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2.
Proc Natl Acad Sci U S A ; 117(4): 2122-2132, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932446

RESUMO

There is a strong need for a new broad-spectrum antiinfluenza therapeutic, as vaccination and existing treatments are only moderately effective. We previously engineered a lectin, H84T banana lectin (H84T), to retain broad-spectrum activity against multiple influenza strains, including pandemic and avian, while largely eliminating the potentially harmful mitogenicity of the parent compound. The amino acid mutation at position 84 from histidine to threonine minimizes the mitogenicity of the wild-type lectin while maintaining antiinfluenza activity in vitro. We now report that in a lethal mouse model H84T is indeed nonmitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus-endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a broad-spectrum antiinfluenza agent.


Assuntos
Antivirais/administração & dosagem , Influenza Humana/tratamento farmacológico , Lectinas/administração & dosagem , Lectinas/genética , Musa/genética , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/genética , Internalização do Vírus/efeitos dos fármacos , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/virologia , Masculino , Camundongos , Musa/química , Musa/metabolismo , Mutação , Engenharia de Proteínas
3.
PLoS Negl Trop Dis ; 13(7): e0007595, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356611

RESUMO

Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.


Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Musa/química , Lectinas de Plantas/farmacologia , Animais , Antivirais/síntese química , Linhagem Celular Tumoral , Ebolavirus/genética , Ebolavirus/imunologia , Escherichia coli , Feminino , Engenharia Genética , Células HEK293 , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Lectinas de Plantas/síntese química , Replicação Viral/efeitos dos fármacos
4.
J Clin Invest ; 129(6): 2555-2570, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31107242

RESUMO

The nuclear protein DEK is an endogenous DNA-binding chromatin factor regulating hematopoiesis. DEK is one of only 2 known secreted nuclear chromatin factors, but whether and how extracellular DEK regulates hematopoiesis is not known. We demonstrated that extracellular DEK greatly enhanced ex vivo expansion of cytokine-stimulated human and mouse hematopoietic stem cells (HSCs) and regulated HSC and hematopoietic progenitor cell (HPC) numbers in vivo and in vitro as determined both phenotypically (by flow cytometry) and functionally (through transplantation and colony formation assays). Recombinant DEK increased long-term HSC numbers and decreased HPC numbers through a mechanism mediated by the CXC chemokine receptor CXCR2 and heparan sulfate proteoglycans (HSPGs) (as determined utilizing Cxcr2-/- mice, blocking CXCR2 antibodies, and 3 different HSPG inhibitors) that was associated with enhanced phosphorylation of ERK1/2, AKT, and p38 MAPK. To determine whether extracellular DEK required nuclear function to regulate hematopoiesis, we utilized 2 mutant forms of DEK: one that lacked its nuclear translocation signal and one that lacked DNA-binding ability. Both altered HSC and HPC numbers in vivo or in vitro, suggesting the nuclear function of DEK is not required. Thus, DEK acts as a hematopoietic cytokine, with the potential for clinical applicability.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Citocinas/genética , Proteínas de Ligação a DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptores de Interleucina-8B
5.
Arthritis Rheumatol ; 70(4): 594-605, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29287303

RESUMO

OBJECTIVE: The nuclear oncoprotein DEK is an autoantigen associated with juvenile idiopathic arthritis (JIA), especially the oligoarticular subtype. DEK is a secreted chemotactic factor. Abundant levels of DEK and DEK autoantibodies are found in inflamed synovium in JIA. We undertook this study to further characterize the nature of DEK autoantibodies in screening serum samples from 2 different cohorts that consisted mostly of patients with JIA. METHODS: DEK autoantibody levels were analyzed in sera from 33 JIA patients, 13 patients with other inflammatory conditions, and 11 healthy controls, as well as in 89 serum samples from JIA patients receiving anti-tumor necrosis factor (anti-TNF) therapy. Recombinant His-tagged full-length DEK protein (1-375 amino acids [aa]) and the 187-375-aa and 1-350-aa His-tagged DEK fragments made in a baculovirus system were used for enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The C-terminal 25-aa fragment of DEK was expressed in a glutathione S-transferase-tagged vector. ELISA results were calculated as area under the curve by the trapezoidal rule. RESULTS: DEK autoantibody levels were significantly higher in patients with polyarticular JIA than in those with oligoarticular JIA, and were higher in patients with polyarticular JIA who had more active disease after cessation of anti-TNF therapy. Immunoblotting against the C-terminal 25-aa fragment of DEK confirmed that this section of the DEK molecule is the most immunogenic domain. CONCLUSION: DEK autoantibody levels are higher in patients with polyarticular JIA than in those with oligoarticular JIA, and higher in patients who have disease flares after cessation of anti-TNF therapy. The C-terminal 25-aa fragment is the most immunogenic portion of DEK. These findings are significant with respect to the nature of DEK autoantibodies, their contribution to JIA pathogenesis, and their implications for JIA management.


Assuntos
Antirreumáticos/imunologia , Artrite Juvenil/sangue , Autoanticorpos/sangue , Proteínas Cromossômicas não Histona/imunologia , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adolescente , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/imunologia , Autoanticorpos/imunologia , Estudos de Casos e Controles , Criança , Feminino , Humanos , Masculino , Exacerbação dos Sintomas , Suspensão de Tratamento
6.
Nat Commun ; 8: 14252, 2017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28165452

RESUMO

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.


Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Artrite Juvenil/terapia , Fatores Quimiotáticos/antagonistas & inibidores , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Armadilhas Extracelulares/imunologia , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adulto , Animais , Artrite Juvenil/imunologia , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/imunologia , Fatores Quimiotáticos/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Proteínas Oncogênicas/imunologia , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Cultura Primária de Células , Líquido Sinovial/química , Líquido Sinovial/citologia , Líquido Sinovial/imunologia , Zimosan/imunologia
7.
Stem Cells ; 31(8): 1447-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23733396

RESUMO

Understanding the factors that regulate hematopoiesis opens up the possibility of modifying these factors and their actions for clinical benefit. DEK, a non-histone nuclear phosphoprotein initially identified as a putative proto-oncogene, has recently been linked to regulate hematopoiesis. DEK has myelosuppressive activity in vitro on proliferation of human and mouse hematopoietic progenitor cells and enhancing activity on engraftment of long-term marrow repopulating mouse stem cells, has been linked in coordinate regulation with the transcription factor C/EBPα, for differentiation of myeloid cells, and apparently targets a long-term repopulating hematopoietic stem cell for leukemic transformation. This review covers the uniqueness of DEK, what is known about how it now functions as a nuclear protein and also as a secreted molecule that can act in paracrine fashion, and how it may be regulated in part by dipeptidylpeptidase 4, an enzyme known to truncate and modify a number of proteins involved in activities on hematopoietic cells. Examples are provided of possible future areas of investigation needed to better understand how DEK may be regulated and function as a regulator of hematopoiesis, information possibly translatable to other normal and diseased immature cell systems.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas Oncogênicas/fisiologia , Animais , Humanos , Proteínas de Ligação a Poli-ADP-Ribose
8.
Proc Natl Acad Sci U S A ; 110(17): 6847-52, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23569252

RESUMO

DEK is a biochemically distinct, conserved nonhistone protein that is vital to global heterochromatin integrity. In addition, DEK can be secreted and function as a chemotactic, proinflammatory factor. Here we show that exogenous DEK can penetrate cells, translocate to the nucleus, and there carry out its endogenous nuclear functions. Strikingly, adjacent cells can take up DEK secreted from synovial macrophages. DEK internalization is a heparan sulfate-dependent process, and cellular uptake of DEK into DEK knockdown cells corrects global heterochromatin depletion and DNA repair deficits, the phenotypic aberrations characteristic of these cells. These findings thus unify the extracellular and intracellular activities of DEK, and suggest that this paracrine loop involving DEK plays a role in chromatin biology.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Oncogênicas/metabolismo , Comunicação Parácrina/fisiologia , Fracionamento Celular , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Proteínas de Ligação a Poli-ADP-Ribose , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética
9.
Sci Rep ; 3: 1045, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23304436

RESUMO

Vimentin, an abundant intermediate filament protein, presumably has an important role in stabilizing intracellular architecture, but its function is otherwise poorly understood. In a vimentin knockout (Vim KO) mouse model, we note that Vim KO mice challenged with intraperitoneal Escherichia coli control bacterial infection better than do wild-type (WT) mice. In vitro, Vim KO phagocytes show significantly increased capacity to mediate bacterial killing by abundant production of reactive oxygen species (ROS) and nitric oxides, likely due to interactions with the p47phox active subunit of NADPH oxidase. In acute colitis induced by dextran sodium sulfate (DSS), Vim KO mice develop significantly less gut inflammation than do WT mice. Further, Vim KO mice have markedly decreased bacterial extravasation in the setting of DSS-induced acute colitis, consistent with decreased intestinal disease. Our results suggest that vimentin impedes bacterial killing and production of ROS, thereby contributing to the pathogenesis of acute colitis.


Assuntos
Colite/metabolismo , Vimentina/metabolismo , Animais , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Escherichia coli/patogenicidade , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Fagocitose , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vimentina/antagonistas & inibidores , Vimentina/genética
10.
Stem Cells Dev ; 21(9): 1449-54, 2012 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21943234

RESUMO

DEK is a biochemically distinct protein that is generally found in the nucleus, where it is vital to global heterochromatin integrity. However, DEK is also secreted by cells (eg, macrophages) and influences other adjacent cells (eg, acts as a chemoattractant for certain mature blood cells). We hypothesized that DEK may modulate functions of hematopoietic stem (HSCs) and progenitor (HPCs) cells. C57Bl/6 mice were used to demonstrate that absolute numbers and cycling status of HPCs (colony forming unit-granulocyte macrophage [CFU-GM], burst forming unit-erythroid [BFU-E], and colony forming unit-granulocyte erythroid macrophage megakaryocyte [CFU-GEMM]) in bone marrow (BM) and spleen were significantly enhanced in DEK -/- as compared with wild-type (WT) control mice. Moreover, purified recombinant DEK protein inhibited colony formation in vitro by CFU-GM, BFU-E, and CFU-GEMM from WT BM cells and human cord blood (CB) cells in a dose-dependent fashion, demonstrating that DEK plays a negative role in HPC proliferation in vitro and in vivo. Suppression was direct acting as determined by inhibition of proliferation of single isolated CD34(+) CB cells in vitro. In contrast, DEK -/- BM cells significantly demonstrated reduced long term competitive and secondary mouse repopulating HSC capacity compared with WT BM cells, demonstrating that DEK positively regulates engrafting capability of self-renewing HSCs. This demonstrates that DEK has potent effects on HSCs, HPCs, and hematopoiesis, information of biological and potential clinical interest.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Proliferação de Células , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Transplante Heterólogo , Transplante Homólogo
11.
Arthritis Rheum ; 63(2): 556-67, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21280010

RESUMO

OBJECTIVE: DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes. Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA. METHODS: DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). RESULTS: DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS. CONCLUSION: These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.


Assuntos
Artrite Juvenil/metabolismo , Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Oncogênicas/metabolismo , Processamento de Proteína Pós-Traducional , Membrana Sinovial/metabolismo , Acetilação , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Artrite Juvenil/imunologia , Artrite Juvenil/patologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Criança , Proteínas Cromossômicas não Histona/imunologia , Humanos , Articulações/metabolismo , Articulações/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Membrana Sinovial/patologia
12.
Mol Cell Biol ; 26(24): 9484-96, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17030615

RESUMO

The nuclear DNA-binding protein DEK is an autoantigen that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing. We demonstrate here that DEK is actively secreted by macrophages and is also found in synovial fluid samples from patients with juvenile arthritis. Secretion of DEK is modulated by casein kinase 2, stimulated by interleukin-8, and inhibited by dexamethasone and cyclosporine A, consistent with a role as a proinflammatory molecule. DEK is secreted in both a free form and in exosomes, vesicular structures in which transcription-modulating factors such as DEK have not previously been found. Furthermore, DEK functions as a chemotactic factor, attracting neutrophils, CD8+ T lymphocytes, and natural killer cells. Therefore, the DEK autoantigen, previously described as a strictly nuclear protein, is secreted and can act as an extracellular chemoattractant, suggesting a direct role for DEK in inflammation.


Assuntos
Artrite Juvenil/metabolismo , Artrite Juvenil/patologia , Autoantígenos/fisiologia , Fatores Quimiotáticos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Oncogênicas/metabolismo , Artrite Juvenil/imunologia , Autoantígenos/metabolismo , Células Cultivadas , Fatores Quimiotáticos/fisiologia , Quimiotaxia de Leucócito , Criança , Proteínas Cromossômicas não Histona/fisiologia , Líquido Extracelular/imunologia , Líquido Extracelular/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Proteínas Oncogênicas/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose
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