RESUMO
BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.
Assuntos
Antígenos CD55 , Núcleo Celular , Cromatina , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Histonas , Células-Tronco Neoplásicas , Neoplasias Ovarianas , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Feminino , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Camundongos , Antígenos CD55/metabolismo , Antígenos CD55/genética , Linhagem Celular Tumoral , Histonas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Metilação , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Transporte ProteicoRESUMO
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Assuntos
Nucleotídeos de Adenina/química , Endorribonucleases/química , Oligorribonucleotídeos/química , Difosfato de Adenosina/química , Adenilil Imidodifosfato/química , Animais , Repetição de Anquirina , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Vírus da Encefalomiocardite , Endorribonucleases/genética , Endorribonucleases/fisiologia , Células HeLa , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Picornaviridae , Estrutura Terciária de Proteína , Espalhamento de Radiação , Relação Estrutura-Atividade , Sus scrofaRESUMO
The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler's murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2'-5' oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/metabolismo , Endorribonucleases/antagonistas & inibidores , Vírus da Hepatite Murina/fisiologia , Oligorribonucleotídeos/metabolismo , Theilovirus/metabolismo , Proteínas Virais/metabolismo , Animais , Antivirais/metabolismo , Endorribonucleases/fisiologia , Células HeLa , Hepatite Viral Animal/metabolismo , Hepatite Viral Animal/virologia , Interações Hospedeiro-Patógeno , Humanos , CamundongosRESUMO
Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.
Assuntos
Infecções por Cardiovirus/metabolismo , Endorribonucleases/antagonistas & inibidores , Evasão da Resposta Imune/fisiologia , Imunidade Inata , Theilovirus/metabolismo , Proteínas Virais/metabolismo , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/patologia , Linhagem Celular , Cricetinae , Endorribonucleases/genética , Endorribonucleases/imunologia , Endorribonucleases/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Estrutura Terciária de Proteína , Especificidade da Espécie , Theilovirus/genética , Theilovirus/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Interactions between cigarette smoke (CS) exposure and viral infection play an important role(s) in the pathogenesis of chronic obstructive pulmonary disease and a variety of other disorders. A variety of lines of evidence suggest that this interaction induces exaggerated inflammatory, cytokine, and tissue remodeling responses. We hypothesized that the 2'-5' oligoadenylate synthetase (OAS)/RNase L system, an innate immune antiviral pathway, plays an important role in the pathogenesis of these exaggerated responses. To test this hypothesis, we characterize the activation of 2'-5' OAS in lungs from mice exposed to CS and viral pathogen-associated molecular patterns (PAMPs)/live virus, alone and in combination. We also evaluated the inflammatory and remodeling responses induced by CS and virus/viral PAMPs in lungs from RNase L null and wild-type mice. These studies demonstrate that CS and viral PAMPs/live virus interact in a synergistic manner to stimulate the production of select OAS moieties. They also demonstrate that RNase L plays a critical role in the pathogenesis of the exaggerated inflammatory, fibrotic, emphysematous, apoptotic, TGF-ß1, and type I IFN responses induced by CS plus virus/viral PAMP in combination. These studies demonstrate that CS is an important regulator of antiviral innate immunity, highlight novel roles of RNase L in CS plus virus induced inflammation, tissue remodeling, apoptosis, and cytokine elaboration and highlight pathways that may be operative in chronic obstructive pulmonary disease and mechanistically related disorders.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Endorribonucleases/metabolismo , Inflamação/enzimologia , Infecções por Orthomyxoviridae/complicações , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Inflamação/etiologia , Inflamação/patologia , Vírus da Influenza A , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/enzimologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4µ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.
Assuntos
Cumarínicos/farmacologia , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Animais , Sítios de Ligação , Cumarínicos/química , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/antagonistas & inibidores , Humanos , Lisina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteólise/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Ribonucleases/antagonistas & inibidores , Ribonucleases/metabolismo , Bases de Schiff/química , Bases de Schiff/metabolismo , Via Secretória/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Activation of RNase L endonuclease activity is part of the mammalian innate immune response to viral infection. The poliovirus RNA genome contains a sequence in its protein-coding region that can act as a competitive inhibitor of RNase L. Mutation, sequence, and functional analysis of this competitive inhibitor RNA (ciRNA) revealed that its activity depends on specific sequences, showed that a loop-loop hairpin interaction forms in the ciRNA, and suggested the presence of a loop E motif. These features lead to the hypothesis that the ciRNA's function is conferred in part by a specific three-dimensional folded RNA architecture. By using a combination of biophysical, mutational, and functional studies, we have mapped features of the three-dimensional architecture of the ciRNA in its unbound form. We show that the loop-loop interaction forms in the free ciRNA and affects the overall structure, perhaps forming long-range tertiary interactions with the loop E motif. Local tight RNA-RNA backbone packing occurs in parts of the structure, but the fold appears to be less stable than many other tightly packed RNAs. This feature may allow the ciRNA to accommodate the translocation of ribosomes and polymerase across this multifunctional region of the viral RNA but also to function as an RNase L inhibitor.
Assuntos
Endorribonucleases/antagonistas & inibidores , RNA/química , Animais , Sequência de Bases , Ligação Competitiva , Endorribonucleases/química , Endorribonucleases/genética , Temperatura Alta , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , RNA/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.
Assuntos
Evasão da Resposta Imune , Interferon Tipo I/imunologia , RNA não Traduzido/imunologia , RNA Viral/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Linhagem Celular , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA não Traduzido/genética , RNA Viral/genética , Virulência , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/patogenicidadeRESUMO
Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3'-to-5' exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases.
Assuntos
COVID-19 , HIV-1 , Humanos , SARS-CoV-2/genética , Exorribonucleases/genética , HIV-1/genética , Simulação de Acoplamento Molecular , Antivirais/farmacologia , Replicação Viral , Catecóis/farmacologia , Ribonuclease H/farmacologia , Proteínas não Estruturais Virais/genética , RNA Viral/genéticaRESUMO
RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 µM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-ß levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.
Assuntos
Antineoplásicos/farmacologia , Infecções por Cardiovirus/imunologia , Endorribonucleases/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Indóis/farmacologia , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Pirróis/farmacologia , eIF-2 Quinase/antagonistas & inibidores , Animais , Infecções por Cardiovirus/enzimologia , Infecções por Cardiovirus/genética , Vírus da Encefalomiocardite , Endorribonucleases/genética , Endorribonucleases/imunologia , Endorribonucleases/metabolismo , Humanos , Imunidade Inata/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Sunitinibe , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
PBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.
Assuntos
Carcinoma de Células Renais/patologia , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Túbulos Renais Proximais/metabolismo , Fator de Transcrição PAX8/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Camundongos Nus , Mutagênese , Fator de Transcrição PAX8/genética , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Ativação Transcricional , Transplante HeterólogoRESUMO
The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2',5'-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Neoplasias da Próstata/enzimologia , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/genética , Nucleotídeos de Adenina/química , Linhagem Celular Tumoral , Clonagem Molecular , Retrovirus Endógenos/genética , Ativação Enzimática , Produtos do Gene env/genética , Humanos , Masculino , Oligorribonucleotídeos/química , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Somatic mutations in TET2 are common in myelodysplastic syndromes (MDS), myeloproliferative, and overlap syndromes. TET2 mutant (TET2MT) clones are also found in asymptomatic elderly individuals, a condition referred to as clonal hematopoiesis of indeterminate potential (CHIP). In various entities of TET2MT neoplasia, we examined the phenotype in relation to the strata of TET2 hits within the clonal hierarchy. Using deep sequencing, 1781 mutations were found in 1205 of 4930 patients; 40% of mutant cases were biallelic. Hierarchical analysis revealed that of TET2MT cases >40% were ancestral, e.g., representing 8% of MDS. Higher (earlier) TET2 lesion rank within the clonal hierarchy (greater clonal burden) was associated with impaired survival. Moreover, MDS driven by ancestral TET2MT is likely derived from TET2MT CHIP with a penetrance of ~1%. Following ancestral TET2 mutations, individual disease course is determined by secondary hits. Using multidimensional analyses, we demonstrate how hits following the TET2 founder defect induces phenotypic shifts toward dysplasia, myeloproliferation, or progression to AML. In summary, TET2MT CHIP-derived MDS is a subclass of MDS that is distinct from de novo disease.
Assuntos
Células Clonais/patologia , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Diferenciação Celular , Evolução Clonal , Células Clonais/metabolismo , Dioxigenases , Progressão da Doença , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , PrognósticoRESUMO
Ribonuclease L (RNase L) is an antiviral endoribonuclease that cleaves hepatitis C virus (HCV) RNA at single-stranded UA and UU dinucleotides throughout the open reading frame (ORF). To determine whether RNase L exerts evolutionary pressure on HCV we examined the frequencies of UA and UU dinucleotides in 162 RNA sequences from the Los Alamos National Labs HCV Database (http://hcv.lanl.gov). Considering the base composition of the HCV ORFs, both UA and UU dinucleotides were less frequent than predicted in each of 162 HCV RNAs. UA dinucleotides were significantly less frequent than predicted at each of the three codon positions while UU dinucleotides were less frequent than predicted predominantly at the wobble position of codons. UA and UU dinucleotides were among the least abundant dinucleotides in HCV RNA ORFs. Furthermore, HCV genotype 1 RNAs have a lower frequency of UA and UU dinucleotides than genotype 2 and 3 RNAs, perhaps contributing to increased resistance of HCV genotype 1 infections to interferon therapy. In vitro, RNase L cleaved both HCV genotype 1 and 2 RNAs efficiently. Thus, RNase L can cleave HCV RNAs efficiently and variably reduced frequencies of UA and UU dinucleotides in HCV RNA ORFs are consistent with the selective pressure of RNase L.
Assuntos
Composição de Bases/genética , Fosfatos de Dinucleosídeos/genética , Endorribonucleases/metabolismo , Hepacivirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Códon/genética , Endorribonucleases/imunologia , Hepacivirus/imunologia , Seleção GenéticaRESUMO
The interferon (IFN)-inducible antiviral state is mediated in part by the 2',5'-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-ß by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-ß by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-ß production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-ß in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-ß induction that could affect tissue protection and survival during viral infections. IMPORTANCE Type I interferons (IFNs) such as IFN-ß are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-ß production in mouse embryonic fibroblasts and in virus-infected mice. Here we report that high basal levels of OAS/RNase L in macrophages reduce, rather than increase, virus induction of IFN-ß. RNA damage and apoptosis caused by RNase L were the likely reasons for the decreased IFN-ß production in virus-infected macrophages. Our studies suggest that during viral infections, the OAS/RNase L pathway can either enhance or suppress IFN production, depending on the cell type. IFN regulation by RNase L is suggested to contribute to tissue protection and survival during viral infections.
Assuntos
2',5'-Oligoadenilato Sintetase/imunologia , Vírus da Encefalomiocardite/imunologia , Endorribonucleases/imunologia , Fibroblastos/imunologia , Interferon beta/imunologia , Interferon beta/metabolismo , Macrófagos/imunologia , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Endorribonucleases/metabolismo , Fibroblastos/virologia , Macrófagos/virologia , CamundongosRESUMO
The interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway blocks infections by some types of viruses through cleavage of viral and cellular single-stranded RNA. Viruses induce type I IFNs that initiate signaling to the OAS genes. OAS proteins are pathogen recognition receptors for the viral pathogen-associated molecular pattern, double-stranded RNA. Double-stranded RNA activates OAS to produce p(x)5'A(2'p5'A)(n); x = 1-3; n > 2 (2-5A) from ATP. Upon binding 2-5A, RNase L is converted from an inactive monomer to a potently active dimeric endoribonuclease for single-stranded RNA. RNase L contains, from N- to C-terminus, a series of 9 ankyrin repeats, a linker, several protein kinase-like motifs, and a ribonuclease domain homologous to Ire1 (involved in the unfolded protein response). In the past few years, it has become increasingly apparent that RNase L and OAS contribute to innate immunity in many ways. For example, small RNA cleavage products produced by RNase L during viral infections can signal to the retinoic acid-inducible-I like receptors to amplify and perpetuate signaling to the IFN-ß gene. In addition, RNase L is now implicated in protecting the central nervous system against viral-induced demyelination. A role in tumor suppression was inferred by mapping of the RNase L gene to the hereditary prostate cancer 1 (HPC1) gene, which in turn led to discovery of the xenotropic murine leukemia-related virus. A broader role in innate immunity is suggested by involvement of RNase L in cytokine induction and endosomal pathways that suppress bacterial infections. These newly described findings about RNase L could eventually provide the basis for developing broad-spectrum antimicrobial drugs.
Assuntos
Endorribonucleases/metabolismo , Imunidade Inata , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Antígenos de Superfície/metabolismo , Doenças Desmielinizantes/prevenção & controle , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Endorribonucleases/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Interferons/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Domínios e Motivos de Interação entre Proteínas , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Viroses/imunologia , Viroses/metabolismoRESUMO
Bacterial hyaluronan lyase enzymes are the major virulence factors that enable greater microbial ingress by cleaving hyaluronan (HA) polymers present predominantly in extracellular space of vertebrates. Based on the premise that effective inhibitors may bind to and stabilize HA thereby protecting it from degradation, here we investigated inhibitory activity of human hyaluronan-binding protein 1 (HABP1) on bacterial hyaluronidase because it is highly specific to HA and localized on the cell surface. Biochemical characterization revealed that HABP1 is a competitive inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of 22 microm. This is thus the first report of an endogenous protein inhibitor that may be used during natural antibacterial defense. Our findings also support a novel multipronged mechanism for the high efficacy of HABP1-mediated inhibition based on structural modeling of enzyme, substrate, and inhibitor. Evidence from docking simulations and contact interface interactions showed that the inherent charge asymmetry of HABP1 plays a key role in the inhibitory activity. This novel role of HABP1 may pave the way for peptide inhibitors as alternatives to synthetic chemicals in antibacterial research.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Transporte/química , Inibidores Enzimáticos/química , Hialuronoglucosaminidase/antagonistas & inibidores , Proteínas Mitocondriais/química , Modelos Moleculares , Streptococcus pneumoniae/enzimologia , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/metabolismo , Proteínas Mitocondriais/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêuticoRESUMO
RNase L is an antiviral endoribonuclease that cleaves viral mRNAs after single-stranded UA and UU dinucleotides. Poliovirus (PV) mRNA is surprisingly resistant to cleavage by RNase L due to an RNA structure in the 3C(Pro) open reading frame (ORF). The RNA structure associated with the inhibition of RNase L is phylogenetically conserved in group C enteroviruses, including PV type 1 (PV1), PV2, PV3, coxsackie A virus 11 (CAV11), CAV13, CAV17, CAV20, CAV21, and CAV24. The RNA structure is not present in other human enteroviruses (group A, B, or D enteroviruses). Coxsackievirus B3 mRNA and hepatitis C virus mRNA were fully sensitive to cleavage by RNase L. HeLa cells expressing either wild-type RNase L or a dominant-negative mutant RNase L were used to examine the effects of RNase L on PV replication. PV replication was not inhibited by RNase L activity, but rRNA cleavage characteristic of RNase L activity was detected late during the course of PV infection, after assembly of intracellular virus. Rather than inhibiting PV replication, RNase L activity was associated with larger plaques and better cell-to-cell spread. Mutations in the RNA structure associated with the inhibition of RNase L did not affect the magnitude of PV replication in HeLa cells expressing RNase L, consistent with the absence of observed RNase L activity until after virus assembly. Thus, PV carries an RNA structure in the 3C protease ORF that potently inhibits the endonuclease activity of RNase L, but this RNA structure does not prevent RNase L activity late during the course of infection, as virus assembly nears completion.
Assuntos
Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Fases de Leitura Aberta/fisiologia , Poliovirus/genética , RNA Viral/fisiologia , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Sequência de Bases , Sequência Conservada , Farmacorresistência Viral/genética , Endorribonucleases/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Poliovirus/fisiologiaRESUMO
RNase L, a principal mediator of innate immunity to viral infections in higher vertebrates, is required for a complete IFN antiviral response against certain RNA stranded viruses. dsRNA produced during viral infections activates IFN-inducible synthetases that produce 5'-phosphorylated, 2',5'-oligoadenylates (2-5A) from ATP. 2-5A activates RNase L in a wide range of different mammalian cell types, thus blocking viral replication. However, 2-5A has unfavorable pharmacologic properties; it is rapidly degraded, does not transit cell membranes, and leads to apoptosis. To obtain activators of RNase L with improved drug-like properties, high-throughput screening was performed on chemical libraries by using fluorescence resonance energy transfer. Seven compounds were obtained that activated RNase L at micromolar concentrations, and structure-activity relationship studies resulted in identification of an additional four active compounds. Two lead compounds were shown to have a similar mechanistic path toward RNase L activation as the natural activator 2-5A. The compounds bound to the 2-5A-binding domain of RNase L (as determined by surface plasmon resonance and confirmed by computational docking), and the compounds induced RNase L dimerization and activation. Interestingly, the low-molecular-weight activators of RNase L had broad-spectrum antiviral activity against diverse types of RNA viruses, including the human pathogen human parainfluenza virus type 3, yet these compounds by themselves were not cytotoxic at the effective concentrations. Therefore, these RNase L activators are prototypes for a previously uncharacterized class of broad-spectrum antiviral agents.
Assuntos
Antivirais/metabolismo , Endorribonucleases/metabolismo , Ativadores de Enzimas/metabolismo , Imunidade Inata/fisiologia , Vírus da Parainfluenza 3 Humana/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Dimerização , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ativadores de Enzimas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Camundongos , Modelos Moleculares , Oligonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Replicação Viral/efeitos dos fármacosRESUMO
Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg(2+) concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function.