RESUMO
Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.
Assuntos
Poli A , Poliadenilação , Regiões 3' não Traduzidas , Humanos , Poli A/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Zinco/metabolismoRESUMO
Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.
Assuntos
Sistemas CRISPR-Cas , Genes Reporter , Precursores de RNA , Fatores de Poliadenilação e Clivagem de mRNA , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Genoma Humano , Células HEK293 , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Poliadenilação , Clivagem do RNA , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genéticaRESUMO
SUMO modifications regulate the function of many proteins and are important in controlling herpesvirus infections. We performed a site-specific proteomic analysis of SUMO1- and SUMO2-modified proteins in Epstein-Barr virus (EBV) latent and lytic infection to identify proteins that change in SUMO modification status in response to EBV reactivation. Major changes were identified in all three components of the TRIM24/TRIM28/TRIM33 complex, with TRIM24 being rapidly degraded and TRIM33 being phosphorylated and SUMOylated in response to EBV lytic infection. Further experiments revealed TRIM24 and TRIM33 repress expression of the EBV BZLF1 lytic switch gene, suppressing EBV reactivation. However, BZLF1 was shown to interact with TRIM24 and TRIM33, resulting in disruption of TRIM24/TRIM28/TRIM33 complexes, degradation of TRIM24 and modification followed by degradation of TRIM33. Therefore, we have identified TRIM24 and TRIM33 as cellular antiviral defence factors against EBV lytic infection and established the mechanism by which BZLF1 disables this defence.
Assuntos
Infecções por Vírus Epstein-Barr , Humanos , Herpesvirus Humano 4/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteômica , Ativação Viral , Latência Viral , Fatores de Transcrição/metabolismo , Proteínas de TransporteRESUMO
The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3'UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3'UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.
Assuntos
Carboxipeptidases/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/metabolismo , Interações Hospedeiro-Parasita/fisiologia , MicroRNAs/metabolismo , Proteínas Virais de Fusão/metabolismo , Linhagem Celular , Infecções por Vírus Epstein-Barr/metabolismo , Humanos , SumoilaçãoRESUMO
Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.
Assuntos
Nucléolo Celular/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Sondas Moleculares/química , Domínios Proteicos , Proteínas Repressoras/metabolismo , Metilação , Mieloma Múltiplo/metabolismo , Nucleossomos/metabolismoRESUMO
Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.
Assuntos
Chaperonas de Histonas/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Evolução Biológica , Sequência Conservada , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Expressão Gênica , Células HEK293 , Chaperonas de Histonas/química , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidade , Oncogenes , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/crescimento & desenvolvimentoRESUMO
OBJECTIVES: Same day discharge after minimally invasive hysterectomy has been shown to be safe and feasible. We designed and implemented a quality improvement perioperative program based on early recovery after surgery principles to improve the rate of same day discharge from 30% to 75% after minimally invasive gynecologic oncology surgery over a 12 month period. METHODS: We enrolled 102 consecutive patients undergoing minimally invasive hysterectomy at a single cancer center during a 12 month period. A pre-intervention cohort of 100 consecutive patients was identified for comparison of clinicodemographic variables and perioperative outcomes. A multidisciplinary team developed a comprehensive perioperative care program and followed quality improvement methodology. Patients were followed up for 30 days after discharge. A statistical process chart was used to monitor the effects of our interventions, and a multivariate analysis was conducted to determine factors associated with same day discharge. RESULTS: Same day discharge rate increased from 29% to 75% after implementation (p<0.001). The post-intervention cohort was significantly younger (59 vs 62 years; p=0.038) and had shorter operative times (180 vs 211 min; p<0.001) but the two groups were similar in body mass index, comorbidity, stage, and intraoperative complications. There was no difference in 30 day perioperative complications, readmissions, reoperations, emergency department visits, or mortality. Overnight admissions were secondary to nausea and vomiting (16%), complications of pre-existing comorbidities (12%), and urinary retention (8%). On multivariate analysis, longer surgery, timing of surgery, and narcotic use on the ward were significantly associated with overnight admission. Overall, 89% of patients rated their experience as 'very good' or 'excellent', and 87% felt that their length of stay was adequate. CONCLUSIONS: Following implementation of a perioperative quality improvement program targeted towards minimally invasive gynecologic oncology surgery, our intervention significantly improved same day discharge rates while maintaining a low 30 day perioperative complication rate and excellent patient experience.
Assuntos
Neoplasias dos Genitais Femininos , Alta do Paciente , Feminino , Neoplasias dos Genitais Femininos/cirurgia , Humanos , Tempo de Internação , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Complicações Pós-Operatórias/epidemiologia , Melhoria de Qualidade , Estudos RetrospectivosRESUMO
Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 µM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/química , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Proteína do Locus do Complexo MDS1 e EVI1/genética , Camundongos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína 4 de Ligação ao Retinoblastoma/química , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here, we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS) cells, nasopharyngeal carcinoma (NPC43) cells, and B (M81) cells led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing that ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit CK2ß, suggesting that CK2ß might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2ß, but not the CK2α catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 was associated with Zp in reporter assays and this was increased by additional CK2ß. Together, the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2ß-mediated interaction.IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Interações Hospedeiro-Patógeno , Ativação Viral , Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection, including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV.IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes, and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.
Assuntos
Antígenos Virais/metabolismo , Dano ao DNA , Herpesvirus Humano 4/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Antígenos Virais/genética , Linhagem Celular Tumoral , Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Células HeLa , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Glicoproteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Transativadores/metabolismo , Ativação Transcricional , Proteínas Virais/metabolismo , Replicação ViralRESUMO
To replicate and persist in human cells, linear double-stranded DNA (dsDNA) viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV), and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumors in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma.IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including â¼10% of gastric carcinomas. EBV encodes â¼80 proteins, many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. In this study, a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and blocks their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it downregulates the DDR, as well as in EBV-positive gastric tumors. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Histonas/metabolismo , Neoplasias Gástricas/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Biblioteca Gênica , Células HEK293 , Humanos , Domínios Proteicos , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/química , Replicação ViralRESUMO
Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.
Assuntos
Proteínas Culina/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Ubiquitinas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Culina/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/química , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Variação Genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/genética , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/química , Ubiquitinas/genética , Proteínas Contendo Repetições de beta-Transducina/antagonistas & inibidores , Proteínas Contendo Repetições de beta-Transducina/química , Proteínas Contendo Repetições de beta-Transducina/genéticaRESUMO
Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.
Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Cromatina/química , Imunoprecipitação/métodos , Proteômica/métodos , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/metabolismo , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Proteínas/química , Proteoma , Reprodutibilidade dos TestesRESUMO
UNLABELLED: Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. IMPORTANCE: Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins from three different herpesviruses that contribute to this block. Several of the proteins we identified had previously unknown functions or were structural components of the virion. Subsets of these proteins from Epstein-Barr virus were studied for their effects on the cell cycle regulatory proteins p53 and p21, thereby identifying two proteins that induce p53 and one that induces p21 (BGLF2). We identified interactions of BGLF2 with two human proteins, both of which regulate p21, suggesting that BGLF2 induces p21 by interfering with the functions of these two host proteins. Our study indicates that multiple herpesvirus proteins contribute to the cell proliferation block, including components of the incoming virions.
Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular , Infecções por Herpesviridae/fisiopatologia , Herpesviridae/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Proteínas Virais/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Herpesviridae/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/genéticaRESUMO
We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.
Assuntos
Benzamidas/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Descoberta de Drogas , Lisina/análogos & derivados , Sondas Moleculares/farmacologia , Piperidinas/farmacologia , Benzamidas/química , Benzamidas/metabolismo , Ligação Competitiva/efeitos dos fármacos , Cristalografia por Raios X , Proteínas de Ligação a DNA/antagonistas & inibidores , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Lisina/antagonistas & inibidores , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Estrutura Molecular , Piperidinas/química , Piperidinas/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/metabolismoRESUMO
Introduction: Upper limb function loss in cervical spinal cord injury (SCI) contributes to substantial disability, and negatively impacts quality of life. Nerve transfer and tendon transfer surgery can provide improved upper limb function. This study assessed the utilization of nerve and tendon transfer surgery for individuals with tetraplegia in Canada. Methods: Data from the Canadian Institute for Health Information's Discharge Abstracts Database and the National Ambulatory Care Reporting System were used to identify the nerve and tendon transfer procedures performed in individuals with tetraplegia (2004-2020). Cases were identified using cervical SCI ICD-10-CA codes and Canadian Classification of Intervention codes for upper extremity nerve and tendon transfers. Data on sex, age at time of procedure, province, and hospital stay duration were recorded. Results: From 2004 to 2020, there were ≤80 nerve transfer procedures (81% male, mean age 38.3 years) and 61 tendon transfer procedures (78% male, mean age 45.0 years) performed (highest in Ontario and British Columbia). Using an estimate of 50% eligibility, an average of 1.3% of individuals underwent nerve transfer and 1.0% underwent tendon transfer. Nerve transfers increased over time (2004-2009, n = <5; 2010-2015, n = 27; 2016-2019, n = 49) and tendon transfers remained relatively constant. Both transfer types were performed as day-surgery or single night stay. Conclusions: Nerve and tendon transfer surgery to improve upper limb function in Canadians with tetraplegia remains low. This study highlights a substantial gap in care for this vulnerable population. Identification of barriers that prevent access to care is required to promote best practice for upper extremity care.
Introduction : La perte de fonction du membre supérieur en cas de lésion de la moelle épinière cervicale (SCI0 contribue à un handicap substantiel avec des répercussions négatives sur la qualité de vie. La chirurgie de transfert des nerfs et des tendons peut apporter une amélioration du fonctionnement du membre supérieur. Cette étude a évalué l'utilisation de la chirurgie de transfert de nerfs et de tendons pour les patients tétraplégiques au Canada. Méthodes : Des données issues de la base de données des résumés de congés de l'Institut canadien d'information sur la santé du système national d'information sur les soins ambulatoires ont été utilisées pour identifier les procédures de transfert de nerfs et de tendons pratiquées sur des patients tétraplégiques entre 2004 et 2020. Les cas ont été identifiés en utilisant les codes de SCI cervicales du CIM-10-CA et des codes canadiens de classification des interventions pour les transferts de nerfs et de tendons du membre supérieur. Les données sur le sexe et l'âge au moment de la procédure, la province et la durée de séjour à l'hôpital ont été consignées. Résultats : Entre 2004 et 2020, il y a eu ≤ 80 procédures de transferts de nerfs (hommes : 81 %, âge moyen : 38,3 ans) et 61 procédures de transfert de tendons (hommes : 78 %, âge moyen : 45,0 ans) pratiquées (principalement en Ontario et en Colombie-Britannique). En estimant une admissibilité de 50 %, une moyenne de 1,3 % des patients a subi un transfert de nerfs et 1,0 % des patients a subi un transfert tendineux. Les transferts de nerfs ont augmenté au fil des années (2004-2009, n = < 5; 2010-2015, n = 27; 2016-2019, n = 49) tandis que le nombre de transferts tendineux est resté relativement stable. Les deux types de transferts ont été pratiqués das le cadre de la chirurgie d'un jour ou avec une hospitalisation d'une seule nuit. Conclusions : La chirurgie de transfert de nerfs et de tendons pour l'amélioration des fonctions des membres supérieurs reste peu utilisée pour les Canadiens tétraplégiques. Cette étude souligne une lacune substantielle des soins pour cette population vulnérable. Il est nécessaire d'identifier les obstacles qui empêchent l'accès aux soins afin de promouvoir une meilleure pratique pour les soins du membre supérieur.
RESUMO
During meiosis, accurate segregation of intact chromosomes is essential for generating healthy gametes. Defects in recombination and/or chromosome synapsis activate the pachytene checkpoint, which delays meiotic cell cycle progression to avoid aberrant chromosome segregation and formation of defective gametes. Here, we characterize the role of the conserved DNA damage checkpoint protein Ddc2/ATRIP in this meiotic surveillance mechanism. We show that deletion of DDC2 relieves the checkpoint-dependent meiotic block that occurs in Saccharomyces cerevisiae mutants defective in various aspects of meiotic chromosome dynamics and results in the generation of faulty meiotic products. Moreover, production of the Ddc2 protein is induced during meiotic prophase, accumulates in checkpoint-arrested mutants and localizes to distinctive chromosomal foci. Formation of meiotic Ddc2 foci requires the generation of Spo11-dependent DNA double-strand breaks (DSBs), and is impaired in an RPA mutant. Chromatin immunoprecipitation analysis reveals that Ddc2 accumulates at meiotic DSB sites, indicating that Ddc2 senses the presence of meiotic recombination intermediates. Furthermore, pachytene checkpoint signaling is defective in the ddc2 mutant. In addition, we show that mammalian ATRIP colocalizes with ATR, TopBP1 and RPA at unsynapsed regions of mouse meiotic chromosomes. Thus, our results point to an evolutionary conserved role for Ddc2/ATRIP in monitoring meiotic chromosome metabolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Masculino , Camundongos , Recombinação Genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.
Assuntos
Glicoproteína da Espícula de Coronavírus , Humanos , Bioensaio , Mutação , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Oxirredução , Estabilidade ProteicaRESUMO
SARS-CoV-2 depends on host cell components for infection and replication. Identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection and replication. If druggable, host factor dependencies may present an attractive strategy for anti-viral therapy. In this study, we performed genome wide CRISPR knockout screens in Vero E6 cells and four human cell lines including Calu-3, UM-UC-4, HEK-293 and HuH-7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while other host genes identified were largely cell line specific, including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, immune-related pathways, and chromatin modification. Notably, the chromatin modifier gene KMT2C in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed.
RESUMO
Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.