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1.
Immunity ; 55(12): 2436-2453.e5, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36462503

RESUMEN

The factors that influence survival during severe infection are unclear. Extracellular chromatin drives pathology, but the mechanisms enabling its accumulation remain elusive. Here, we show that in murine sepsis models, splenocyte death interferes with chromatin clearance through the release of the DNase I inhibitor actin. Actin-mediated inhibition was compensated by upregulation of DNase I or the actin scavenger gelsolin. Splenocyte death and neutrophil extracellular trap (NET) clearance deficiencies were prevalent in individuals with severe COVID-19 pneumonia or microbial sepsis. Activity tracing by plasma proteomic profiling uncovered an association between low NET clearance and increased COVID-19 pathology and mortality. Low NET clearance activity with comparable proteome associations was prevalent in healthy donors with low-grade inflammation, implicating defective chromatin clearance in the development of cardiovascular disease and linking COVID-19 susceptibility to pre-existing conditions. Hence, the combination of aberrant chromatin release with defects in protective clearance mechanisms lead to poor survival outcomes.


Asunto(s)
COVID-19 , Sepsis , Animales , Ratones , Actinas , Cromatina , Desoxirribonucleasa I , ADN , Neutrófilos , Proteómica
2.
Cell ; 163(3): 734-45, 2015 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-26456112

RESUMEN

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.


Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas de Complejo Poro Nuclear/química , Proteínas Nucleares/química , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Carioferinas/química , Carioferinas/metabolismo , Modelos Moleculares , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae
3.
Semin Cell Dev Biol ; 68: 34-41, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28669824

RESUMEN

The nuclear pore complex (NPC) forms a permeability barrier between the nucleus and the cytoplasm. Molecules that are able to cross this permeability barrier encounter different disordered phenylalanine glycine rich nucleoporins (FG-Nups) that act as a molecular filter and regulate the selective NPC crossing of biomolecules. In this review, we provide a current overview regarding the interaction mechanism between FG-Nups and the carrier molecules that recognize and enable the transport of cargoes through the NPC aiming to understand the general molecular mechanisms that facilitate the nucleocytoplasmic transport.


Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Humanos
4.
Angew Chem Int Ed Engl ; 58(14): 4720-4724, 2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30703278

RESUMEN

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time-resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinß) with drastically different binding mechanisms, coupled folding-binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG-Importinß fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB-Importinß complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.


Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Termodinámica , Agua/metabolismo , beta Carioferinas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Conformación Proteica , Agua/química , beta Carioferinas/química
5.
Chembiochem ; 17(16): 1518-24, 2016 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-27223658

RESUMEN

Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.


Asunto(s)
Aminoácidos/química , Ciclooctanos/química , Colorantes Fluorescentes/química , Proteínas/química , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Fluorescente , Estructura Molecular
6.
Chemistry ; 21(35): 12266-70, 2015 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-26177861

RESUMEN

trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped-flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.


Asunto(s)
Aminoácidos/química , Ciclooctanos/química , Línea Celular , Estructura Molecular
7.
Elife ; 122024 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-39009040

RESUMEN

Background: Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin. Methods: Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors. Results: We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01-2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 µg/mL, p=0.004). Conclusions: Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin. Funding: LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust). Clinical trial number: NCT04359654.


Asunto(s)
Antiinflamatorios , Tratamiento Farmacológico de COVID-19 , COVID-19 , Desoxirribonucleasa I , Humanos , Masculino , Femenino , Desoxirribonucleasa I/administración & dosificación , Desoxirribonucleasa I/uso terapéutico , Persona de Mediana Edad , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Anciano , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Trampas Extracelulares/efectos de los fármacos , SARS-CoV-2 , Proteína C-Reactiva/análisis , Proteína C-Reactiva/metabolismo , Dexametasona/administración & dosificación , Dexametasona/uso terapéutico , Adulto , Nebulizadores y Vaporizadores , Resultado del Tratamiento , Administración por Inhalación
8.
J Clin Invest ; 133(14)2023 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-37192000

RESUMEN

Increased levels and diversity of human endogenous retrovirus (HERV) transcription characterize most cancer types and are linked with disease outcomes. However, the underlying processes are incompletely understood. Here, we show that elevated transcription of HERVH proviruses predicted survival of lung squamous cell carcinoma (LUSC) and identified an isoform of CALB1, encoding calbindin, ectopically driven by an upstream HERVH provirus under the control of KLF5, as the mediator of this effect. HERVH-CALB1 expression was initiated in preinvasive lesions and associated with their progression. Calbindin loss in LUSC cell lines impaired in vitro and in vivo growth and triggered senescence, consistent with a protumor effect. However, calbindin also directly controlled the senescence-associated secretory phenotype (SASP), marked by secretion of CXCL8 and other neutrophil chemoattractants. In established carcinomas, CALB1-negative cancer cells became the dominant source of CXCL8, correlating with neutrophil infiltration and worse prognosis. Thus, HERVH-CALB1 expression in LUSC may display antagonistic pleiotropy, whereby the benefits of escaping senescence early during cancer initiation and clonal competition were offset by the prevention of SASP and protumor inflammation at later stages.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Retrovirus Endógenos , Neoplasias Pulmonares , Humanos , Calbindinas/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Senescencia Celular/genética , Retrovirus Endógenos/genética , Neoplasias Pulmonares/genética , Provirus/genética
9.
Cell Stem Cell ; 30(6): 781-799.e9, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37267914

RESUMEN

Somatic mutations commonly occur in hematopoietic stem cells (HSCs). Some mutant clones outgrow through clonal hematopoiesis (CH) and produce mutated immune progenies shaping host immunity. Individuals with CH are asymptomatic but have an increased risk of developing leukemia, cardiovascular and pulmonary inflammatory diseases, and severe infections. Using genetic engineering of human HSCs (hHSCs) and transplantation in immunodeficient mice, we describe how a commonly mutated gene in CH, TET2, affects human neutrophil development and function. TET2 loss in hHSCs produce a distinct neutrophil heterogeneity in bone marrow and peripheral tissues by increasing the repopulating capacity of neutrophil progenitors and giving rise to low-granule neutrophils. Human neutrophils that inherited TET2 mutations mount exacerbated inflammatory responses and have more condensed chromatin, which correlates with compact neutrophil extracellular trap (NET) production. We expose here physiological abnormalities that may inform future strategies to detect TET2-CH and prevent NET-mediated pathologies associated with CH.


Asunto(s)
Dioxigenasas , Neutrófilos , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas , Células Madre Hematopoyéticas/fisiología , Médula Ósea , Hematopoyesis/genética , Mutación , Proteínas de Unión al ADN/genética , Dioxigenasas/genética
10.
Cardiovasc Res ; 118(13): 2737-2753, 2022 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-34648022

RESUMEN

At the frontline of the host defence response, neutrophil antimicrobial functions have adapted to combat infections and injuries of different origins and magnitude. The release of web-like DNA structures named neutrophil extracellular traps (NETs) constitutes an important mechanism by which neutrophils prevent pathogen dissemination or deal with microorganisms of a bigger size. At the same time, nuclear and granule proteins with microbicidal activity bind to these DNA structures promoting the elimination of entrapped pathogens. However, these toxic properties may produce unwanted effects in the host, when neutrophils uncontrollably release NETs upon persistent inflammation. As a consequence, NET accumulation can produce vessel occlusion, tissue damage, and prolonged inflammation associated with the progression and exacerbation of multiple pathologic conditions. This review outlines recent advances in understanding the mechanisms of NET release and functions in sterile disease. We also discuss mechanisms of physiological regulation and the importance of neutrophil heterogeneity in NET formation and composition.


Asunto(s)
Trampas Extracelulares , Humanos , Neutrófilos/metabolismo , Inflamación/metabolismo , ADN/metabolismo
11.
Nat Commun ; 13(1): 4658, 2022 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-35945238

RESUMEN

The mechanisms linking systemic infection to hyperinflammation and immune dysfunction in sepsis are poorly understood. Extracellular histones promote sepsis pathology, but their source and mechanism of action remain unclear. Here, we show that by controlling fungi and bacteria captured by splenic macrophages, neutrophil-derived myeloperoxidase attenuates sepsis by suppressing histone release. In systemic candidiasis, microbial capture via the phagocytic receptor SIGNR1 neutralizes myeloperoxidase by facilitating marginal zone infiltration and T cell death-dependent histone release. Histones and hyphae induce cytokines in adjacent CD169 macrophages including G-CSF that selectively depletes mature Ly6Ghigh neutrophils by shortening their lifespan in favour of immature Ly6Glow neutrophils with a defective oxidative burst. In sepsis patient plasma, these mediators shorten mature neutrophil lifespan and correlate with neutrophil mortality markers. Consequently, high G-CSF levels and neutrophil lifespan shortening activity are associated with sepsis patient mortality. Hence, by exploiting phagocytic receptors, pathogens degrade innate and adaptive immunity through the detrimental impact of downstream effectors on neutrophil lifespan.


Asunto(s)
Neutrófilos , Sepsis , Factor Estimulante de Colonias de Granulocitos/metabolismo , Histonas/metabolismo , Humanos , Longevidad , Macrófagos/metabolismo , Peroxidasa/metabolismo , Linfocitos T/metabolismo
12.
Stem Cell Reports ; 16(3): 428-436, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33581053

RESUMEN

We document here that intensive care COVID-19 patients suffer a profound decline in hemoglobin levels but show an increase of circulating nucleated red cells, suggesting that SARS-CoV-2 infection either directly or indirectly induces stress erythropoiesis. We show that ACE2 expression peaks during erythropoiesis and renders erythroid progenitors vulnerable to infection by SARS-CoV-2. Early erythroid progenitors, defined as CD34-CD117+CD71+CD235a-, show the highest levels of ACE2 and constitute the primary target cell to be infected during erythropoiesis. SARS-CoV-2 causes the expansion of colony formation by erythroid progenitors and can be detected in these cells after 2 weeks of the initial infection. Our findings constitute the first report of SARS-CoV-2 infectivity in erythroid progenitor cells and can contribute to understanding both the clinical symptoms of severe COVID-19 patients and how the virus can spread through the circulation to produce local inflammation in tissues, including the bone marrow.


Asunto(s)
COVID-19/virología , Células Precursoras Eritroides/virología , Eritropoyesis/fisiología , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , Línea Celular , Chlorocebus aethiops , Células Precursoras Eritroides/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/virología , Células Vero
13.
Nat Commun ; 11(1): 5566, 2020 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-33149141

RESUMEN

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.


Asunto(s)
Trampas Extracelulares/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interferón Tipo I/metabolismo , Pulmón/microbiología , Mycobacterium tuberculosis/inmunología , Neutrófilos/inmunología , Neumonía/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Bases de Datos Genéticas , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Interferón Tipo I/genética , Interferón gamma/genética , Interferón gamma/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/patogenicidad , Neumonía/genética , Neumonía/metabolismo , Neumonía/patología , RNA-Seq , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/microbiología
14.
Cell Rep ; 22(13): 3660-3671, 2018 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-29590630

RESUMEN

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an "archetypal-fuzzy" complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lower than in the archetypal-fuzzy complex between FG-Nup153 and NTRs. Unexpectedly, this behavior appears not to be encoded selectively into CRM1 but rather into the FG-Nup214 sequence. The same distinct binding mechanisms are unperturbed in O-linked ß-N-acetylglucosamine-modified FG-Nups. Our results have implications for differential roles of distinctly spatially distributed FG-Nup⋅NTR interactions in the cell.


Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Glicina/metabolismo , Humanos , Modelos Moleculares , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Fenilalanina/metabolismo , Unión Proteica , Conformación Proteica
15.
Nat Protoc ; 10(5): 780-91, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25906116

RESUMEN

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.


Asunto(s)
Aminoácidos/química , Química Clic/métodos , Colorantes Fluorescentes/química , Proteínas/química , Alquinos/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Azidas/química , Carbocianinas/química , Química Clic/instrumentación , Codón de Terminación , Reacción de Cicloadición , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Mamíferos , Mutagénesis Sitio-Dirigida , Proteínas/genética , Receptor de Insulina/química , Receptor de Insulina/genética
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