RESUMEN
Extracellular vesicles (EVs) are preeminent carriers of biomarkers and have become the subject of intense biomedical research for medical diagnostics using biosensors. To create effective EV-based immunoassays, it is imperative to develop surface chemistry approaches with optimal EV detection targeting transmembrane protein biomarkers that are not affected by cell-to-cell variability. Here, we developed a series of immunoassays for the detection of EVs derived from mouse monocyte cells using surface plasmon resonance (SPR) biosensors. We chemically immobilized antibodies onto mixed self-assembled monolayers of oligo ethylene glycol (OEG) alkanethiolates with carboxylic and hydroxylic terminal groups. The effects of antibody clonality (monoclonal vs polyclonal) and antibody surface coverage in targeting EVs via CD81 tetraspanins were investigated. We determined binding kinetic parameters, establishing trends from steric hindrance effects and epitope recognition properties of antibodies. Our results indicate that a 40% surface coverage of polyclonal antibodies covalently linked onto a mixed SAM with 10% of terminated -COOH groups yields a promising approach for EV detection with a linear range of 1.9 × 108-1.9 × 109 EVs/mL and a limit of detection of 5.9 × 106 EVs/mL. This optimal immunoassay exhibits a 1.92 nM equilibrium dissociation constant for bound EVs, suggesting a high binding affinity when CD81 is targeted. Our study provides important insights into surface chemistry development for EV detection targeted via transmembrane protein biomarkers using antibodies, which has promising applications for disease diagnostics.
Asunto(s)
Vesículas Extracelulares , Resonancia por Plasmón de Superficie , Resonancia por Plasmón de Superficie/métodos , Vesículas Extracelulares/química , Animales , Inmunoensayo/métodos , Ratones , Tetraspanina 28/análisis , Tetraspanina 28/química , Tetraspanina 28/metabolismo , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/química , Técnicas Biosensibles/métodos , Proteínas de la Membrana/químicaRESUMEN
Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.
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Endocitosis , Exosomas , Tetraspanina 30 , Exosomas/metabolismo , Humanos , Tetraspanina 30/metabolismo , Biomarcadores/metabolismo , Sinteninas/metabolismo , Sinteninas/genética , Tetraspanina 28/metabolismo , Membrana Celular/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Tetraspanina 29/metabolismoRESUMEN
Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.
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Biomarcadores , Técnicas Biosensibles , Polimerizacion , Humanos , Femenino , Embarazo , Biomarcadores/análisis , Biomarcadores/sangre , Técnicas Biosensibles/métodos , Preeclampsia/diagnóstico , Preeclampsia/sangre , Tetraspanina 28/análisis , Tetraspanina 28/metabolismo , Inmunoadsorbentes/química , Límite de Detección , Fluorescencia , Ensayo de Inmunoadsorción Enzimática , Eclampsia/diagnósticoRESUMEN
Introduction: To understand the immune system within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), it is crucial to elucidate the characteristics of molecules associated with T cell activation. Methods: We conducted an in-depth analysis using single-cell RNA sequencing data obtained from tissue samples of 19 NSCLC patients. T cells were classified based on the Tumor Proportion Score (TPS) within the tumor region, and molecular markers associated with activation and exhaustion were analyzed in T cells from high TPS areas. Results: Notably, tetraspanins CD81 and CD82, belonging to the tetraspanin protein family, were found to be expressed in activated T cells, particularly in cytotoxic T cells. These tetraspanins showed strong correlations with activation and exhaustion markers. In vitro experiments confirmed increased expression of CD81 and CD82 in IL-2-stimulated T cells. T cells were categorized into CD81highCD82high and CD81lowCD82low groups based on their expression levels, with CD81highCD82high T cells exhibiting elevated activation markers such as CD25 and CD69 compared to CD81lowCD82low T cells. This trend was consistent across CD3+, CD8+, and CD4+ T cell subsets. Moreover, CD81highCD82high T cells, when stimulated with anti-CD3, demonstrated enhanced secretion of cytokines such as IFN-γ, TNF-α, and IL-2, along with an increase in the proportion of memory T cells. Bulk RNA sequencing results after sorting CD81highCD82high and CD81lowCD82low T cells consistently supported the roles of CD81 and CD82. Experiments with overexpressed CD81 and CD82 showed increased cytotoxicity against target cells. Discussion: These findings highlight the multifaceted roles of CD81 and CD82 in T cell activation, cytokine production, memory subset accumulation, and target cell cytolysis. Therefore, these findings suggest the potential of CD81 and CD82 as promising candidates for co-stimulatory molecules in immune therapeutic strategies for cancer treatment within the intricate TME.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Antígenos CD/metabolismo , Linfocitos Infiltrantes de Tumor , Interleucina-2/metabolismo , Microambiente Tumoral , Neoplasias Pulmonares/metabolismo , Citocinas/metabolismo , Tetraspaninas/metabolismo , Tetraspanina 28 , Proteína Kangai-1/metabolismoRESUMEN
Extracellular vesicles, especially exosomes, have attracted attention in the last few decades as novel cancer biomarkers. Exosomal membrane proteins provide easy-to-reach targets and can be utilized as information sources of their parent cells. In this study, a MagLev-based, highly sensitive, and versatile biosensor platform for detecting minor differences in the density of suspended objects is proposed for exosome detection. The developed platform utilizes antibody-functionalized microspheres to capture exosomal membrane proteins (ExoMPs) EpCAM, CD81, and CD151 as markers for cancerous exosomes, exosomes, and non-small cell lung cancer (NSCLC)-derived exosomes, respectively. Initially, the platform was utilized for protein detection and quantification by targeting solubilized ExoMPs, and a dynamic range of 1-100 nM, with LoD values of 1.324, 0.638, and 0.722 nM for EpCAM, CD81, and CD151, were observed, respectively. Then, the sensor platform was tested using exosome isolates derived from NSCLC cell line A549 and MRC5 healthy lung fibroblast cell line. It was shown that the sensor platform is able to detect and differentiate exosomal biomarkers derived from cancerous and non-cancerous cell lines. Overall, this innovative, simple, and rapid method shows great potential for the early diagnosis of lung cancer through exosomal biomarker detection.
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Molécula de Adhesión Celular Epitelial , Exosomas , Neoplasias Pulmonares , Exosomas/química , Humanos , Neoplasias Pulmonares/patología , Molécula de Adhesión Celular Epitelial/metabolismo , Tetraspanina 28/metabolismo , Tetraspanina 28/análisis , Técnicas Biosensibles/métodos , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Biomarcadores de Tumor/análisis , Tetraspanina 24 , Células A549RESUMEN
The tetraspanin CD81 is one of the main entry receptors for Hepatitis C virus, which is a major causative agent to develop liver cirrhosis and hepatocellular carcinoma (HCC). Here, we identify CD81 as one of few surface proteins that are downregulated in HCV expressing hepatoma cells, discovering a functional role of CD81 beyond mediating HCV entry. CD81 was downregulated at the mRNA level in hepatoma cells that replicate HCV. Kinetics of HCV expression were increased in CD81-knockout cells and accompanied by enhanced cellular growth. Furthermore, loss of CD81 compensated for inhibition of pro-survival TBK1-signaling in HCV expressing cells. Analysis of functional phenotypes that could be associated with pro-survival signaling revealed that CD81 is a negative regulator of NF-κB. Interaction of the NF-κB subunits p50 and p65 was increased in cells lacking CD81. Similarly, we witnessed an overall increase in the total levels of phosphorylated and cellular p65 upon CD81-knockout in hepatoma cells. Finally, translocation of p65 in CD81-negative hepatoma cells was markedly induced upon stimulation with TNFα or PMA. Altogether, CD81 emerges as a regulator of pro-survival NF-κB signaling. Considering the important and established role of NF-κB for HCV replication and tumorigenesis, the downregulation of CD81 by HCV and the associated increase in NF-κB signaling might be relevant for viral persistence and chronic infection.
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Carcinoma Hepatocelular , Hepatitis C , Neoplasias Hepáticas , Humanos , Hepacivirus/genética , FN-kappa B , Neoplasias Hepáticas/patología , Tetraspanina 28/metabolismoRESUMEN
Probing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi-functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site-specifically binds, and forms a complex with CD81, enabling in-situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.
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Péptidos , Tetraspanina 28 , Humanos , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Tetraspanina 28/metabolismo , Tetraspanina 28/química , Metástasis de la Neoplasia , Movimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Animales , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismoRESUMEN
CD81 is a cell surface transmembrane protein of the tetraspanin family, which critically regulates signal transduction and immune response. Growing evidence has shown that CD81 plays important roles in tumorigenesis and influences immunotherapy response. Here, combining bio-informatics and functional analysis, we find that CD81 is a risk factor in lung squamous cell carcinoma (LUSC), whereas a protective factor in lung adenocarcinoma. In LUSC with high expression of CD81, the autophagy and JAK-STAT signaling pathway are activated. Meanwhile, the expression level of CD81 is negatively correlated with tumor mutational load (TMB), microsatellite instability (MSI), and neoantigen (NEO). Furthermore, patients with LUSC and high expression of CD81 do not respond to immunotherapy drugs, but can respond to chemotherapy drugs. Importantly, depletion of CD81 suppresses the proliferation of LUSC cell, and enhances the sensitivity to cisplatin. Our findings suggest that CD81 represents a potential target for cisplatin-based chemotherapy in patients with LUSC.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Cisplatino , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Pulmón/patología , Tetraspanina 28/metabolismoRESUMEN
BACKGROUND: Hepatitis C virus (HCV) infection is a global health threat to the public, and vaccines against it are not yet available. The HCV envelope glycoprotein E2 is a key target for anti-HCV vaccines. The majority of previous studies have focused on the hypervariable region and the glycosylation sites of the_HCV structural protein. This study aims to investigate a conserved domain of HCV E2 glycoprotein and explore its potential to induce an immune response against HCV. METHODS: HCV E2 conserved domain (encompassing amino acids 505-702) was prepared in Escherichia coli (E. coli). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HCV or healthy controls. Interferon-gamma (IFN-γ) enzyme-linked immunosorbent spot assay was conducted to examine the HCV E2-specific immune response as reflected by IFN-γ-secreting cells/106 PBMCs. RESULTS: HCV E2 conserved domain was highly conserved among 25 HCV subtypes, and its recombinant soluble production in E. coli was recognized by anti-HCV E2 monoclonal antibodies. This study characterized in vitro direct interaction between bacterially expressed HCV E2 conserved domain and human CD81 (hCD81). Furthermore, the recombinant HCV E2_conserved domain markedly induced the production of IFN-γ by PBMCs from patients with HCV. Its stimulated specific immune response was significantly different from non-specific peptide controls or PBMCs isolated from healthy controls. CONCLUSIONS: HCV E2 conserved domain directly binds hCD81 and activates the production of IFN-γ in the PBMCs of patients with HCV. Therefore, the conserved domain of HCV E2 glycoprotein may be a new candidate for developing an HCV vaccine.
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Hepatitis C , Vacunas , Humanos , Escherichia coli/genética , Hepacivirus/fisiología , Hepatitis C/metabolismo , Interferón gamma/metabolismo , Leucocitos Mononucleares/metabolismo , Tetraspanina 28/química , Tetraspanina 28/metabolismoRESUMEN
Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.
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Hepacivirus , Hepatitis C , Humanos , Hepacivirus/fisiología , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Internalización del Virus , Proteínas Portadoras , Receptores ErbB/metabolismo , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismoRESUMEN
The role of tetraspanin CD81 in malignant transformation is best studied in colorectal cancer, and it appears that other transcripts beside the fully coding mRNA may also be dysregulated in malignant cells. Recent data from a comprehensive pan-cancer transcriptome analysis demonstrated differential activity of two alternative CD81 gene promoters in malignant versus nonmalignant gut mucosa. The promoter active in gut mucosa gives rise to transcripts CD81-203 and CD81-213, while the promoter active in colon and rectal cancer gives rise to transcripts CD81-205 and CD81-215. Our study aimed to explore the biomarker potential of the transcripts from the alternative CD81 gene promoters in colon cancer, as well as to investigate their structure and potential function using in silico tools. The analysis of the transcripts' expression in several colon cell lines cultivated in 2D and 3D and a set of colon cancer and healthy gut mucosa samples by qPCR and RNA sequencing suggested their low expression and stromal origin. Expression patterns in tumor and nontumor tissue along with in silico data suppose that the transcript CD81-215 may be a noncoding RNA of stromal origin with possible involvement in signaling related to malignant transformation.
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Neoplasias del Colon , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Perfilación de la Expresión Génica , Transducción de Señal , Tetraspanina 28/genética , Tetraspanina 28/metabolismoRESUMEN
The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.
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Vesículas Extracelulares , Tetraspanina 28 , Tetraspanina 29 , Tetraspanina 30 , Movimiento Celular , Vesículas Extracelulares/metabolismo , Proteómica , Tetraspanina 28/metabolismo , Humanos , Células MCF-7 , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismoRESUMEN
Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 104 particles·mL-1 to 6.1 × 107 particles·mL-1, and a low detection limit (0.6-1.8) × 104 particles·mL-1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.
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Técnicas Biosensibles , Vesículas Extracelulares , Neoplasias Pulmonares , Humanos , Resonancia por Plasmón de Superficie/métodos , Técnicas Biosensibles/métodos , Tecnicas de Microbalanza del Cristal de Cuarzo , Inmunoensayo , Tetraspaninas , Vesículas Extracelulares/química , Biomarcadores , Tetraspanina 28 , Tetraspanina 30/análisis , Tetraspanina 29/análisisRESUMEN
Metastases are reduced in CD81KO mice. In addition, a unique anti-CD81 antibody, 5A6, inhibits metastasis in vivo and invasion and migration in vitro. Here, we probed the structural components of CD81 required for the antimetastatic activity induced by 5A6. We found that the removal of either cholesterol or the intracellular domains of CD81 did not affect inhibition by the antibody. We show that the uniqueness of 5A6 is due not to increased affinity but rather to its recognition of a specific epitope on the large extracellular loop of CD81. Finally, we present a number of CD81 membrane-associated partners that may play a role in mediating the 5A6 antimetastatic attributes, including integrins and transferrin receptors.
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Anticuerpos , Integrinas , Animales , Ratones , Tetraspanina 28RESUMEN
Background: The global outbreak of COVID-19, and the limited availability of clinical treatments, forced researchers around the world to search for the pathogenesis and potential treatments. Understanding the pathogenesis of SARS-CoV-2 is crucial to respond better to the current coronavirus disease 2019 (COVID-19) pandemic. Methods: We collected sputum samples from 20 COVID-19 patients and healthy controls. Transmission electron microscopy was used to observe the morphology of SARS-CoV-2. Extracellular vesicles (EVs) were isolated from sputum and the supernatant of VeroE6 cells, and were characterized by transmission electron microscopy, nanoparticle tracking analysis and Western-Blotting. Furthermore, a proximity barcoding assay was used to investigate immune-related proteins in single EV, and the relationship between EVs and SARS-CoV-2. Result: Transmission electron microscopy images of SARS-COV-2 virus reveal EV-like vesicles around the virion, and western blot analysis of EVs extracted from the supernatant of SARS-COV-2-infected VeroE6 cells showed that they expressed SARS-COV-2 protein. These EVs have the infectivity of SARS-COV-2, and the addition can cause the infection and damage of normal VeroE6 cells. In addition, EVs derived from the sputum of patients infected with SARS-COV-2 expressed high levels of IL6 and TGF-ß, which correlated strongly with expression of the SARS-CoV-2 N protein. Among 40 EV subpopulations identified, 18 differed significantly between patients and controls. The EV subpopulation regulated by CD81 was the most likely to correlate with changes in the pulmonary microenvironment after SARS-CoV-2 infection. Single extracellular vesicles in the sputum of COVID-19 patients harbor infection-mediated alterations in host and virus-derived proteins. Conclusions: These results demonstrate that EVs derived from the sputum of patients participate in virus infection and immune responses. This study provides evidence of an association between EVs and SARS-CoV-2, providing insight into the possible pathogenesis of SARS-CoV-2 infection and the possibility of developing nanoparticle-based antiviral drugs.
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COVID-19 , Vesículas Extracelulares , Humanos , COVID-19/metabolismo , SARS-CoV-2 , Integrinas/metabolismo , Esputo , Proteómica/métodos , Vesículas Extracelulares/metabolismo , Tetraspanina 28RESUMEN
Preeclampsia (PE) seriously affects pregnant women and fetuses' health and causes maternal near-misses. CD81 has been confirmed to be a novel PE biomarker with great potential. Herein, a hypersensitive dichromatic biosensor based on the plasmonic enzyme-linked immunosorbent assay (plasmonic ELISA) is proposed initially for the application of CD81 in early screening for PE. In this work, a novel chromogenic substrate [(HAuCl4)-(N-methylpyrrolidone)-(Na3C6H5O7)] is designed based on the H2O2 dual catalysis reduction pathway of Au ions. The two reduction pathways of Au ions are controlled by H2O2 which ensures that the synthesis and growth of AuNPs are sensitive to H2O2. The amount of H2O2 correlates with the concentration of CD81 and directs the production of different-sized AuNPs in this sensor. Blue solutions are generated when analytes are present. When analytes are absent, solutions turn red. Therefore, due to different absorption peaks in red and blue, bimodal detection can be performed, and then two detection signals can be generated, one on signal at 550 nm and another off signal at 600 nm. This method exhibits a linear response to the logarithmic CD81 concentrations in the range of 0.1-1000 pg mL-1 with detection limits of 86 fg mL-1 and 152 fg mL-1 at two wavelengths. The false positive rate is low due to the nonspecific coloration caused by serum, which produces a more intense color contrast. The results indicate that the proposed dichromatic sensor could be used as a visual sensing platform for the direct detection of CD81 in biological samples and demonstrate its potential in preeclampsia diagnosis.
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Nanopartículas del Metal , Preeclampsia , Humanos , Femenino , Embarazo , Preeclampsia/diagnóstico , Oro , Peróxido de Hidrógeno , Ensayo de Inmunoadsorción Enzimática , Límite de Detección , Tetraspanina 28RESUMEN
The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.
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Hipertensión , Preeclampsia , Embarazo , Ratones , Humanos , Ratas , Femenino , Animales , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Simportadores del Cloruro de Sodio/genética , Simportadores del Cloruro de Sodio/metabolismo , Lipopolisacáridos/toxicidad , Lipopolisacáridos/metabolismo , Cloruros/metabolismo , Preeclampsia/inducido químicamente , Preeclampsia/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Placenta/metabolismo , Túbulos Renales Distales/metabolismo , Hipertensión/metabolismo , Sodio/metabolismo , Potasio/metabolismo , Tetraspanina 28/metabolismoRESUMEN
CD81, a transmembrane protein belonging to the tetraspanin family, has recently been suggested as a therapeutic target for cancers. Here, we screened peptides that bind to the tetraspanin CD81 protein, and evaluated their inhibitory activity in cancer cell migration. To screen for CD81-binding peptides (CD81-BP), a peptide array membrane was prepared from the amino acid sequence of the EWI-2 protein, a major partner of CD81, before binding to fluorescently labeled CD81. As a result, four candidate CD81-BPs were identified and characterized. In particular, the CFMKRLRK peptide (called P152 in this study) was found to be the best candidate that preferentially binds to the extracellular loop of CD81, with an estimated dissociation constant of 0.91 µM. Since CD81 was reported to promote cancer cell migration, an initial step in metastasis, the Boyden chamber assay, was next performed to assess the effect of CD81-BP candidates on the migration of MDA-MB-231 human breast cancer cells. Interestingly, our result indicated that P152 could suppress MDA-MB-231 cell migration at the level comparable to that of an anti-human CD81 antibody (5A6). Thus, we propose these CD81-BPs with the anti-migration property against cancer cells for the development of novel therapeutic strategies.
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Antígenos CD , Neoplasias , Humanos , Antígenos CD/metabolismo , Tetraspanina 28/metabolismo , Detección Precoz del Cáncer , Tetraspaninas , Péptidos/farmacología , Movimiento CelularRESUMEN
Extracellular vesicles (EVs) are implicated in the pathogenesis of rheumatoid arthritis (RA) but little is known about the composition of specific small EV (sEV) subpopulations. This study aimed to characterize the CD63, CD81 and CD9 tetraspanin profile in the membrane of single EVs in plasma from treatment naïve RA patients and assess potential discrepancies between methotrexate (MTX) responder groups. EVs isolated from plasma were characterized using transmission electron microscopy, and detection of surface markers (CD63, CD81 and CD9) on single EVs was performed on the ExoView platform. All RA patients (N = 8) were newly diagnosed, treatment naïve, females, ACPA positive and former smokers. The controls (N = 5) were matched for age and gender. After three months of MTX treatment, responders (N = 4) were defined as those with ΔDAS28 > 1.2 and DAS28 ≤ 3.2 post-treatment. The isolated EVs were 50-200 nm in size. The RA patients had a higher proportion of both CD9 and CD81 single positive sEVs compared to healthy controls, while there was a decrease in CD81/CD9 double positive sEVs in patients. Stratification of RA patients into MTX responders and non-responders revealed a distinctly higher proportion of CD81 single positive sEVs in the responder group. The proportion of CD81/CD9 double positive sEVs (anti-CD9 captured) was lower in the non-responders, but increased upon 3 months of MTX treatment. Our exploratory study revealed distinct tetraspanin profiles in RA patients suggesting their implication in RA pathophysiology and MTX treatment response.
Asunto(s)
Artritis Reumatoide , Vesículas Extracelulares , Femenino , Humanos , Tetraspanina 29/metabolismo , Tetraspanina 28 , Tetraspaninas , Vesículas Extracelulares/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismoRESUMEN
Migrasomes are newly discovered cell organelles forming by local swelling of retraction fibers. The migrasome formation critically depends on tetraspanin proteins present in the retraction fiber membranes and is modulated by the membrane tension and bending rigidity. It remained unknown how and in which time sequence these factors are involved in migrasome nucleation, growth, and stabilization, and what are the possible intermediate stages of migrasome biogenesis. Here using live cell imaging and a biomimetic system for migrasomes and retraction fibers, we reveal that migrasome formation is a two-stage process. At the first stage, which in biomimetic system is mediated by membrane tension, local swellings largely devoid of tetraspanin 4 form on the retraction fibers. At the second stage, tetraspanin 4 molecules migrate toward and onto these swellings, which grow up to several microns in size and transform into migrasomes. This tetraspanin 4 recruitment to the swellings is essential for migrasome growth and stabilization. Based on these findings we propose that the major role of tetraspanin proteins is in stabilizing the migrasome structure, while the migrasome nucleation and initial growth stages can be driven by membrane mechanical stresses.