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1.
J Biol Chem ; 295(25): 8537-8549, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32371391

RESUMO

Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth.

3.
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
4.
Sci Rep ; 9(1): 11259, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375789

RESUMO

Centromere genomics remain poorly characterized in cancer, due to technologic limitations in sequencing and bioinformatics methodologies that make high-resolution delineation of centromeric loci difficult to achieve. We here leverage a highly specific and targeted rapid PCR methodology to quantitatively assess the genomic landscape of centromeres in cancer cell lines and primary tissue. PCR-based profiling of centromeres revealed widespread heterogeneity of centromeric and pericentromeric sequences in cancer cells and tissues as compared to healthy counterparts. Quantitative reductions in centromeric core and pericentromeric markers (α-satellite units and HERV-K copies) were observed in neoplastic samples as compared to healthy counterparts. Subsequent phylogenetic analysis of a pericentromeric endogenous retrovirus amplified by PCR revealed possible gene conversion events occurring at numerous pericentromeric loci in the setting of malignancy. Our findings collectively represent a more comprehensive evaluation of centromere genetics in the setting of malignancy, providing valuable insight into the evolution and reshuffling of centromeric sequences in cancer development and progression.

5.
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
6.
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
7.
PLoS One ; 14(2): e0212970, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30818388

RESUMO

Human endogenous retroviruses are remnants of ancient germline infections that make up approximately 8% of the modern human genome. The HERV-K (HML-2) family is one of the most recent entrants into the human germline, these viruses appear to be transcriptionally active, and HERV-K viral like particles (VLPs) are found in cell lines from a number of human malignancies. HERV-K VLPs were first found to be produced in teratocarcinoma cell lines, and since then teratocarcinoma has been thought of as the classical model for HERV-Ks, with the NCCIT teratocarcinoma cell line particularly known to produce VLPs. Treatment for teratocarcinoma has progressed since its discovery, with improved prognosis for patients. Since the introduction of platinum based therapy, first year survival has greatly improved even with disseminated disease; however, it is estimated that 20% to 30% of patients present with metastatic germ cell tumor relapse following initial treatments. Also, the toxicity associated with the use of chemotherapeutic agents used to treat germ cell tumors is still a major concern. In this study, we show that the depletion of the HERV-K accessory protein Np9 increases the sensitivity of NCCIT teratocarcinoma cells to bleomycin and cisplatin. While decreasing the expression of Np9 had only a modest effect on the baseline viability of the cells, the reduced expression of Np9 increased the sensitivity of the teratocarcinoma cells to environmental (serum starvation) and chemical (chemotherapeutic) stresses. Np9 is also essential to the migration of NCCIT teratocarcinoma cells: in a wound closure assay, reduced expression of Np9 resulted in cells migrating into the wound at a slower rate, whereas reintroduction of Np9 resulted in NCCIT cells migrating back into the wound in a manner similar to the control. These findings support the implication that the HERV-K accessory protein Np9 has oncogenic potential.


Assuntos
Retrovirus Endógenos/fisiologia , Produtos do Gene env/fisiologia , Teratocarcinoma/fisiopatologia , Teratocarcinoma/virologia , Antineoplásicos/farmacologia , Bleomicina/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidade , Produtos do Gene env/genética , Humanos , Masculino , Teratocarcinoma/patologia , Neoplasias Testiculares/patologia , Neoplasias Testiculares/fisiopatologia , Neoplasias Testiculares/virologia
8.
Neoplasia ; 20(12): 1209-1218, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30412857

RESUMO

DNA damage repair alterations play a critical role in ovarian cancer tumorigenesis. Mechanistic drivers of the DNA damage response consequently present opportunities for therapeutic targeting. The chromatin-binding DEK oncoprotein functions in DNA double-strand break repair. We therefore sought to determine the role of DEK in epithelial ovarian cancer. DEK is overexpressed in both primary epithelial ovarian cancers and ovarian cancer cell lines. To assess the impact of DEK expression levels on cell growth, small interfering RNA and short hairpin RNA approaches were utilized. Decreasing DEK expression in ovarian cancer cell lines slows cell growth and induces apoptosis and DNA damage. The biologic effects of DEK depletion are enhanced with concurrent chemotherapy treatment. The in vitro effects of DEK knockdown are reproduced in vivo, as DEK depletion in a mouse xenograft model results in slower tumor growth and smaller tumors compared to tumors expressing DEK. These findings provide a compelling rationale to target the DEK oncoprotein and its pathways as a therapeutic strategy for treating epithelial ovarian cancer.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Oncogênicas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Apoptose/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
mBio ; 9(1)2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487235

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.IMPORTANCEListeria monocytogenes is an intracellular bacterial pathogen that is capable of invading numerous host cells during infection. L. monocytogenes can cross the blood-brain barrier, leading to life-threatening meningitis. Here we show that an L. monocytogenes surface protein, InlF, is necessary for optimal colonization of the brain in mice. Furthermore, in the absence of vimentin, a cytosolic intermediate filament protein that is also present on the surface of brain endothelial cells, colonization of the brain was significantly impaired. We further show that InlF binds vimentin to mediate invasion of host cells. This work identifies InlF as a bacterial surface protein with specific relevance for infection of the brain and underscores the significance of host cell surface vimentin interactions in microbial pathogenesis.


Assuntos
Encéfalo/parasitologia , Endocitose , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/parasitologia , Vimentina/metabolismo , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Listeriose/patologia , Camundongos , Ratos
10.
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
11.
Genome Res ; 27(12): 2040-2049, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29141960

RESUMO

The centromere is the structural unit responsible for the faithful segregation of chromosomes. Although regulation of centromeric function by epigenetic factors has been well-studied, the contributions of the underlying DNA sequences have been much less well defined, and existing methodologies for studying centromere genomics in biology are laborious. We have identified specific markers in the centromere of 23 of the 24 human chromosomes that allow for rapid PCR assays capable of capturing the genomic landscape of human centromeres at a given time. Use of this genetic strategy can also delineate which specific centromere arrays in each chromosome drive the recruitment of epigenetic modulators. We further show that, surprisingly, loss and rearrangement of DNA in centromere 21 is associated with trisomy 21. This new approach can thus be used to rapidly take a snapshot of the genetics and epigenetics of each specific human centromere in nondisjunction disorders and other biological settings.


Assuntos
Centrômero , Genômica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , Proteína B de Centrômero/metabolismo , Instabilidade Cromossômica , Cromossomos Humanos Par 21 , DNA , DNA Satélite , Síndrome de Down/genética , Epigênese Genética , Feminino , Rearranjo Gênico , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Cariótipo , Masculino , Deleção de Sequência
12.
J Virol ; 91(23)2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28931682

RESUMO

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.


Assuntos
Retrovirus Endógenos/efeitos dos fármacos , Retrovirus Endógenos/genética , Genoma Viral/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Transcrição Reversa/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral , Retrovirus Endógenos/enzimologia , Retrovirus Endógenos/patogenicidade , Humanos , Inibidores de Integrase/farmacologia , Lamivudina/farmacologia , Inibidores de Proteases/farmacologia , Estavudina/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Zidovudina/farmacologia
13.
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
14.
PLoS One ; 11(9): e0162641, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27657497

RESUMO

BACKGROUND: NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. METHODOLOGY/PRINCIPAL FINDINGS: The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. CONCLUSION/SIGNIFICANCE: Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal inflammation. Our findings provide the first evidence for Vim-dependent mechanisms underlying the pathogenic triad of bacterial meningitis.

15.
Nat Microbiol ; 1: 16025, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-27572444

RESUMO

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.


Assuntos
Aderência Bacteriana , Salmonella typhimurium/fisiologia , Shigella flexneri/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Vimentina/metabolismo , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/fisiologia , Animais , Antígenos de Bactérias/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Queratina-18/metabolismo , Camundongos , Ligação Proteica , Transporte Proteico
16.
ACS Cent Sci ; 2(12): 943-953, 2016 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-28058284

RESUMO

Nonlamellar lipid arrangements, including cubosomes, appear in unhealthy cells, e.g., when they are subject to stress, starvation, or viral infection. The bioactivity of cubosomes-nanoscale particles exhibiting bicontinuous cubic structures-versus more common vesicles is an unexplored area due to lack of suitable model systems. Here, glycodendrimercubosomes (GDCs)-sugar-presenting cubosomes assembled from Janus glycodendrimers by simple injection into buffer-are proposed as mimics of biological cubic membranes. The bicontinuous cubic GDC architecture has been demonstrated by electron tomography. The stability of these GDCs in buffer enabled studies on lectin-dependent agglutination, revealing significant differences compared with the vesicular glycodendrimersome (GDS) counterpart. In particular, GDCs showed an increased activity toward concanavalin A, as well as an increased sensitivity and selectivity toward two variants of banana lectins, a wild-type and a genetically modified variant, which is not exhibited by GDSs. These results suggest that cells may adapt under unhealthy conditions by undergoing a transformation from lamellar to cubic membranes as a method of defense.

17.
Cell ; 163(3): 746-58, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26496612

RESUMO

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.


Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/genética , Fármacos Anti-HIV/química , Sequência de Carboidratos , Engenharia Genética , Mitógenos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Musa/química
18.
J Virol ; 89(14): 7187-201, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25926654

RESUMO

UNLABELLED: Human endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied. IMPORTANCE: Retroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.


Assuntos
DNA Viral/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiologia , Montagem de Vírus , Linhagem Celular , DNA Viral/genética , Transferência Genética Horizontal , Genes Reporter , Humanos , Transcrição Reversa , Transcrição Genética , Transdução Genética
19.
Genome Biol ; 16: 74, 2015 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-25886262

RESUMO

BACKGROUND: Approximately 8% of the human genome consists of sequences of retroviral origin, a result of ancestral infections of the germ line over millions of years of evolution. The most recent of these infections is attributed to members of the human endogenous retrovirus type-K (HERV-K) (HML-2) family. We recently reported that a previously undetected, large group of HERV-K (HML-2) proviruses, which are descendants of the ancestral K111 infection, are spread throughout human centromeres. RESULTS: Studying the genomes of certain cell lines and the DNA of healthy individuals that seemingly lack K111, we discover new HERV-K (HML-2) members hidden in pericentromeres of several human chromosomes. All are related through a common ancestor, termed K222, which is a virus that infected the germ line approximately 25 million years ago. K222 exists as a single copy in the genomes of baboons and high order primates, but not New World monkeys, suggesting that progenitor K222 infected the primate germ line after the split between New and Old World monkeys. K222 exists in modern humans at multiple loci spread across the pericentromeres of nine chromosomes, indicating it was amplified during the evolution of modern humans. CONCLUSIONS: Copying of K222 may have occurred through recombination of the pericentromeres of different chromosomes during human evolution. Evidence of recombination between K111 and K222 suggests that these retroviral sequences have been templates for frequent cross-over events during the process of centromere recombination in humans.


Assuntos
Centrômero/virologia , DNA Viral/isolamento & purificação , Retrovirus Endógenos/genética , Evolução Molecular , Sequência de Bases , Centrômero/genética , Cromossomos Humanos/genética , Cromossomos Humanos/virologia , Biologia Computacional , Primers do DNA , DNA Viral/genética , Retrovirus Endógenos/classificação , Retrovirus Endógenos/isolamento & purificação , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA
20.
Blood Cells Mol Dis ; 54(1): 123-31, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25128083

RESUMO

DEK is important in regulating cellular processes including proliferation, differentiation and maintenance of stem cell phenotype. The translocation t(6;9) in Acute Myeloid Leukemia (AML), which fuses DEK with NUP214, confers a poor prognosis and a higher risk of relapse. The over-expression of DEK in AML has been reported, but different studies have shown diminished levels in pediatric and promyelocytic leukemias. This study has characterized DEK expression, in silico, using a large multi-center cohort of leukemic and normal control cases. Overall, DEK was under-expressed in AML compared to normal bone marrow (NBM). Studying specific subtypes of AML confirmed either no significant change or a significant reduction in DEK expression compared to NBM. Importantly, the similarity of DEK expression between AML and NBM was confirmed using immunohistochemistry analysis of tissue mircorarrays. In addition, stratification of AML patients based on median DEK expression levels indicated that DEK showed no effect on the overall survival of patients. DEK expression during normal hematopoiesis did reveal a relationship with specific cell types implicating a distinct function during myeloid differentiation. Whilst DEK may play a potential role in hematopoiesis, it remains to be established whether it is important for leukemagenesis, except when involved in the t(6;9) translocation.


Assuntos
Proteínas Cromossômicas não Histona/biossíntese , Proteínas de Ligação a DNA/biossíntese , Bases de Dados Genéticas , Regulação Leucêmica da Expressão Gênica , Hematopoese , Leucemia Mieloide Aguda/metabolismo , Proteínas Oncogênicas/biossíntese , Animais , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 9/genética , Estudos de Coortes , Proteínas de Ligação a DNA/genética , Intervalo Livre de Doença , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Estudos Multicêntricos como Assunto , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Taxa de Sobrevida , Translocação Genética
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