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1.
Mol Cell ; 61(1): 138-52, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26669262

RESUMO

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5'→3' processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm.


Assuntos
Proteínas Argonautas/metabolismo , Núcleo Celular/enzimologia , Citosol/enzimologia , Exonucleases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exonucleases/química , Exonucleases/genética , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
2.
EMBO J ; 36(17): 2626-2641, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765164

RESUMO

Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes.


Assuntos
Inativação Gênica , Heterocromatina/genética , RNA Longo não Codificante/genética , Regulação Fúngica da Expressão Gênica , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
3.
Mol Cell ; 47(6): 970-9, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22902560

RESUMO

Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other cochaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be byproducts of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Proteínas Argonautas/biossíntese , Proteínas Argonautas/metabolismo , Linhagem Celular , Metilação de DNA , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a Tacrolimo/deficiência
4.
Clin Chem Lab Med ; 57(7): 1053-1062, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-30422797

RESUMO

Background Extracellular vesicle (EV)-associated microRNAs (miRNAs) have been suggested as promising biomarkers for blood-based cancer diagnosis. However, one of the major limitations for the use of EVs with diagnostic purpose is the lack of standardized EV-profiling techniques. In this regard, the objective of our study was to design an integrated next-generation sequencing (NGS)-based workflow for analyzing the signature of EV-associated miRNA in the plasma of platinum-resistant ovarian cancer patients. Methods For EV-extraction, different enrichment methods were compared (ExoQuick vs. exoRNeasy). NGS was performed with the Illumina platform. Results We established an integrated NGS-based workflow, including EV-enrichment with the ExoQuick system, which resulted in an optimal RNA-yield and consistent small RNA libraries. We applied this workflow in a pilot cohort of clinically documented platinum-sensitive (n=15) vs. platinum-resistant (n=15) ovarian cancer patients, resulting in a panel of mature EV-associated miRNAs (including ovarian cancer associated miR-181a, miR-1908, miR-21, miR-486 and miR-223), which were differentially abundant in the plasma of platinum-resistant patients. Conclusions This is the first study, analyzing the profile of EV-associated miRNAs in platinum-resistant ovarian cancer patients. We provide rationale to further validate these miRNA candidates in an independent set of patients, in order to characterize their biomarker potential as predictors for platinum-resistance.


Assuntos
Biomarcadores Tumorais/sangue , Vesículas Extracelulares/metabolismo , MicroRNAs/sangue , Neoplasias Ovarianas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexos de Coordenação/química , Complexos de Coordenação/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Vesículas Extracelulares/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Projetos Piloto , Platina/química , Intervalo Livre de Progressão , Projetos de Pesquisa , Análise de Sequência de RNA , Fluxo de Trabalho
5.
J Environ Manage ; 233: 30-38, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30554022

RESUMO

A central challenge in the Mississippi River Basin is how to continue to support profitable agricultural production, provide water supply, flood control, transportation, and other benefits, while reducing the current burden of environmental degradation. Several practices have been shown to reduce nutrient runoff and water pollution, and improve soil fertility, while often yielding profits for farmers. Yet many of these beneficial practices remain underutilized. Participants at an expert workshop identified five candidate financial mechanisms that could increase adoption of these beneficial farming practices in four focal Midwest states in the next five years: crop insurance premium subsidies, transformation of the private service provider business model, expansion and targeting of 2019 U.S. Farm Bill funding, development of new state funds, and direction of post-disaster federal funds towards habitat restoration, particularly in floodplains. This study provides rough approximations of the change in nutrient runoff and greenhouse gas (GHG) emissions, the annualized costs, and the nutrient and GHG reductions per dollar likely to result from deployment of each financial mechanism. Based upon these approximations, the adoption of these programs could reduce annual nitrate flows at the outlet of the Ohio and Upper Mississippi River Basins by 25%, surpassing the intermediate 2025 target (20% reduction) and achieving more than half of the long-term target (45% reduction) set by the Mississippi River/Gulf of Mexico Hypoxia Task Force. These approximations also illustrate that these five mechanisms could provide the same GHG reductions (∼43 Tg CO2e yr-1) as taking 12 coal-fired energy plants offline. The total cost of these five financial mechanisms is estimated at ∼$2.6 billion, or 64 g of nitrates and ∼17 kg of CO2e per dollar spent. These proposed solutions all face political, financial, cultural or institutional challenges, but with industry support, creative political action, and continued communication of both private and public benefits, they can create meaningful nutrient reductions and rebuild soils by 2022.


Assuntos
Motivação , Solo , Golfo do México , Mississippi , Ohio
6.
Nature ; 480(7376): 264-7, 2011 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-22121019

RESUMO

Repetitive-element-derived Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility. A third Piwi family member, Miwi (also known as Piwil1), is expressed in specific postnatal germ cells and associates with a unique set of piRNAs of unknown function. Here we show that Miwi is a small RNA-guided RNase (slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation causes male infertility, and mutant germ cells show increased accumulation of LINE1 retrotransposon transcripts. We provide evidence for Miwi slicer activity directly cleaving transposon messenger RNAs, offering an explanation for the continued maintenance of repeat-derived piRNAs long after transposon silencing is established in germline stem cells. Furthermore, our study supports a slicer-dependent silencing mechanism that functions without piRNA amplification. Thus, Piwi proteins seem to act in a two-pronged mammalian transposon silencing strategy: one promotes transcriptional repression in the embryo, the other reinforces silencing at the post-transcriptional level after birth.


Assuntos
Proteínas Argonautas/metabolismo , Biocatálise , Elementos de DNA Transponíveis/genética , Inativação Gênica , Elementos Nucleotídeos Longos e Dispersos/genética , RNA Interferente Pequeno/biossíntese , Animais , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Infertilidade Masculina/genética , Masculino , Camundongos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Espermatogênese/genética , Especificidade por Substrato
7.
Nucleic Acids Res ; 42(10): 6208-18, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24782529

RESUMO

The Piwi-interacting RNA (piRNA)-interacting Piwi protein is involved in transcriptional silencing of transposable elements in ovaries of Drosophila melanogaster. Here we characterized the genome-wide effect of nuclear Piwi elimination on the presence of the heterochromatic H3K9me3 mark and HP1a, as well as on the transcription-associated mark H3K4me2. Our results demonstrate that a significant increase in the H3K4me2 level upon nuclear Piwi loss is not accompanied by the alterations in H3K9me3 and HP1a levels for several germline-expressed transposons, suggesting that in this case Piwi prevents transcription by a mechanism distinct from H3K9 methylation. We found that the targets of Piwi-dependent chromatin repression are mainly related to the elements that display a higher level of H3K4me2 modification in the absence of silencing, i.e. most actively transcribed elements. We also show that Piwi-guided silencing does not significantly influence the chromatin state of dual-strand piRNA-producing clusters. In addition, host protein-coding gene expression is essentially not affected due to the nuclear Piwi elimination, but we noted an increase in small nuclear spliceosomal RNAs abundance and propose Piwi involvement in their post-transcriptional regulation. Our work reveals new aspects of transposon silencing in Drosophila, indicating that transcription of transposons can underpin their Piwi dependent silencing, while canonical heterochromatin marks are not obligatory for their repression.


Assuntos
Proteínas Argonautas/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Inativação Gênica , Animais , Proteínas Argonautas/genética , Núcleo Celular/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Histonas/metabolismo , Ovário/metabolismo , RNA Nuclear Pequeno/metabolismo , Retroelementos
8.
RNA ; 18(12): 2128-34, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23086923

RESUMO

Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcripts has been long sought. A Phospholipase D-like protein, Zucchini, that is essential for piRNA processing has been proposed to be a nuclease acting in piRNA biogenesis. Here we describe the crystal structure of Zucchini from Drosophila melanogaster and show that it is very similar to the bacterial endonuclease, Nuc. The structure also reveals that homodimerization induces major conformational changes assembling the active site. The active site is situated on the dimer interface at the bottom of a narrow groove that can likely accommodate single-stranded nucleic acid substrates. Furthermore, biophysical analysis identifies protein segments essential for dimerization and provides insights into regulation of Zucchini's activity.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Endorribonucleases/química , Endorribonucleases/metabolismo , RNA Interferente Pequeno/biossíntese , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Endorribonucleases/genética , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipase D/química , Fosfolipase D/genética , Multimerização Proteica , Homologia de Sequência de Aminoácidos , Eletricidade Estática
9.
Proc Natl Acad Sci U S A ; 108(26): 10579-84, 2011 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-21670278

RESUMO

In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.


Assuntos
Cromatina/metabolismo , Ribonucleoproteínas/metabolismo , Espermatogênese , Animais , Cromossomos Artificiais Bacterianos , Masculino , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologia
10.
Blood ; 118(10): 2774-83, 2011 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-21757614

RESUMO

The human plasma protein ß(2)-glycoprotein I (ß(2)-GPI) is the major target of autoantibodies associated with antiphospholipid syndrome. However, the biologic function of this abundant protein is still unclear. Here we identify ß(2)-GPI as a complement regulator. ß(2)-GPI circulates in the plasma in an inactive circular form. On surface binding, such as to apoptotic cells, ß(2)-GPI changes conformation to an elongated form that acquires C3/C3b binding activities. ß(2)-GPI apparently changes conformation of C3, so that the regulator factor H attaches and induces subsequent degradation by the protease factor I. ß(2)-GPI also mediates further cleavage of C3/C3b compared with factor H alone. Our data provide important insights into innate immune regulation by plasma protein ß(2)-GPI, which may be exploited in the prevention and therapy of autoimmune disease antiphospholipid syndrome.


Assuntos
Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , Complemento C3/imunologia , Complemento C3b/imunologia , beta 2-Glicoproteína I/metabolismo , Síndrome Antifosfolipídica/fisiopatologia , Apoptose , Western Blotting , Ativação do Complemento , Complemento C3/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Humanos , Imunoprecipitação , Conformação Molecular , Ligação Proteica , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , beta 2-Glicoproteína I/imunologia
11.
Proc Natl Acad Sci U S A ; 107(26): 11841-6, 2010 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-20534472

RESUMO

Piwi-interacting RNAs (piRNAs) are essential for silencing of transposable elements in the germline, but their biogenesis is poorly understood. Here we demonstrate that MOV10L1, a germ cell-specific putative RNA helicase, is associated with Piwi proteins. Genetic disruption of the MOV10L1 RNA helicase domain in mice renders both MILI and MIWI2 devoid of piRNAs. Absence of a functional piRNA pathway in Mov10l1 mutant testes causes loss of DNA methylation and subsequent derepression of retrotransposons in germ cells. The Mov10l1 mutant males are sterile owing to complete meiotic arrest. This mouse mutant expresses Piwi proteins but lacks piRNAs, suggesting that MOV10L1 is required for piRNA biogenesis and/or loading to Piwi proteins.


Assuntos
RNA Helicases/genética , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas , Sequência de Bases , Proteínas de Ciclo Celular , Metilação de DNA , Primers do DNA/genética , Fertilidade , Masculino , Meiose , Camundongos , Camundongos Knockout , Mutação , Proteínas/metabolismo , RNA Helicases/deficiência , Retroelementos/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Espermatócitos/metabolismo , Espermatogênese , Espermatogônias/metabolismo , Testículo/metabolismo
12.
Front Cell Neurosci ; 17: 1214003, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37964793

RESUMO

Aging of the peripheral nervous system (PNS) is associated with structural and functional changes that lead to a reduction in regenerative capacity and the development of age-related peripheral neuropathy. Myelin is central to maintaining physiological peripheral nerve function and differences in myelin maintenance, degradation, formation and clearance have been suggested to contribute to age-related PNS changes. Recent proteomic studies have elucidated the complex composition of the total myelin proteome in health and its changes in neuropathy models. However, changes in the myelin proteome of peripheral nerves during aging have not been investigated. Here we show that the proteomes of myelin fractions isolated from young and old nerves show only subtle changes. In particular, we found that the three most abundant peripheral myelin proteins (MPZ, MBP, and PRX) do not change in old myelin fractions. We also show a tendency for high-abundance myelin proteins other than these three to be downregulated, with only a small number of ribosome-related proteins significantly downregulated and extracellular matrix proteins such as collagens upregulated. In addition, we illustrate that the peripheral nerve myelin proteome reported in this study is suitable for assessing myelin degradation and renewal during peripheral nerve degeneration and regeneration. Our results suggest that the peripheral nerve myelin proteome is relatively stable and undergoes only subtle changes in composition during mouse aging. We proffer the resultant dataset as a resource and starting point for future studies aimed at investigating peripheral nerve myelin during aging. Said datasets are available in the PRIDE archive under the identifier PXD040719 (aging myelin proteome) and PXD041026 (sciatic nerve injury proteome).

13.
Hamostaseologie ; 43(3): 215-218, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34327693

RESUMO

Immune thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder characterized by severely reduced activity of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) due to autoantibodies. This leads to the development of pathogenic multimers of VWF, causing a thrombotic microangiopathy with decreased number of platelets, hemolysis, and life-threatening tissue ischemia of mostly brain, heart, and kidneys. Standard treatment of iTTP involves daily plasma exchange to remove ultra large multimers of VWF, inhibitors, substituting ADAMTS13, and the accompaniment of an immunosuppressive treatment with steroids. Recently, caplacizumab was approved for iTTP. Caplacizumab is a nanobody binding the A1 domain of VWF, blocking its interaction with glycoprotein Ib-IX-V platelet receptor and therefore preventing platelet aggregation. VWF activities may serve as therapeutic drug monitoring of caplacizumab, whereas ADAMTS13 activities may be used for biomarkers to guide caplacizumab treatment modalities and overall treatment duration. Additional immunosuppressive treatment by inhibiting autoantibody formation (e.g., the use of Rituximab, a chimeric monoclonal antibody directed against the B-cell antigen CD20) is a further treatment option. Infections are well-known causes for an acute episode for patients with iTTP. The novel SARS-CoV-2 virus is mainly associated with acute respiratory distress as well as diffuse endothelial inflammation and increased coagulopathy. However, little is known about an infection with SARS-CoV-2 virus triggering iTTP relapses. We herein report the case of an acute iTTP episode accompanying a SARS-CoV-2 infection.


Assuntos
COVID-19 , Púrpura Trombocitopênica Idiopática , Púrpura Trombocitopênica Trombótica , Trombose , Humanos , Púrpura Trombocitopênica Trombótica/diagnóstico , Púrpura Trombocitopênica Trombótica/terapia , Fator de von Willebrand/metabolismo , COVID-19/complicações , SARS-CoV-2 , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Imunossupressores/uso terapêutico , Trombose/tratamento farmacológico , Autoanticorpos , Proteína ADAMTS13/uso terapêutico
14.
J Biol Chem ; 285(49): 38473-85, 2010 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-20855886

RESUMO

Group A streptococci (GAS) utilize soluble human complement regulators to evade host complement attack. Here, we characterized the binding of the terminal complement complex inhibitor complement Factor H-related protein 1 (CFHR1) and of the C3 convertase regulator Factor H to the streptococcal collagen-like proteins (Scl). CFHR1 and Factor H, but no other member of the Factor H protein family (CFHR2, CFHR3, or CFHR4A), bound to the two streptococcal proteins Scl1.6 and Scl1.55, which are expressed by GAS serotypes M6 and M55. The two human regulators bound to the Scl1 proteins via their conserved C-terminal attachment region, i.e. CFHR1 short consensus repeats 3-5 (SCR3-5) and Factor H SCR18-20. Binding was affected by ionic strength and by heparin. CFHR1 and the C-terminal attachment region of Factor H did not bind to Scl1.1 and Scl2.28 proteins but did bind to intact M1-type and M28-type GAS, which express Scl1.1 and Scl2.28, respectively, thus arguing for the presence of an additional binding mechanism to CFHR1 and Factor H. Furthermore mutations within the C-terminal heparin-binding region and Factor H mutations that are associated with the acute renal disease atypical hemolytic uremic syndrome blocked the interaction with the two streptococcal proteins. Binding of CFHR1 affected the complement regulatory functions of Factor H on the level of the C3 convertase. Apparently, streptococci utilize two types of complement regulator-acquiring surface proteins; type A proteins, as represented by Scl1.6 and Scl1.55, bind to CFHR1 and Factor H via their conserved C-terminal region and do not bind the Factor H-like protein 1 (FHL-1). On the contrary, type B proteins, represented by M-, M-like, and the fibronectin-binding protein Fba proteins, bind Factor H and FHL-1 via domain SCR7 and do not bind CFHR1. In conclusion, binding of CFHR1 is at the expense of Factor H-mediated regulatory function at the level of C3 convertase and at the gain of a regulator that controls complement at the level of the C5 convertase and formation of the terminal complement complex.


Assuntos
Proteínas de Bactérias/metabolismo , Colágeno/metabolismo , Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Doença Aguda , Síndrome Hemolítico-Urêmica Atípica , Proteínas de Bactérias/genética , Colágeno/genética , Convertases de Complemento C3-C5/genética , Convertases de Complemento C3-C5/metabolismo , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Infecções Estreptocócicas/genética , Streptococcus pyogenes/genética
15.
J Thromb Haemost ; 19(9): 2335-2347, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34242469

RESUMO

BACKGROUND: Coronavirus disease 19 (COVID-19)-associated coagulopathy is a hallmark of disease severity and poor prognosis. The key manifestations of this prothrombotic syndrome-microvascular thrombosis, stroke, and venous and pulmonary clots-are also observed in severe and catastrophic antiphospholipid syndrome. Antiphospholipid antibodies (aPL) are detectable in COVID-19 patients, but their association with the clinical course of COVID-19 remains unproven. OBJECTIVES: To analyze the presence and relevance of lipid-binding aPL in hospitalized COVID-19 patients. METHODS: Two cohorts of 53 and 121 patients from a single center hospitalized for PCR-proven severe acute respiratory syndrome-coronavirus 2 infection were analyzed for the presence of aPL and clinical severity of COVID-19. RESULTS: We here demonstrate that lipid-binding aPL are common in COVID-19. COVID-19 patients with lipid-binding aPL have higher median concentrations of C-reactive protein and D-dimer, and are more likely to have a critical clinical course and fatal outcome. Lipid-binding aPL isolated from COVID-19 patients target the recently described cell surface complex of lysobisphosphatidic acid (LBPA) with the protein C receptor (EPCR) to induce prothrombotic and inflammatory responses in monocytes and endothelial cells. We show that B1a cells producing lipid-reactive aPL of the IgG isotype circulate in the blood of COVID-19 patients. In vivo, COVID-19 aPL accelerate thrombus formation in an experimental mouse model dependent on the recently delineated signaling pathway involving EPCR-LBPA. CONCLUSIONS: COVID-19 patients rapidly expand B1a cells secreting pathogenic lipid-binding aPL with broad thrombotic and inflammatory effects. The association with markers of inflammation and coagulation, clinical severity, and mortality suggests a causal role of aPL in COVID-19-associated coagulopathy.


Assuntos
Síndrome Antifosfolipídica , COVID-19 , Animais , Anticorpos Antifosfolipídeos , Células Endoteliais , Humanos , Camundongos , SARS-CoV-2
16.
PLoS Pathog ; 4(12): e1000250, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19112495

RESUMO

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a-to our knowledge-new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite SbiratioC3ratioFactor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and beta(2)-glycoprotein I and interferes with innate immune recognition.


Assuntos
Proteínas de Bactérias/farmacologia , Proteínas de Transporte/farmacologia , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Inativadores do Complemento/farmacologia , Multimerização Proteica , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Complemento C3b/química , Fator H do Complemento/química , Inativadores do Complemento/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Spodoptera , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo
17.
Clin Transl Gastroenterol ; 11(7): e00211, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32764204

RESUMO

INTRODUCTION: Frailty is a common but often underestimated complication in patients with liver cirrhosis. The Clinical Frailty Scale (CFS) allows the assessment of frailty within a short period of time but has only been investigated in a Canadian cohort of outpatients. The aim of the current study was to evaluate the ability of the CFS to predict mortality in outpatients and nonelectively hospitalized German patients. METHODS: Two hundred outpatients and 99 nonelectively hospitalized patients with liver cirrhosis were prospectively enrolled. Outpatients/inpatients were followed for a median of 364/28 days regarding the primary outcome of death or liver transplantation. Eighty-seven patients of the outpatient cohort and 64 patients of the inpatient cohort had available computed tomography-scans for the quantification of muscle mass. RESULTS: Median CFS was 3 in the outpatient and the inpatient cohort. Twenty-one (10.5%) outpatients were at least prefrail (CFS > 3) and 26 (26.3%) inpatients were frail (CFS > 4). For every one-unit increase, there was an independent association between the CFS and mortality in the outpatient cohort (hazard ratio 1.534, P = 0.007). This association remained significant after controlling for muscle mass in the subcohort with available computed tomography scans. In the inpatient cohort, frailty (CFS > 4) was an independent predictor for 28-day mortality after controlling for acute-on-chronic liver failure, albumin, and infections (odds ratio 4.627, P = 0.045). However, this association did not reach significance in a subcohort after controlling for muscle mass. DISCUSSION: Especially in outpatients, CFS is a useful predictor regarding increased mortality independent of the muscle mass.


Assuntos
Insuficiência Hepática Crônica Agudizada/epidemiologia , Fragilidade/diagnóstico , Cirrose Hepática/mortalidade , Índice de Gravidade de Doença , Insuficiência Hepática Crônica Agudizada/etiologia , Idoso , Feminino , Seguimentos , Fragilidade/etiologia , Alemanha/epidemiologia , Mortalidade Hospitalar , Hospitalização , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/cirurgia , Transplante de Fígado/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/diagnóstico por imagem , Estudos Prospectivos , Medição de Risco/métodos , Tomografia Computadorizada por Raios X
18.
Aging Cell ; 17(6): e12833, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30168637

RESUMO

The regenerative capacity of peripheral nerves declines during aging, contributing to the development of neuropathies, limiting organism function. Changes in Schwann cells prompt failures in instructing maintenance and regeneration of aging nerves; molecular mechanisms of which have yet to be delineated. Here, we identified an altered inflammatory environment leading to a defective Schwann cell response, as an underlying mechanism of impaired nerve regeneration during aging. Chronic inflammation was detected in intact uninjured old nerves, characterized by increased macrophage infiltration and raised levels of monocyte chemoattractant protein 1 (MCP1) and CC chemokine ligand 11 (CCL11). Schwann cells in the old nerves appeared partially dedifferentiated, accompanied by an activated repair program independent of injury. Upon sciatic nerve injury, an initial delayed immune response was followed by a persistent hyperinflammatory state accompanied by a diminished repair process. As a contributing factor to nerve aging, we showed that CCL11 interfered with Schwann cell differentiation in vitro and in vivo. Our results indicate that increased infiltration of macrophages and inflammatory signals diminish regenerative capacity of aging nerves by altering Schwann cell behavior. The study identifies CCL11 as a promising target for anti-inflammatory therapies aiming to improve nerve regeneration in old age.


Assuntos
Envelhecimento/patologia , Inflamação/patologia , Regeneração Nervosa , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Aspirina/farmacologia , Aspirina/uso terapêutico , Quimiocina CCL11/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Compressão Nervosa , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Nervos Periféricos/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/patologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia
19.
Semin Arthritis Rheum ; 32(5): 273-84, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12701038

RESUMO

OBJECTIVES: To assess the prevalence, clinical characteristics, and treatment options of patients with interstitial lung disease (ILD) in polymyositis and dermatomyositis (PM/DM). PATIENTS AND METHODS: Sixty-three consecutive patients with PM/DM underwent standardized screening. Patients with ILD were monitored prospectively, and graded immunosuppression was administered according to the rate of clinical progression. RESULTS: ILD was diagnosed in 20 of 63 patients (32%). Generally, the clinical and serologic findings of the anti-Jo1 syndrome were present. Follow-up evaluation disclosed either a progressive or a nonprogressive course. The 10 patients with progressive ILD were distinguished from the nonprogressive group by extensive ground-glass opacities on high-resolution computed tomography (HRCT) and by bronchoalveolar lavage (BAL) neutrophilia. Intravenous pulse cyclophosphamide prevented further progression in all 10 patients and led to some functional improvement. In the 10 patients without rapidly progressive lung disease, immunosuppression of moderate intensity stabilized pulmonary findings during a median 35 months of follow-up. CONCLUSIONS: The prevalence of ILD in our patients with PM/DM was 32%; this emphasizes the need for pulmonary screening in all PM/DM patients. Progressive disease, featuring ground-glass opacities on HRCT and an inflammatory BAL cell profile, is amenable to intensive immunosuppression. Conversely, patients who do not have these HRCT and BAL features appear to have a low risk of pulmonary deterioration. RELEVANCE: Because the treatment for ILD seems to depend on the rate of clinical progression, future therapeutic trials of lung disease in PM/DM should stratify patients accordingly.


Assuntos
Ciclofosfamida/administração & dosagem , Dermatomiosite/complicações , Imunossupressores/administração & dosagem , Doenças Pulmonares Intersticiais/diagnóstico , Polimiosite/complicações , Adulto , Idoso , Feminino , Humanos , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos Prospectivos , Pulsoterapia
20.
Aviat Space Environ Med ; 74(10): 1058-60, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14556567

RESUMO

BACKGROUND: Recent studies suggest that healthy sport divers may develop clinically silent brain damage, based on the association between a finding of multiple brain lesions on MRI and the presence of right-to-left shunt, a pathway for venous gas bubbles to enter the arterial system. METHODS: We performed echocontrast transcranial Doppler sonography in 42 sport divers to determine the presence of a right-to-left shunt. Cranial MRI was carried out using a 1.5 T magnet. A lesion was counted if it was hyperintense on both T2-weighted and T2-weighted fluid attenuated inversion recovery sequences. To test the hypothesis that the occurrence of postdive arterial gas emboli is related to brain lesions on MRI, we measured postdive intravascular bubbles in a subset of 15 divers 30 min after open water scuba dives. RESULTS: Echocontrast transcranial Doppler sonography revealed a right-to-left shunt in 16 of the divers (38%). Only one hyperintensive lesion of the central white matter was found and that was in a diver with no evidence of a right-to-left shunt. Postdive arterial gas emboli were detected in 3 out of 15 divers; they had a right-to-left shunt, but no pathologic findings on cranial magnetic resonance imaging. CONCLUSIONS: Our data support the theory that right-to-left shunts can serve as a pathway for venous gas bubbles into the arterial circulation. However, we could not confirm an association between brain lesions and the presence of a right-to-left shunt in sport divers.


Assuntos
Encefalopatias/fisiopatologia , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Artérias Cerebrais/anormalidades , Doença da Descompressão/fisiopatologia , Mergulho/efeitos adversos , Embolia Aérea/fisiopatologia , Adulto , Encefalopatias/etiologia , Meios de Contraste/administração & dosagem , Doença da Descompressão/etiologia , Ecoencefalografia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Ultrassonografia Doppler
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