RESUMO
Mass-tolerant open search methods allow the high-throughput analysis of modified peptides by mass spectrometry. These techniques have paved the way to unbiased analysis of post-translational modifications in biological contexts, as well as of chemical modifications produced during the manipulation of protein samples. In this work, we have analyzed in-depth a wide variety of samples of different biological origin, including cells, extracellular vesicles, secretomes, centrosomes and tissue preparations, using Comet-ReCom, a recently improved version of the open search engine Comet-PTM. Our results demonstrate that glutamic acid residues undergo intensive methyl esterification when protein digestion is performed using in-gel techniques, but not using gel-free approaches. This effect was highly specific to Glu and was not found for other methylable residues such as Asp.
Assuntos
Ácido Glutâmico , Metanol , Metanol/química , Metilação , Humanos , Ácido Glutâmico/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , AnimaisRESUMO
Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.
Assuntos
Linfócitos T CD8-Positivos , Citosol , Ativação Linfocitária , Mitocôndrias , Biossíntese de Proteínas , Receptores de Antígenos de Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Ativação Linfocitária/imunologia , Citosol/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/imunologia , Citoesqueleto/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Open-search methods allow unbiased, high-throughput identification of post-translational modifications in proteins at an unprecedented scale. The performance of current open-search algorithms is diminished by experimental errors in the determination of the precursor peptide mass. In this work we propose a semi-supervised open search approach, called ReCom, that minimizes this effect by taking advantage of a priori known information from a reference database, such as Unimod or a database provided by the user. We present a proof-of-concept study using Comet-ReCom, an improved version of Comet-PTM. Comet-ReCom increased identification performance of Comet-PTM by 68%. This increased performance of Comet-ReCom to score the MS/MS spectrum comes in parallel with a significantly better assignation of the monoisotopic peak of the precursor peptide in the MS spectrum, even in cases of peptide coelution. Our data demonstrate that open searches using ultra-tolerant mass windows can benefit from using a semi-supervised approach that takes advantage from previous knowledge on the nature of protein modifications. SIGNIFICANCE: The present study introduces a novel approach to ultra-tolerant database search, which employs prior knowledge of post-translational modifications (PTMs) to improve identification of modified peptides. This method addresses the limitations related to experimental errors and precursor mass assignation of previous open-search methods. Thus, it enables the study of the biological significance of a wider variety of PTMs, including unknown or unexpected modifications that may have gone unnoticed using non-supervised search methods.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Espectrometria de Massas em Tandem/métodos , Peptídeos/metabolismo , Proteínas/metabolismo , Algoritmos , Processamento de Proteína Pós-Traducional , Bases de Dados de Proteínas , SoftwareRESUMO
The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.
Assuntos
Linfócitos T CD4-Positivos , Proteínas Associadas aos Microtúbulos , Mitocôndrias , Células Jurkat , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Mitocôndrias/metabolismo , Tubulina (Proteína)/metabolismo , Citoesqueleto/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD28/metabolismo , Potencial da Membrana Mitocondrial , Sinapses ImunológicasRESUMO
Air pollutants increase the risk and mortality of myocardial infarction (MI). The aim of this study was to assess the inflammatory changes in circulating immune cells and microRNAs in MIs related to short-term exposure to air pollutants. We studied 192 patients with acute coronary syndromes and 57 controls with stable angina. For each patient, air pollution exposure in the 24-h before admission, was collected. All patients underwent systematic circulating inflammatory cell analyses. According to PM2.5 exposure, 31 patients were selected for microRNA analyses. STEMI patients exposed to PM2.5 showed a reduction of CD4+ regulatory T cells. Furthermore, in STEMI patients the exposure to PM2.5 was associated with an increase of miR-146a-5p and miR-423-3p. In STEMI and NSTEMI patients PM2.5 exposure was associated with an increase of miR-let-7f-5p. STEMI related to PM2.5 short-term exposure is associated with changes involving regulatory T cells, miR-146a-5p and miR-423-3p.
Assuntos
Poluentes Atmosféricos , Poluição do Ar , MicroRNAs , Infarto do Miocárdio , Poluentes Atmosféricos/toxicidade , Biomarcadores , Humanos , MicroRNAs/genética , Infarto do Miocárdio/genéticaRESUMO
Exosomes are extracellular vesicles (EVs) containing different biomolecules with biological activity, such as proteins, miRNA, long noncoding RNA, and DNA. EVs are efficient platforms for intercellular communication, especially during immune responses, but also in some pathological contexts, such as tumor cell growth. The precise assessment of EV content is relevant for the selection of specific vesicles with specialized biological activities, whose content is hardly visualized due to their small size. We describe herein a protocol for the determination of the content of individual EVs through microscopy imaging and user-friendly analysis using TIRF microscopy.
Assuntos
DNA/análise , Exossomos/química , Proteínas/análise , RNA/análise , Comunicação Celular , DNA/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Humanos , Microscopia de Fluorescência , Proteínas/metabolismo , RNA/metabolismoRESUMO
BACKGROUND: Spontaneous Coronary Artery Dissection (SCAD) is an important cause of acute coronary syndromes, particularly in young to middle-aged women. Differentiating acute SCAD from coronary atherothrombosis remains a major clinical challenge. METHODS: A case-control study was used to explore the usefulness of circulating miRNAs to discriminate both clinical entities. The profile of miRNAs was evaluated using an unbiased human RT-PCR platform and confirmed using individual primers. miRNAs were evaluated in plasma samples from acute SCAD and atherothrombotic acute myocardial infarction (AT-AMI) from two independent cohorts; discovery cohort (SCAD n = 15, AT-AMI n = 15), and validation cohort (SCAD n = 11, AT-AMI n = 41) with 9 healthy control subjects. Plasma levels of IL-8, TGFB1, TGBR1, Endothelin-1 and MMP2 were analysed by ELISA assays. FINDINGS: From 15 differentially expressed miRNAs detected in cohort 1, we confirmed in cohort 2 the differential expression of 4 miRNAs: miR-let-7f-5p, miR-146a-5p, miR-151a-3p and miR-223-5p, whose expression was higher in SCAD compared to AT-AMI. The combined expression of these 4 miRNAs showed the best predictive value to distinguish between both entities (AUC: 0.879, 95% CI 0.72-1.0) compared to individual miRNAs. Functional profiling of target genes identified an association with blood vessel biology, TGF-beta pathway and cytoskeletal traction force. ELISA assays showed high plasma levels of IL-8, TGFB1, TGFBR1, Endothelin-1 and MMP2 in SCAD patients compared to AT-AMI. INTERPRETATION: We present a novel signature of plasma miRNAs in patients with SCAD. miR-let-7f-5p, miR-146a-5p, miR-151a-3p and miR-223-5p discriminate SCAD from AT-AMI patients and also shed light on the pathological mechanisms underlying this condition. FUNDING: Spanish Ministry of Economy and Competitiveness (MINECO): Plan Nacional de Salud SAF2017-82886-R, Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV). Fundación BBVA a equipos de Investigación Científica 2018 and from Caixa Banking Foundation under the project code HR17-00016 to F.S.M. Instituto de Salud Carlos III (AES 2019): PI19/00565 to F.R, PI19/00545 to P.M. CAM (S2017/BMD-3671-INFLAMUNE-CM) from Comunidad de Madrid to FSM and PM. The UK SCAD study was supported by BeatSCAD, the British Heart Foundation (BHF) PG/13/96/30608 the NIHR rare disease translational collaboration and the Leicester NIHR Biomedical Research Centre.
Assuntos
Biomarcadores , Anomalias dos Vasos Coronários/etiologia , Anomalias dos Vasos Coronários/fisiopatologia , Regulação da Expressão Gênica , MicroRNAs/genética , Doenças Vasculares/congênito , Adulto , Estudos de Casos e Controles , MicroRNA Circulante , Anomalias dos Vasos Coronários/diagnóstico , Anomalias dos Vasos Coronários/metabolismo , Diagnóstico Diferencial , Suscetibilidade a Doenças , Feminino , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Interferência de RNA , RNA Mensageiro , Curva ROC , Doenças Vasculares/diagnóstico , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo , Doenças Vasculares/fisiopatologiaRESUMO
Coronavirus Disease 2019 (COVID-19) pneumonia is a life-threatening infectious disease, especially for elderly patients with multiple comorbidities. Despite enormous efforts to understand its underlying etiopathogenic mechanisms, most of them remain elusive. In this study, we compared differential plasma miRNAs and cytokines profiles between COVID-19 and other community-acquired pneumonias (CAP). A first screening and subsequent validation assays in an independent cohort of patients revealed a signature of 15 dysregulated miRNAs between COVID-19 and CAP patients. Additionally, multivariate analysis displayed a combination of 4 miRNAs (miR-106b-5p, miR-221-3p, miR-25-3p and miR-30a-5p) that significantly discriminated between both pathologies. Search for targets of these miRNAs, combined with plasma protein measurements, identified a differential cytokine signature between COVID-19 and CAP that included EGFR, CXCL12 and IL-10. Significant differences were also detected in plasma levels of CXCL12, IL-17, TIMP-2 and IL-21R between mild and severe COVID-19 patients. These findings provide new insights into the etiopathological mechanisms underlying COVID-19.
Assuntos
COVID-19/imunologia , MicroRNA Circulante/sangue , Citocinas/sangue , Pneumonia/imunologia , Biomarcadores/sangue , COVID-19/sangue , Estudos de Coortes , Infecções Comunitárias Adquiridas/sangue , Infecções Comunitárias Adquiridas/imunologia , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Pneumonia/sangueRESUMO
miRNAs have been associated with psoriasis since just over a decade. However, we are far from a complete understanding of their role during the development of this disease. Our objective was to characterize the cutaneous expression of miRNAs not previously described in psoriasis, the changes induced following the treatment with biologicals and their association with disease improvement. Next generation sequencing was performed from five skin samples from psoriasis patients (lesional and non-lesional skin) and five controls, and from this cohort, 12 microRNAs were selected to be analyzed in skin samples from 44 patients with plaque psoriasis. In 15 patients, an additional sample was obtained after three months of biological treatment. MiR-9-5p, miR-133a-3p and miR-375 were downregulated in the lesional skin of psoriasis patients. After treatment, expression of miR-133a-3p, miR-375, miR-378a and miR-135b in residual lesions returned towards the levels observed in non-lesional skin. The decrease in miR-135b levels after treatment with biologics was associated with both the improvement of patients evaluated through Psoriasis Area and Severity Index score and the decrease in local inflammatory response. Moreover, basal expression of miR-135b along with age was associated with the improvement of psoriasis, suggesting its possible usefulness as a prognostic biomarker.