Interestingly, microbial metabolites have been shown to play a role in shaping the host immune response, thus assisting with adaptations that have significant implications for human health and disease. New investigations have uncovered roles for metabolites in modulating almost every aspect of the immune system. In this minireview, we survey these recent findings, which taken together reveal nuanced interactions that we are just beginning to understand.


Assuntos
Bactérias/metabolismo , Microbioma Gastrointestinal , Imunidade Inata , Imunidade nas Mucosas , Mucosa Intestinal/microbiologia , Animais , Anticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Bactérias/imunologia , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Neuroimunomodulação , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo
19.
Arch Virol ; 165(5): 1163-1176, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32232673

RESUMO

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.


Assuntos
Anticorpos/metabolismo , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Endocitose , Potyvirus/genética , Virossomos/genética , Animais , Anticorpos/imunologia , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Proteína Estafilocócica A/genética
20.
PLoS Comput Biol ; 16(4): e1007779, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339164

RESUMO

Antibodies are capable of potently and specifically binding individual antigens and, in some cases, disrupting their functions. The key challenge in generating antibody-based inhibitors is the lack of fundamental information relating sequences of antibodies to their unique properties as inhibitors. We develop a pipeline, Antibody Sequence Analysis Pipeline using Statistical testing and Machine Learning (ASAP-SML), to identify features that distinguish one set of antibody sequences from antibody sequences in a reference set. The pipeline extracts feature fingerprints from sequences. The fingerprints represent germline, CDR canonical structure, isoelectric point and frequent positional motifs. Machine learning and statistical significance testing techniques are applied to antibody sequences and extracted feature fingerprints to identify distinguishing feature values and combinations thereof. To demonstrate how it works, we applied the pipeline on sets of antibody sequences known to bind or inhibit the activities of matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes that promote cancer progression and undesired inflammation under pathological conditions, against reference datasets that do not bind or inhibit MMPs. ASAP-SML identifies features and combinations of feature values found in the MMP-targeting sets that are distinct from those in the reference sets.


Assuntos
Anticorpos , Biologia Computacional/métodos , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Software , Algoritmos , Anticorpos/química , Anticorpos/metabolismo , Bases de Dados de Proteínas , Humanos , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/metabolismo , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo
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