RESUMO
Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Mutagênese/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinolinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mad2/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.
Assuntos
5-Metilcitosina/análogos & derivados , Reparo do DNA/fisiologia , DNA de Cadeia Simples/fisiologia , 5-Metilcitosina/metabolismo , Ácido Apurínico/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , Replicação do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases , Escherichia coli/metabolismo , Polinucleotídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.
Assuntos
Evolução Biológica , HIV-1/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Entropia , Aptidão Genética , Infecções por HIV/virologia , Humanos , Mutação , Fases de Leitura Aberta , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genéticaRESUMO
Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBα, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription in vivo. REV-ERBα condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBα. This work demonstrates physiological circadian protein condensates containing REV-ERBα whose IDR is required for hub formation and the control of rhythmic gene expression.
Assuntos
Relógios Circadianos , Camundongos , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Fígado/metabolismo , Expressão GênicaRESUMO
Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.
Assuntos
Proteína BRCA2/metabolismo , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Exodesoxirribonucleases/metabolismo , Fatores de Transcrição/química , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Células HeLa , Humanos , Ácido Mevalônico , Camundongos , Complexos Multiproteicos , Mutação , Ligação Proteica , Conformação Proteica , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Recombinação GenéticaRESUMO
BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.
Assuntos
Quebras de DNA de Cadeia Simples , DNA Primase/metabolismo , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Neoplasias/enzimologia , Nucleotidiltransferases/metabolismo , Reparo de DNA por Recombinação , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA Primase/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Feminino , Células HEK293 , Humanos , Camundongos Nus , Enzimas Multifuncionais/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chemotherapy with cisplatin becomes limiting due to toxicity and secondary malignancies. In principle, therapeutics could be improved by targeting translesion synthesis (TLS) polymerases (Pols) that promote replication through intrastrand cross-links, the major cisplatin-induced DNA adduct. However, to specifically target malignancies with minimal adverse effects on normal cells, a good understanding of TLS mechanisms in normal versus cancer cells is paramount. We show that in normal cells, TLS through cisplatin intrastrand cross-links is promoted by Polη- or Polι-dependent pathways, both of which require Rev1 as a scaffolding component. In contrast, cancer cells require Rev1-Polζ. Our findings that a recently identified Rev1 inhibitor, JH-RE-06, purported to specifically disrupt Rev1 interaction with Polζ to block TLS through cisplatin adducts in cancer cells, abrogates Rev1's ability to function with Y family Pols as well, implying that by inactivating Rev1-dependent TLS in normal cells, this inhibitor will exacerbate the toxicity and tumorigenicity of chemotherapeutics with cisplatin.
Assuntos
Cisplatino , Dano ao DNA , Cisplatino/farmacologia , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.
Assuntos
DNA/genética , RNA/genética , Saccharomyces cerevisiae/genética , Adolescente , Adulto , DNA/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Instabilidade Genômica/genética , Humanos , Pessoa de Meia-Idade , RNA/ultraestrutura , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Adulto JovemRESUMO
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , DNA Primase/metabolismo , DNA Primase/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , Humanos , Células K562 , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/fisiologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/fisiologia , Fatores de Transcrição/genéticaRESUMO
Genetic alterations are often acquired during prolonged propagation of pluripotent stem cells (PSCs). This ruins the stem cell quality and hampers their full applications. Understanding how PSCs maintain genomic integrity would provide the clues to overcome the hurdle. It has been known that embryonic stem cells (ESCs) utilize high-fidelity pathways to ensure genomic stability, but the underlying mechanisms remain largely elusive. Here, we show that many DNA damage response and repair genes display differential alternative splicing in mouse ESCs compared to differentiated cells. Particularly, Rev1 and Polq, two key genes for mutagenic translesion DNA synthesis (TLS) and microhomology-mediated end joining (MMEJ) repair pathways, respectively, display a significantly higher rate of cryptic exon (CE) inclusion in ESCs. The frequent CE inclusion disrupts the normal protein expressions of REV1 and POLθ, thereby suppressing the mutagenic TLS and MMEJ. Further, we identify an ESC-specific RNA binding protein DPPA5A which stimulates the CE inclusion in Rev1 and Polq. Depletion of DPPA5A in mouse ESCs decreased the CE inclusion of Rev1 and Polq, induced the protein expression, and stimulated the TLS and MMEJ activity. Enforced expression of DPPA5A in NIH3T3 cells displayed reverse effects. Mechanistically, we found that DPPA5A directly regulated CE splicing of Rev1. DPPA5A associates with U2 small nuclear ribonucleoprotein of the spliceosome and binds to the GA-rich motif in the CE of Rev1 to promote CE inclusion. Thus, our study uncovers a mechanism to suppress mutagenic TLS and MMEJ pathways in ESCs.
Assuntos
Mutagênicos , Nucleotidiltransferases , Animais , Camundongos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , DNA , Dano ao DNARESUMO
Physiological root resorption is a common occurrence during the development of deciduous teeth in children. Previous research has shown that the regulation of the inflammatory microenvironment through autophagy in DDPSCs is a significant factor in this process. However, it remains unclear why there are variations in the autophagic status of DDPSCs at different stages of physiological root resorption. To address this gap in knowledge, this study examines the relationship between the circadian clock of DDPSCs, the autophagic status, and the periodicity of masticatory behavior. Samples were collected from deciduous teeth at various stages of physiological root resorption, and DDPSCs were isolated and cultured for analysis. The results indicate that the circadian rhythm of important autophagy genes, such as Beclin-1 and LC3, and the clock gene REV-ERBα in DDPSCs, disappears under mechanical stress. Additionally, the study found that REV-ERBα can regulate Beclin-1 and LC3. Evidence suggests that mechanical stress is a trigger for the regulation of autophagy via REV-ERBα. Overall, this study highlights the importance of mechanical stress in regulating autophagy of DDPSCs via REV-ERBα, which affects the formation of the inflammatory microenvironment and plays a critical role in physiological root resorption in deciduous teeth.
Assuntos
Relógios Circadianos , Reabsorção da Raiz , Criança , Humanos , Reabsorção da Raiz/genética , Proteína Beclina-1/genética , Ritmo Circadiano/genética , Células-Tronco , Dente DecíduoRESUMO
The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Heterocromatina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Instabilidade Cromossômica , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Heterocromatina/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Transdução de SinaisRESUMO
BACKGROUND: While the daily rhythm of allergic rhinitis (AR) has long been recognized, the molecular mechanism underlying this phenomenon remains enigmatic. OBJECTIVE: We aimed to investigate the role of circadian clock in AR development and to clarify the mechanism by which the daily rhythm of AR is generated. METHODS: AR was induced in mice with ovalbumin. Toluidine blue staining, liquid chromatography-tandem mass spectrometry analysis, real-time quantitative PCR, and immunoblotting were performed with AR and control mice. RESULTS: Ovalbumin-induced AR is diurnally rhythmic and associated with clock gene disruption in nasal mucosa. In particular, Rev-erbα is generally downregulated and its rhythm retained, but with a near-12-hour phase shift. Furthermore, global knockout of core clock gene Bmal1 or Rev-erbα increases the susceptibility of mice to AR and blunts AR rhythmicity. Importantly, nasal solitary chemosensory cells (SCCs) are rhythmically activated, and inhibition of the SCC pathway leads to attenuated AR and a loss of its rhythm. Moreover, rhythmic activation of SCCs is accounted for by diurnal expression of ChAT (an enzyme responsible for the synthesis of acetylcholine) and temporal generation of the neurotransmitter acetylcholine. Mechanistically, Rev-erbα trans-represses Chat through direct binding to a specific response element, generating a diurnal oscillation in this target gene. CONCLUSION: SCCs, under the control of Rev-erbα, are a driver of AR rhythmicity; targeting SCCs should be considered as a new avenue for AR management.
RESUMO
REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin in vitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin in vivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.
Assuntos
Adenocarcinoma , Fenômenos Biológicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Platina/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Adenocarcinoma/tratamento farmacológico , Reparo do DNA/genéticaRESUMO
Cooling patients to sub-physiological temperatures is an integral part of modern medicine. We show that cold exposure induces temperature-specific changes to the higher-order chromatin and gene expression profiles of human cells. These changes are particularly dramatic at 18°C, a temperature synonymous with that experienced by patients undergoing controlled deep hypothermia during surgery. Cells exposed to 18°C exhibit largely nuclear-restricted transcriptome changes. These include the nuclear accumulation of mRNAs encoding components of the negative limbs of the core circadian clock, most notably REV-ERBα. This response is accompanied by compaction of higher-order chromatin and hindrance of mRNPs from engaging nuclear pores. Rewarming reverses chromatin compaction and releases the transcripts into the cytoplasm, triggering a pulse of negative limb gene proteins that reset the circadian clock. We show that cold-induced upregulation of REV-ERBα is sufficient to trigger this reset. Our findings uncover principles of the cellular cold response that must be considered for current and future applications involving therapeutic deep hypothermia.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Ritmo Circadiano/fisiologia , Temperatura Baixa , RNA Mensageiro/metabolismo , Linhagem Celular , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/genética , Técnicas de Inativação de Genes , Heterocromatina , Humanos , Hipotermia/cirurgia , Ativação Transcricional , Transcriptoma , Regulação para CimaRESUMO
IMPORTANCE: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.
Assuntos
Transporte Ativo do Núcleo Celular , Infecções por HIV , HIV-1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Processamento Alternativo , Núcleo Celular/metabolismo , Produtos do Gene rev/genética , HIV-1/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Splicing de RNA , RNA Viral/genética , RNA Viral/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismoRESUMO
Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirusï¼APVï¼ has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus.
Assuntos
Avipoxvirus , Vírus da Varíola das Aves Domésticas , Genoma Viral , Filogenia , Vírus da Reticuloendoteliose , Sequenciamento Completo do Genoma , Animais , Genoma Viral/genética , Vírus da Reticuloendoteliose/genética , Vírus da Reticuloendoteliose/isolamento & purificação , Avipoxvirus/genética , Avipoxvirus/classificação , Avipoxvirus/isolamento & purificação , Vírus da Varíola das Aves Domésticas/genética , Vírus da Varíola das Aves Domésticas/isolamento & purificação , China , Embrião de Galinha , Integração Viral/genética , Galinhas/virologia , Fases de Leitura Aberta/genética , Doenças das Aves Domésticas/virologia , Análise de Sequência de DNA , DNA Viral/genética , Varíola Aviária/virologia , Infecções por Poxviridae/virologia , Infecções por Poxviridae/veterináriaRESUMO
Allergens and Th2 cytokines affect the homeostatic environment in the airways, leading to increased mucus production by goblet cells associated with altered adherens junctional complex (AJC) and tight junction (TJ) proteins responsible for maintaining epithelial barrier function. Circadian clock-dependent regulatory mechanisms such as inflammation and epithelial barrier function are gaining more attention due to their therapeutic potential against allergic inflammatory lung diseases. Currently, there are no studies to support whether REV-ERBα activation can attenuate Th2 cytokine-induced epithelial barrier dysfunction in human bronchial epithelial cells. We hypothesized that Th2 cytokine-induced epithelial barrier dysfunction may be protected by activating REV-ERBα. Treatment with Th2 cytokines or HDM significantly reduced the cell impedance, as confirmed by transepithelial electrical resistance (TEER). However, pre-treatment with SR10067 attenuated Th2 cytokine-induced barrier dysfunction, such as decreased permeability, improved TEER, localization of AJC and TJ proteins, and mRNA and protein levels of selected epithelial barrier and circadian clock targets. Overall, we showed for the first time that REV-ERBα activation regulates altered epithelial barrier function that may have direct implications for the treatment of asthma and other allergic diseases.
RESUMO
BACKGROUND: Radioresistance is a key clinical constraint on the efficacy of radiotherapy in lung cancer patients. REV1 DNA directed polymerase (REV1) plays an important role in repairing DNA damage and maintaining genomic stability. However, its role in the resistance to radiotherapy in lung cancer is not clear. This study aims to clarify the role of REV1 in lung cancer radioresistance, identify the intrinsic mechanisms involved, and provide a theoretical basis for the clinical translation of this new target for lung cancer treatment. METHODS: The effect of targeting REV1 on the radiosensitivity was verified by in vivo and in vitro experiments. RNA sequencing (RNA-seq) combined with nontargeted metabolomics analysis was used to explore the downstream targets of REV1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the content of specific amino acids. The coimmunoprecipitation (co-IP) and GST pull-down assays were used to validate the interaction between proteins. A ubiquitination library screening system was constructed to investigate the regulatory proteins upstream of REV1. RESULTS: Targeting REV1 could enhance the radiosensitivity in vivo, while this effect was not obvious in vitro. RNA sequencing combined with nontargeted metabolomics revealed that the difference result was related to metabolism, and that the expression of glycine, serine, and threonine (Gly/Ser/Thr) metabolism signaling pathways was downregulated following REV1 knockdown. LC-MS/MS demonstrated that REV1 knockdown results in reduced levels of these three amino acids and that cystathionine γ-lyase (CTH) was the key to its function. REV1 enhances the interaction of CTH with the E3 ubiquitin ligase Rad18 and promotes ubiquitination degradation of CTH by Rad18. Screening of the ubiquitination compound library revealed that the ubiquitin-specific peptidase 9 X-linked (USP9X) is the upstream regulatory protein of REV1 by the ubiquitin-proteasome system, which remodels the intracellular Gly/Ser/Thr metabolism. CONCLUSION: USP9X mediates the deubiquitination of REV1, and aberrantly expressed REV1 acts as a scaffolding protein to assist Rad18 in interacting with CTH, promoting the ubiquitination and degradation of CTH and inducing remodeling of the Gly/Ser/Thr metabolism, which leads to radioresistance. A novel inhibitor of REV1, JH-RE-06, was shown to enhance lung cancer cell radiosensitivity, with good prospects for clinical translation.
Assuntos
Neoplasias Pulmonares , Nucleotidiltransferases , Tolerância a Radiação , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Camundongos , Animais , DNA Polimerase Dirigida por DNARESUMO
Artificial light at night (ALAN) is an emerging environmental pollutant that threatens public health. Recently, ALAN has been identified as a risk factor for obesity; however, the role of ALAN and its light wavelength in hepatic lipid metabolic homeostasis remains undetermined. We showed that chronic dim (~5 lx) ALAN (dLAN) exposure significantly promoted hepatic lipid accumulation in obese or diabetic mice, with the most severe effect of blue light and little effect of green or red light. These metabolic phenotypes were attributed to blue rather than green or red dLAN interfering with hepatic lipid metabolism, especially lipogenesis and lipolysis. Further studies found that blue dLAN disrupted hepatic lipogenesis and lipolysis processes by inhibiting hepatic REV-ERBs. Mechanistically, feeding behavior mediated the regulation of dLAN on hepatic REV-ERBs. In addition, different effects of light wavelengths at night on liver REV-ERBs depended on the activation of the corticosterone (CORT)/glucocorticoid receptor (GR) axis. Blue dLAN could activate the CORT/GR axis significantly while other wavelengths could not. Notably, we demonstrated that exogenous melatonin could effectively inhibit hepatic lipid accumulation and restore the hepatic GR/REV-ERBs axis disrupted by blue dLAN. These findings demonstrate that dLAN promotes hepatic lipid accumulation in mice via a short-wavelength-dependent manner, and exogenous melatonin is a potential therapeutic approach. This study strengthens the relationship between ALAN and hepatic lipid metabolism and provides insights into directing ambient light.