RESUMO
Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60-80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence. A major familial form of ALS/FTD, C9orf72 (C9), is due to a hexanucleotide repeat expansion (HRE) of (GGGGCC), a Pur binding element. We show by circular dichroism that RNA oligonucleotides containing this purine-rich sequence consist largely of parallel G-quadruplexes. TZIP peptide binds this repeat sequence in both DNA and RNA. It binds the RNA element, including the G-quadruplexes, with a high degree of specificity versus a random oligonucleotide. In addition, TZIP binds both linear and G-quadruplex repeat RNA to form higher order G-quadruplex secondary structures. This change in conformational form by Pur-based peptide represents a new mechanism for regulating G quadruplex secondary structure within the C9 repeat. TZIP modulation of C9 RNA structural configuration may alter interaction of the complex with other proteins. This Pur-based mechanism provides new targets for therapy, and it may help to explain Pura alleviation of certain cellular pathological aspects of ALS/FTD.
Assuntos
Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteínas de Ligação a DNA/química , Peptídeos/farmacologia , Fatores de Transcrição/química , Proteína C9orf72/química , Dicroísmo Circular , Expansão das Repetições de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Quadruplex G/efeitos dos fármacos , Humanos , Modelos Moleculares , Mimetismo Molecular , Peptídeos/síntese química , RNA/química , RNA/metabolismo , Termodinâmica , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: HIV-1 infection of the brain and related cognitive impairment remain prevalent in HIV-1-infected individuals despite combination antiretroviral therapy. Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a newly identified host restriction factor that blocks the replication of HIV-1 and other retroviruses in myeloid cells. Cell cycle-regulated phosphorylation at residue Thr592 and viral protein X (Vpx)-mediated degradation of SAMHD1 have been shown to bypass SAMHD1 restriction in vitro. Herein, we investigated expression and phosphorylation of SAMHD1 in vivo in relation to macrophage infection and proliferation during the neuropathogenesis of HIV-1 and simian immunodeficiency virus (SIV) encephalitis. METHODS: Using brain and other tissues from uninfected and SIV-infected macaques with or without encephalitis, we performed immunohistochemistry, multilabel fluorescence microscopy and western blot to examine the expression, localization and phosphorylation of SAMHD1. RESULTS: The number of SAMHD1 nuclei increased in encephalitic brains despite the presence of Vpx. Many of these cells were perivascular macrophages, although subsets of SAMHD1 microglia and endothelial cells were also observed. The SAMHD1 macrophages were shown to be both infected and proliferating. Moreover, the presence of cycling SAMHD1 brain macrophages was confirmed in the tissue of HIV-1-infected patients with encephalitis. Finally, western blot analysis of brain-protein extracts from SIV-infected macaques showed that SAMHD1 protein exists in the brain mainly as an inactive Thr592-phosphorylated form. CONCLUSION: The ability of SAMHD1 to act as a restriction factor for SIV/HIV in the brain is likely bypassed in proliferating brain macrophages through the phosphorylation-mediated inactivation, not Vpx-mediated degradation of SAMHD1.
Assuntos
Encefalite Viral/patologia , Infecções por HIV/patologia , HIV/crescimento & desenvolvimento , Macrófagos/virologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Animais , Western Blotting , Encéfalo/imunologia , Encéfalo/patologia , Encéfalo/virologia , Proliferação de Células , Expressão Gênica , HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Imuno-Histoquímica , Fatores Imunológicos/metabolismo , Macaca , Macrófagos/imunologia , Masculino , Microscopia de Fluorescência , Fosforilação , Processamento de Proteína Pós-Traducional , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
The PURA gene encodes Pur-alpha, a 322 amino acid protein with repeated nucleic acid binding domains that are highly conserved from bacteria through humans. PUR genes with a single copy of this domain have been detected so far in spirochetes and bacteroides. Lower eukaryotes possess one copy of the PUR gene, whereas chordates possess 1 to 4 PUR family members. Human PUR genes encode Pur-alpha (Pura), Pur-beta (Purb) and two forms of Pur-gamma (Purg). Pur-alpha is a protein that binds specific DNA and RNA sequence elements. Human PURA, located at chromosome band 5q31, is under complex control of three promoters. The entire protein coding sequence of PURA is contiguous within a single exon. Several studies have found that overexpression or microinjection of Pura inhibits anchorage-independent growth of oncogenically transformed cells and blocks proliferation at either G1-S or G2-M checkpoints. Effects on the cell cycle may be mediated by interaction of Pura with cellular proteins including Cyclin/Cdk complexes and the Rb tumor suppressor protein. PURA knockout mice die shortly after birth with effects on brain and hematopoietic development. In humans environmentally induced heterozygous deletions of PURA have been implicated in forms of myelodysplastic syndrome and progression to acute myelogenous leukemia. Pura plays a role in AIDS through association with the HIV-1 protein, Tat. In the brain Tat and Pura association in glial cells activates transcription and replication of JC polyomavirus, the agent causing the demyelination disease, progressive multifocal leukoencephalopathy. Tat and Pura also act to stimulate replication of the HIV-1 RNA genome. In neurons Pura accompanies mRNA transcripts to sites of translation in dendrites. Microdeletions in the PURA locus have been implicated in several neurological disorders. De novo PURA mutations have been related to a spectrum of phenotypes indicating a potential PURA syndrome. The nucleic acid, G-rich Pura binding element is amplified as expanded polynucleotide repeats in several brain diseases including fragile X syndrome and a familial form of amyotrophic lateral sclerosis/fronto-temporal dementia. Throughout evolution the Pura protein plays a critical role in survival, based on conservation of its nucleic acid binding properties. These Pura properties have been adapted in higher organisms to the as yet unfathomable development of the human brain.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos/genética , Animais , Sequência de Bases , Ciclo Celular , Proteínas de Ciclo Celular/genética , Sequência Conservada/genética , Replicação do DNA , Células Dendríticas/metabolismo , HIV-1/genética , Humanos , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Mensageiro/metabolismoRESUMO
The recent observation of interviral recombination between members of two distinct classes of DNA viruses has opened the gates to a new field of human disease development. In all cases studied thus far interviral recombination is a rare event that requires special circumstances for intracellular interaction of participating viral genomes. The rarity and special requirements do not detract from the potential clinical significance of resulting recombinants, as exemplified by recombination between JC viral and Epstein-Barr viral genomes. This significance depends largely upon the mechanisms of recombination that would generate specific forms of recombinant viral genomes. At this time little is known regarding mechanisms of interviral recombination. DNA break-induced replication seems presently to be a highly plausible means of initiating formation of different, potentially active recombination products. Generalizing interviral recombination to a variety of viruses will open a fertile field for discovery as multiple diseases of mysterious etiology are investigated.
RESUMO
The aim of the present study was to investigate if macrophage proliferation occurs in the brain during simian immunodeficiency virus (SIV) infection of adult macaques. We examined the expression of the Ki-67 proliferation marker in the brains of uninfected and SIV-infected macaques with or without encephalitis. Double-label immunohistochemistry using antibodies against the pan-macrophage marker CD68 and Ki-67 showed that there was a significant increase in CD68+Ki-67+ cells in macaques with SIV encephalitis (SIVE) compared to uninfected and SIV-infected animals without encephalitis, a trend that was also confirmed in brain samples from patients with HIV encephalitis. Multi-label immunofluorescence for CD163 and Ki-67 confirmed that the vast majority of Ki-67+ nuclei were localized to CD163+ macrophages in perivascular cuffs and lesions. The proliferative capacity of Ki-67+ perivascular macrophages (PVM) was confirmed by their nuclear incorporation of bromodeoxyuridine. Examining SIVE lesions, using double-label immunofluorescence with antibodies against SIV-Gag-p28 and Ki-67, showed that the population of Ki-67+ cells were productively infected and expanded proportionally with lesions. Altogether, this study shows that there are subpopulations of resident PVM that express Ki-67 and are SIV-infected, suggesting a mechanism of macrophage accumulation in the brain via PVM proliferation.
Assuntos
Proliferação de Células , Encefalite/etiologia , Infecções por HIV/complicações , Macrófagos/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Vasos Sanguíneos/fisiopatologia , Encéfalo/fisiopatologia , Encefalite/fisiopatologia , Humanos , Antígeno Ki-67 , Macaca mulatta , Masculino , Receptores de Superfície CelularRESUMO
Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Neurônios Motores/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Antibacterianos/farmacologia , Arsenitos/farmacologia , Encéfalo/citologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/efeitos dos fármacos , DNA Helicases , Proteínas de Ligação a DNA/genética , Doxiciclina/farmacologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína FUS de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Compostos de Sódio/farmacologia , Fatores de Transcrição/genéticaRESUMO
We document a unique DNA recombination between polyomavirus JC (JC virus [JCV]) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression, JCV can infect the brain, causing a demyelinating disease, progressive multifocal leukoencephalopathy. Rearrangements in the archetype noncoding control region are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be coinfected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed patients with multiple sclerosis, those susceptible to progressive multifocal leukoencephalopathy. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical noncoding control region sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types.
Assuntos
Herpesvirus Humano 4/genética , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/virologia , Recombinação Genética/genética , Sequência de Bases , Linhagem Celular , Células Cultivadas , Humanos , Hospedeiro Imunocomprometido , Vírus JC/patogenicidade , Dados de Sequência Molecular , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/virologiaRESUMO
Progressive multifocal leukoencephalopathy is a neurodegenerative disease caused by demyelination in the brain. The demyelination is due to infection of oligodendroglial cells by polyomavirus JC, a circular DNA virus. The virus resides as an archetype form in uroepithelial cells and bone marrow of more than 70% of adults, in whom it seldom causes overt symptoms. The JC viral form infecting the brain differs from the archetype. This viral form contains two deletions and a duplication in the non-coding control region that are thought to be derived from the archetype. These rearrangements are necessary for neurovirulence. This review considers how these rearrangements occur in the context of transit to the brain and adaptation to infect glial cells.
RESUMO
We examined the expression of the mannose receptor CD206 by perivascular macrophages (PVM) in normal human and monkey brains and in brains of HIV-infected humans and of monkeys infected with simian immunodeficiency virus (SIV). Depletion of brain PVM in SIV-infected monkeys by intrathecal injection of liposome-encapsulated bisphosphonates eliminated CD206-expressing cells in the brain, confirming their perivascular location and phagocytic capacity. In vivo labeling with bromodeoxyuridine in normal uninfected and SIV-infected macaques in combination with CD206 immunostaining revealed a CD206+-to-CD206- shift within pre-existing PVM during SIV brain infection and neuroinflammation. These findings identify CD206 as a unique marker of human and macaque PVM, and underscore the utility of this marker in studying the origin, turnover and functions of these cells in AIDS.
Assuntos
Encefalite/metabolismo , Infecções por HIV/metabolismo , HIV-1 , Lectinas Tipo C/biossíntese , Lectinas de Ligação a Manose/biossíntese , Receptores de Superfície Celular/biossíntese , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia , Animais , Encefalite/genética , Encefalite/patologia , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/patologia , HIV-1/genética , Humanos , Lectinas Tipo C/genética , Macaca mulatta , Macrófagos/metabolismo , Macrófagos/virologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Fenótipo , Receptores de Superfície Celular/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/genéticaRESUMO
Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence.
Assuntos
Encéfalo/imunologia , Regulação Viral da Expressão Gênica/imunologia , Tolerância Imunológica , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/imunologia , RNA não Traduzido/imunologia , Encéfalo/patologia , Encéfalo/virologia , Progressão da Doença , Humanos , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/patologia , Leucoencefalopatia Multifocal Progressiva/virologia , Linfócitos/imunologia , Linfócitos/virologia , Mutação , Neuroglia/imunologia , Neuroglia/virologia , Conformação de Ácido Nucleico , Oligodendroglia/imunologia , Oligodendroglia/virologia , RNA não Traduzido/genética , Replicação Viral/imunologiaRESUMO
The Pur proteins are an ancient family of sequence-specific single-stranded nucleic acid-binding proteins. They bind a G-rich element in either single- or double-stranded nucleic acids and are capable of displacing the complementary C-rich strand. Recently several reports have described Pur family member knockouts, mutations, and disease aberrations. Together with a recent crystal structure of Purα, these data reveal conserved structural features of these proteins that have been adapted to serve functions unique to higher eukaryotes. In humans Pur proteins are critical for myeloid cell development, muscle development, and brain development, including trafficking of mRNA to neuronal dendrites. Pur family members have been implicated in diseases as diverse as cancer, premature aging, and fragile-X mental retardation syndrome.
Assuntos
Proteínas de Ligação a DNA , Síndrome do Cromossomo X Frágil , Desenvolvimento Muscular , Neoplasias , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Polyomavirus JC (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a frequently fatal infection of the brain afflicting nearly 4% of AIDS patients in the USA. Human immunodeficiency virus type 1 (HIV-1) Tat, acting together with cellular proteins at the JCV non-coding control region (NCCR), can stimulate JCV DNA transcription and replication. Tat in the brain is secreted by HIV-1-infected cells and incorporated by oligodendroglia, cells capable of infection by JCV. Thus far the effects of Tat on JCV have been studied primarily with protein encoded by the HIV-1 B clade most common in North America. Here, we determine the abilities of Tat from different HIV-1 clades to alter JCV early and late gene transcription and DNA replication initiated at the JCV origin. Tat from all clades tested stimulates both JCV early and late gene promoters, with clade B Tat being significantly most effective. Tat proteins from the HIV-1 clades display parallel patterns of differences in their effects on HIV-1 and JCV transcription, suggesting that Tat effects in both cases are mediated by the same cellular proteins. Clade B Tat is most effective at directing Smad mediators of tumour growth factor beta and cellular partner Purα to the NCCR. Tat proteins from all non-B clades inhibit initiation of JCV DNA replication. The effectiveness of HIV-1 clade B Tat at promoting JCV transcriptional and replicative processes highlights a need for further investigation to determine which molecular aspects of Tat from distinct HIV-1 substrains can contribute to the course of PML development in neuroAIDS.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Produtos do Gene tat/metabolismo , HIV-1/classificação , HIV-1/genética , Vírus JC/efeitos dos fármacos , Neuroglia/virologia , Sequência de Aminoácidos , Linhagem Celular , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Produtos do Gene tat/genética , Humanos , Dados de Sequência Molecular , Mutação , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
Comment on: Wilk A, et al. Cell Cycle 2012; 11:2660-71.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Fenofibrato/toxicidade , Fatores de Transcrição Forkhead/metabolismo , Hipolipemiantes/toxicidade , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , HumanosRESUMO
BACKGROUND: Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals. RESULTS: Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts. CONCLUSIONS: Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts.
Assuntos
Infecções por Citomegalovirus/metabolismo , Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição , Ativação Transcricional , Regiões 5' não Traduzidas , Animais , Linhagem Celular Tumoral , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Interações Hospedeiro-Patógeno , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos/virologia , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Ligação Proteica , RNA/genética , TransfecçãoRESUMO
Purα is a nucleic acid-binding protein with DNA-unwinding activity, which has recently been shown to have a role in the cellular response to DNA damage. We have investigated the function of Purα in Ultraviolet-C (UVC) radiation-induced DNA damage and nucleotide excision repair (NER). Mouse embryo fibroblasts from PURA(-/-) knockout mice, which lack Purα, showed enhanced sensitivity to UVC irradiation as assessed by assays for cell viability and clonogenicity compared to Purα positive control cultures. In reporter plasmid reactivation assays to measure the removal of DNA adducts induced in vitro by UVC, the Purα-negative cells were less efficient in DNA damage repair. Purα-negative cells were also more sensitive to UVC-induced DNA damage measured by Comet assay and showed a decreased ability to remove UVC-induced cyclobutane pyrimidine dimers. In wild-type mouse fibroblasts, expression of Purα is induced following S-phase checkpoint activation by UVC in a similar manner to the NER factor TFIIH. Moreover, co-immunoprecipitation experiments showed that Purα physically associates with TFIIH. Thus, Purα has a role in NER and the repair of UVC-induced DNA damage.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/efeitos da radiação , Proteínas do Tecido Nervoso/metabolismo , Raios Ultravioleta , Animais , Células Cultivadas , Ensaio Cometa , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/efeitos da radiaçãoRESUMO
Conflict procedures can be used to study the receptor mechanisms underlying the anxiolytic effects of benzodiazepines and other GABA(A) receptor modulators. In the present study, we first determined the efficacy and binding affinity of the benzodiazepine diazepam and recently synthesized GABA(A) receptor modulators JY-XHe-053, XHe-II-053, HZ-166, SH-053-2'F-S-CH3 and SH-053-2'F-R-CH3 at GABA(A) receptors containing α1, α2, α3 and α5 subunits. Results from these studies suggest that each compound displayed lower efficacy at GABA(A) receptors containing α1 subunits and varying degrees of efficacy and affinity at GABA(A) receptors containing α2, α3 and α5 subunits. Next, we assessed their anxiolytic effects using a rhesus monkey conflict procedure in which behavior was maintained under a fixed-ratio schedule of food delivery in the absence (non-suppressed responding) and presence (suppressed responding) of response-contingent electric shock. Relatively non-selective compounds, such as diazepam and JY-XHe-053 produced characteristic increases in rates of suppressed responding at low to intermediate doses and decreased the average rates of non-suppressed responding at higher doses. XHe-II-053 and HZ-166 also produced increases in suppressed responding at low to intermediate doses, but were ineffective at decreasing rates of non-suppressed responding, consistent with their relatively low efficacy at GABA(A) receptors containing α1 and α5 subunits. In contrast, SH-053-2'F-S-CH3 and SH-053-2'F-R-CH3 produced only partial increases in suppressed responding and were ineffective on non-suppressed responding, consistent with their profiles as partial agonists at GABA(A) receptors containing α2, α3 and α5 subunits. These behavioral effects suggest that the anxiolytic and rate-reducing effects of GABA(A) receptor positive modulators are dependent on their relative efficacy and affinity at different GABA(A) receptor subtypes.
Assuntos
Alcinos/farmacologia , Ansiolíticos/farmacologia , Benzodiazepinas/farmacologia , Conflito Psicológico , Diazepam/análogos & derivados , Moduladores GABAérgicos/farmacologia , Imidazóis/farmacologia , Alcinos/química , Animais , Benzodiazepinas/química , Ligação Competitiva , Linhagem Celular , Diazepam/química , Diazepam/farmacologia , Feminino , Humanos , Imidazóis/química , Técnicas In Vitro , Macaca mulatta , Masculino , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ensaio Radioligante , Ratos , Receptores de GABA-A/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Xenopus laevisRESUMO
In the past three years, remarkable discoveries have added three new human polyomaviruses (KI virus (KIV), WU virus (WUV) and Merkel cell virus (MCV)) to a class that previously had only two disease-causing members (BK virus (BKV) and JC virus (JCV)) identified. Two monkey polyomaviruses, simian virus (SV)40 and B-cell lymphotropic polyomavirus (LPV) are also present in humans. KIV and WUV lack the agnoprotein coding sequence and regulatory micro (mi)RNA clusters of BKV, JCV and SV40. MCV lacks the agnoprotein sequence but generates miRNAs. KIV, WUV and MCV are all widespread in humans. Although they have distinctive tissue tropisms, all these viruses are probably acquired in childhood. Of these viruses, only MCV has thus far been strongly linked to cancer. Marshalled evidence from diverse sources implicates MCV as an etiological agent of Merkel cell carcinoma. This review compares the structural features of the new and previously known polyomaviruses, with the aim of identifying approaches to molecular pathology.
Assuntos
MicroRNAs/fisiologia , Infecções por Polyomavirus/virologia , Polyomavirus/genética , Polyomavirus/patogenicidade , Proteínas Virais/fisiologia , Fatores de Virulência/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Genes Virais , Genoma Viral , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Polyomavirus/isolamento & purificação , Sintenia , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Increased androgen receptor (AR) levels support resistance to apoptosis and hormone therapy in advanced prostate cancer (PC). We recently linked the overexpression of AR in androgen-independent LNCaP cells (AI-cells) and tissues from castration-resistant patients to decreased nuclear levels of Pur-alpha (Puralpha) and loss from a protein complex bound to repressor sequences (ARS) in the 5'-UTR of AR. Strategies to regain control of increased AR transcription may overcome resistance of AI-cells and improve treatment outcomes. METHODS: MTT, real-time PCR, Western blot, ChIP, flow cytometry, and caspase 3/7 activation measured the effect on growth and targets of LBH589/bicalutamide treatment of AI-cells and androgen-dependent LNCaP cells (AD). RESULTS: Within 16 hr of treatment of AI-cells with low concentrations of the histone deacetylase inhibitor LBH589, a shift of cytoplasmic Puralpha restored the nuclear levels and the binding of Puralpha to the ARS. This was followed by a decline in AR-mRNA and protein reaching levels of parental AD-cells. The fraction of AI-cells in G1 increased and the cells in S phase decreased similar to AD-cells, and there was a modest caspase activation. Most notably, treatment of bicalutamide-resistant AI-cells with 10 nM LBH589 combined with 12.5 microM bicalutamide synergistically inhibited cell growth and induced a fivefold higher level of caspase 3/7 activation than observed in AD-cells. CONCLUSIONS: Low-dose LBH589 restores Puralpha binding to ARS and down-regulates AR transcription. Biologically, LBH589 reverses the resistance of AI-cells to bicalutamide and to apoptosis. The combination may restore the hormonal response of castration-resistant PC patients.
Assuntos
Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Nitrilas/farmacologia , Receptores Androgênicos/genética , Compostos de Tosil/farmacologia , Fatores de Transcrição/metabolismo , Regiões 5' não Traduzidas , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Epigênese Genética/efeitos dos fármacos , Humanos , Indóis , Masculino , Orquiectomia , Panobinostat , Neoplasias da Próstata , Receptores Androgênicos/biossínteseRESUMO
JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a fatal, demyelinating disease of the brain affecting people with AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type 1 (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine transforming growth factor (TGF)-beta in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary central nervous system cell type infected by JCV in PML. This study demonstrated that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-beta, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulated JCV early gene transcription in KG-1 oligodendroglial cells when expressed via transfection or added exogenously. Using chromatin immunoprecipitation, it was shown that exogenous Tat enhanced binding of SMAD2, -3 and -4 and their binding partner Fast1 to the JCV CR in living cells. When SMAD2, -3 and -4 were expressed together, Tat, expressed from plasmid pTat, stimulated transcription from both early and late gene promoters, with the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and JCV large T-antigen were all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results revealed a positive reinforcement system in which the SMAD mediators of the TGF-beta system act cooperatively with Tat to stimulate JCV gene transcription.
Assuntos
Regulação Viral da Expressão Gênica , Vírus JC/fisiologia , Proteínas Smad/metabolismo , Transcrição Gênica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Encéfalo/patologia , Linhagem Celular , Imunoprecipitação da Cromatina , DNA Viral/metabolismo , HIV-1/fisiologia , Humanos , Leucoencefalopatia Multifocal Progressiva/patologia , Leucoencefalopatia Multifocal Progressiva/virologia , Oligodendroglia/virologia , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Pur-alpha is a ubiquitous multifunctional protein that is strongly conserved throughout evolution, binds to both DNA and RNA and functions in the initiation of DNA replication, control of transcription and mRNA translation. In addition, it binds to several cellular regulatory proteins including the retinoblastoma protein, E2F-1, Sp1, YB-1, cyclin T1/Cdk9 and cyclin A/Cdk2. These observations and functional studies provide evidence that Puralpha is a major player in the regulation of the cell cycle and oncogenic transformation. Puralpha also binds to viral proteins such as the large T-antigen of JC virus (JCV) and the Tat protein of human immunodeficiency virus-1 (HIV-1) and plays a role in the cross-communication of these viruses in the opportunistic polyomavirus JC (JCV) brain infection, progressive multifocal leukoencephalopathy (PML). The creation of transgenic mice with inactivation of the PURA gene that encodes Puralpha has revealed that Puralpha is critical for postnatal brain development and has unraveled an essential role of Puralpha in the transport of specific mRNAs to the dendrites and the establishment of the postsynaptic compartment in the developing neurons. Finally, the availability of cell cultures from the PURA knockout mice has allowed studies that have unraveled a role for Puralpha in DNA repair.