RESUMO
The ability of cells to induce the appropriate transcriptional response to inflammatory stimuli is crucial for the timely induction of host defense mechanisms. Although a role for tumor suppressor p14ARF (ARF) in the innate immune response was previously demonstrated, the underlying mechanism is still unclear. ARF is a potent upregulator of protein SUMOylation; however, no association of this function with the immune system has been made. In this study, we show the unique role of ARF in IFN-γ-induced immune response using human cell lines. Through a systematic search of proteins SUMOylated by ARF, we identified PIAS1, an inhibitor of IFN-activated transcription factor STAT1, as a novel ARF-binding partner and SUMOylation target. In response to IFN-γ treatment, ARF promoted PIAS1 SUMOylation to inhibit the ability of PIAS1 to attenuate IFN-γ response. Wild-type, but not ARF mutants unable to enhance PIAS1 SUMOylation, prevented the PIAS1-mediated inhibition of IFN-γ response. Conversely, the SUMO-deconjugase SENP1 deSUMOylated PIAS1 to reactivate PIAS1 that was inhibited by ARF. These findings suggest that PIAS1 function is negatively modulated by SUMO modification and that SUMOylation by ARF is required to inhibit PIAS1 activity and restore IFN-γ-induced transcription. In the presence of ARF, in which case PIAS1 is inhibited, depletion of PIAS1 did not have an additive effect on IFN-γ response, suggesting that ARF-mediated enhancement of IFN-γ response is mainly due to PIAS1 inhibition. Our findings reveal a novel function of ARF to inhibit PIAS1 by enhancing SUMOylation to promote the robust induction of IFN-γ response.
Assuntos
Imunidade Inata/imunologia , Interferon gama/imunologia , Proteínas Inibidoras de STAT Ativados/imunologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/imunologia , Sumoilação/imunologia , Proteína Supressora de Tumor p14ARF/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Inflamação/imunologia , Fator de Transcrição STAT1/imunologia , Transcrição Gênica/imunologia , Regulação para Cima/imunologiaRESUMO
Therapeutic peptides suffer from major drawbacks such as peptide degradation in vivo due to proteolysis. Gold nanoparticles (AuNPs) are an effective carrier for therapeutic peptides that improve their stability in vivo, while also enabling nonspecific adsorption of complementary proteins to enhance their effectiveness. Using p53 peptide as a model known to disrupt the intracellular MDM2-p53 protein-protein interaction which tags the endogenous p53 proteins for degradation, we conjugated p53 peptides to AuNPs (AuNP-p53) and examined the functionality of AuNP-p53 to release the endogenous p53 proteins from being tagged for degradation, thereby increasing the level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells. We found that AuNPs did not just protect conjugated p53 peptides from trypsin degradation, but also helped to recruit 56.5% and 26.4% of total MDM2 and p53 proteins in the cells to form a protein corona around AuNP-p53. The proximity of MDM2/p53 complexes and p53 peptide on the surface of AuNP-p53 facilitated the action of p53 peptides to cause a sustained elevation of the p53 level in AML2 cells up to 6 h, which was not possible with free p53 peptide alone at the same concentration. Even a 20-fold higher concentration of free p53 peptide caused only a short-lived elevated p53 level of 1 h. The outcome of this study highlights the utility of combining conjugated ligands and complementary protein adsorption on nanoparticles to improve the biological functionality of the therapeutic ligands.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Peptídeos/química , Coroa de Proteína/química , Proteína Supressora de Tumor p53/química , Linhagem Celular Tumoral , Humanos , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing promoter, CpGrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (EGFP) after 8 weeks of culturing. RESULTS: While the promoters gave similar transient expression levels, CpGfree clones had significantly higher average stable expression possibly due to increased resistance to early silencing during integration into the chromosome. A greater proportion of cells in clones generated using the CpGfree promoter were still expressing detectable levels of EGFP after 8 weeks but the relative expression levels measured at week 8 to those measured at week 0 did not improve compared to clones generated using the CpGrich promoter. Chromatin immunoprecipitation assays indicated that the repression of the CpGfree promoter was likely linked to histone deacetylation and methylation. Use of histone deacetylase inhibitors also managed to recover some of the lost expression. CONCLUSION: Using a promoter without CpG dinucleotides could mitigate the early gene silencing but did not improve longer-term expression stability as silencing due to histone modifications could still take place. The results presented here would aid in promoter selection and design for improved protein production in CHO and other mammalian cells.
Assuntos
Ilhas de CpG/genética , Melhoramento Genético/métodos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animais , Células CHO , Cricetulus , Estabilidade de Medicamentos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Acetylation of histones, the major protein component of eukaryotic chromosomes, contributes to the epigenetic regulation of gene expression and is also involved in cancer development. A recent study revealed the correlation between tumour formation and acetylation level of lysine K18 on histone H3. In this study, we developed two colorimetric in vitro assays using gold nanoparticles (AuNPs) for identification of lysine K18 acetylation on histone H3 peptide. In assay I, citrate ion-capped AuNP without further modification was employed. Simply mixing the K18 peptide with AuNP solution leads to distinct particle aggregation, relative to that by non-acetylated or lysine K14 acetylated control peptides. In assay II, an AuNP-peptide-antibody composite was synthesized and used as both the sensing probe and the transducing element. By mixing the sample peptides with the composite solution followed by PBS screening, different aggregation behaviours were observed between the K18 acetylated target peptide and the control sequences. Both assays are capable of identifying the acetylated peptides, and also differentiating the distinctive acetylation positions that differ merely by a distance of three amino acids.
Assuntos
Técnicas Biossensoriais , Histonas/química , Lisina/química , Nanopartículas Metálicas/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Acetilação , Sequência de Aminoácidos , Anticorpos/química , Cromatina/química , Cromatina/metabolismo , Colorimetria/métodos , Colorimetria/normas , Floculação , Ouro/química , Histonas/metabolismo , Lisina/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismoRESUMO
Integration, one of the hallmarks of retrovirus replication, is mediated by a nucleoprotein complex called the preintegration complex (PIC), in which viral DNA is associated with many protein components that are required for completion of the early phase of infection. A striking feature of the PIC is its powerful integration activity in vitro. The PICs from a freshly isolated cytoplasmic extract of infected cells are able to insert viral DNA into exogenously added target DNA in vitro. Therefore, a PIC-based in vitro assay is a reliable system for assessing protein factors influencing retroviral integration. In this study, we applied a microtiter plate-based in vitro assay to a screening study using a protein library that was produced by the wheat germ cell-free protein synthesis system. Using a library of human E3 ubiquitin ligases, we identified RFPL3 as a potential stimulator of human immunodeficiency virus, type 1 (HIV-1) PIC integration activity in vitro. This enhancement of PIC activity by RFPL3 was likely to be attributed to its N-terminal RING domain. To further understand the functional role of RFPL3 in HIV infection, we created a human cell line overexpressing RFPL3. Immunoprecipitation analysis revealed that RFPL3 was associated with the human immunodeficiency virus, type 1 PICs in infected cells. More importantly, single-round HIV-1 infection was enhanced significantly by RFPL3 expression. Our proteomic approach displays an advantage in the identification of new cellular proteins affecting the integration activity of the PIC and, therefore, contributes to the understanding of functional interaction between retroviral integration complexes and host factors.
Assuntos
Proteínas de Transporte/fisiologia , HIV-1/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Células HEK293 , Humanos , Vírus da Leucemia Murina de Moloney/fisiologia , Ligação Proteica , Titulometria , Integração ViralRESUMO
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/fisiologia , Regulação para Cima/fisiologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/efeitos da radiação , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiaçãoRESUMO
X-box binding protein 1 (XBP-1) is a key regulator required for cellular unfolded protein response (UPR) and plasma cell differentiation. In addition, involvement of XBP-1 in host cell-virus interaction and transcriptional regulation of viruses, such as human T-lymphotropic virus type 1 (HTLV-1), has been revealed recently. Two XBP-1 isoforms, XBP-1U and XBP-1S, which share an identical N-terminal domain, are present in cells. XBP-1S is a transcription activator while XBP-1U is the inactive isoform. Although the transactivation domain of XBP-1S has been identified within the XBP-1S-specific C-terminus, molecular mechanism of the transcriptional activation by XBP-1S still remains unknown. Here we report the interaction between p300/CBP-associated factor (PCAF) and XBP-1S through the C-terminal domain of XBP-1S. No binding between XBP-1U and PCAF is detected. In a cell-based reporter assay, overexpression of PCAF further stimulates the XBP-1S-mediated cellular and HTLV-1 transcription while knockdown of PCAF exhibits the opposite effect. Expression of endogenous XBP-1S cellular target genes, such as BiP and CHOP, is significantly inhibited when PCAF is knocked down. Furthermore, PCAF is recruited to the promoters of XBP-1S target genes in vivo, in a XBP-1S-dependent manner. Collectively, our results demonstrate that PCAF mediates the XBP-1S-dependent transcription through the interaction with XBP-1S.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas de Ligação a DNA/química , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/química , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-BoxRESUMO
X-box binding protein 1 (XBP-1), a basic leucine zipper transcription factor, plays a key role in the cellular unfolded protein response (UPR). There are two XBP-1 isoforms in cells, spliced XBP-1S and unspliced XBP-1U. XBP-1U has been shown to bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. Here we identify XBP-1S as a transcription activator of HTLV-1. Compared to XBP-1U, XBP-1S demonstrates stronger activating effects on both basal and Tax-activated HTLV-1 transcription in cells. Our results show that both XBP-1S and XBP-1U interact with Tax and bind to the HTLV-1 LTR in vivo. In addition, elevated mRNA levels of the gene for XBP-1 and several UPR genes were detected in the HTLV-1-infected C10/MJ and MT2 T-cell lines, suggesting that HTLV-1 infection may trigger the UPR in host cells. We also identify Tax as a positive regulator of the expression of the gene for XBP-1. Activation of the UPR by tunicamycin showed no effect on the HTLV-1 LTR, suggesting that HTLV-1 transcription is specifically regulated by XBP-1. Collectively, our study demonstrates a novel host-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Proteínas Nucleares/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Infecções por HTLV-I/genética , Humanos , Proteínas Nucleares/genética , RNA Viral/análise , Fatores de Transcrição de Fator Regulador X , Sequências Repetidas Terminais , Fatores de Transcrição , Transcrição Gênica , Ativação Transcricional , Proteína 1 de Ligação a X-BoxRESUMO
Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70-80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3' untranslated region (3'UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3'UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3'UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-gamma in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3'UTR in CHO cells.
Assuntos
Regiões 3' não Traduzidas , Interferon gama/biossíntese , Fatores de Regulação Miogênica/genética , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Eritropoetina/genética , Eritropoetina/metabolismo , Expressão Gênica , Humanos , Interferon gama/genética , Modelos Biológicos , Fatores de Regulação Miogênica/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , TransgenesRESUMO
We have previously shown the use of gold nanoparticles (AuNPs) functionalized with DNA (AuNP-DNA) to increase insulin mRNA translation in a cell-free system. In this study, we translate the concept into a whole cell system to demonstrate functionality despite the additional complexity of intracellular delivery and mRNA translation inside living cells. We selected an insulin-secreting pancreatic islet cell line, RIN-5F, as our model and designed a DNA oligomer (insDNA) that is complementary to the 3'-untranslated region of insulin mRNA for conjugation to AuNPs (AuNP-insDNA). AuNP-insDNA was stable in the extracellular environment of RIN-5F cells for up to 24 h, without eliciting any cell toxicity. Upon cellular entry, AuNP-insDNA was able to sustain enhanced insulin secretion from 6 to 12 h post-incubation, peaking at 10 h with an enhancement factor of 1.69-fold. This enhancement was not observed when insDNA was removed or replaced with poly thymine or poly adenine DNAs. The enhanced insulin secreted was 100% functional and capable of binding to its insulin receptor. The outcome of this study demonstrated the feasibility of AuNP-DNA to enhance the synthesis of proteins in whole cells and could serve as a new direction of invoking a patient's own beta cells to increase insulin secretion for treatment of diabetes.
RESUMO
The emergence of antibiotic resistant bacteria is one of the biggest threats to human health worldwide. In 2017, World Health Organization listed the world's most dangerous antibiotic-resistant bacteria or "superbugs," such as carbapenem-resistant Pseudomonas aeruginosa and Escherichia coli, indicating the highest priority needs for new antibiotics. The possibility that such infectious diseases may soon be untreatable, due to decreased antibiotic efficacy, creates an urgent need for novel and alternative antimicrobials. Antimicrobial peptides are naturally occurring small molecules found in the innate immunity of mammals, plants and bacteria, and are potentially therapeutic candidates against drug-resistant bacteria. In this study, we examine the antimicrobial activities of the cytotoxic peptides derived from the basic region (BR) of the human hexamethylene bisacetamide-inducible protein 1 (HEXIM1). We found that, when fused with a cell penetrating peptide, the HEXIM1 BR peptide and its derivative, BR-RRR12, exhibited inhibitory activities against selected "superbugs." Negligible effects on the viability of human keratinocyte cell line were observed when the bactericidal dosages of HEXIM1 BR peptides were used. Different killing kinetics were observed between the membrane permeabilizing antimicrobial peptides and HEXIM1 BR peptides, suggesting that a different antimicrobial mechanism might be utilized by the HEXIM1 BR peptides. Using an in vitro translation system based on E. coli lysates, we found that HEXIM1 BR peptides blocked bacterial translation. Taken together, we identify the HEXIM1 BR peptide as a novel antimicrobial peptide with potent inhibitory activity against antibiotic-resistant "superbugs."
RESUMO
X-box binding protein 1 (XBP-1) is a key regulator of the cellular secretory pathway and unfolded protein response (UPR). It has been shown that the spliced form of XBP-1, XBP-1S, functions as a transcription activator and up-regulates many genes associated with protein secretion and biosynthesis of endoplasmic reticula. Since the production of some recombinant proteins is widely believed to be limited by the secretory capacity of the host cell, an increase in protein production may be achieved by overexpressing XBP-1S. In this study, the effects of XBP-1S on the productivity of monoclonal antibody (MAb), interferon gamma (IFNgamma), and erythropoietin (EPO) are examined in Chinese hamster ovary (CHO) and NS0 cell lines. Results show that XBP-1S may become a determinative factor only when accumulation of recombinant proteins exceeds the secretory capacity of the host cell. In transient transfection systems where a bottleneck in protein secretion was achieved, overexpression of XBP-1S improved protein titers by up to 2.5-fold. In contrast, overexpression of XBP-1S had no detectable effects on protein productivity of stable cell lines that did not exhibit any secretory bottleneck. We conclude that overexpression of XBP-1S is an effective strategy in enhancing recombinant protein production when the secretory pathway of the host cell is saturated by high-level synthesis of recombinant proteins.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mieloma Múltiplo/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Fatores de Transcrição de Fator Regulador X , Regulação para CimaRESUMO
X-box binding protein 1 (XBP-1) is a key regulator of the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Cells contain two protein isoforms of XBP-1, the active isoform (XBP-1S) and the inactive isoform (XBP-1U). Induction of UPR leads to the generation of XBP-1S while XBP-1U is dominant in ER stress-free cells. XBP-1S is a transcriptional activator and regulates the expression of a subset of UPR genes. Importantly, recent studies have demonstrated the essential role of XBP-1S in various human diseases, such as viral infections. Many viruses have evolved to manipulate UPR/XBP-1 of the infected cells to promote viral survival and replication. In this review, we will summarize the current findings on the involvement of XBP-1 in viral infection/ replication and discuss the potential anti-viral strategies by targeting XBP-1.
Assuntos
Antivirais/farmacologia , Viroses/tratamento farmacológico , Proteína 1 de Ligação a X-Box/metabolismo , Antivirais/uso terapêutico , Estresse do Retículo Endoplasmático , Humanos , Dobramento de Proteína , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Viroses/metabolismo , Viroses/virologia , Replicação ViralRESUMO
DNA-conjugated gold nanoparticles (AuNPs) have been shown to enhance the translation of mRNA. However, the specific sequence on the DNA dictates the specific mRNA to be enhanced. This study describes poly(thymine)-functionalized AuNPs (AuNP-p(T)DNA) capable of enhancing the translation of any mRNA template that is incorporated into pcDNA6 vector with bovine growth hormone (BGH) polyadenylation signal (P(A)). We demonstrated this by incorporating four genes: green fluorescence protein (GFP), general control nonderepressible 5 (GCN5), cAMP-responsive element binding protein 1 (CREB1), and X-box-binding protein 1-spliced (XBP-1S) separately into pcDNA6 vector with BGH P(A) before their expression in HeLa lysate. The addition of AuNP-p(T)DNA to HeLa lysate containing GFP, GCN5, CREB1, and XBP-1S mRNA increased protein synthesis 1.80, 1.99, 1.95, and 2.20 times, respectively. Similar translation enhancement was also observed in a multiplex reaction containing the mRNA of three genes together in the lysate. Complementary p(T)DNA hybridization to the poly(A) tail of the mRNA was critical as the removal of either p(T)DNA or BGH P(A) in XBP-1S mRNA or the replacement of p(T)DNA with p(A)DNA reduced the translation back to baseline level. Finally, an optimum length of 25 nucleotides for the DNA oligomer and a AuNP-p(T)DNA:mRNA ratio of 0.658 achieved a 3.08-fold translation enhancement. The AuNP-p(T)DNA nanoconstruct could be incorporated into commercial cell-free protein synthesis kits as a universal translation enhancer.
Assuntos
Nanopartículas Metálicas , Animais , Bovinos , DNA , Ouro , Oligonucleotídeos , RNA MensageiroRESUMO
Cyclin-dependent kinase inhibitors (CDKIs) have been shown to block human immunodeficiency virus and herpes simplex virus. It is hypothesized that CDKIs block viral replication by inhibiting transcription of specific cellular genes. Here we find that three CDKIs, flavopiridol, purvalanol A, and methoxy-roscovitine, block Moloney murine leukemia virus (MLV) transcription events. Using gene expression microarray technology to examine the inhibitory effects of CDKIs, we observed a cellular gene, the pre-B-cell leukemia transcription factor 1 (Pbx1) gene, down-regulated by CDKI treatment. The PBX consensus element (PCE), TGATTGAC, is conserved in the long terminal repeats of several murine retroviruses, including Moloney MLV. Mutations in the PCE completely inhibited viral transcription whereas overexpression of PBX1 and a PBX1-associated protein, PREP1, enhanced viral transcription. The interaction between the PCE and PBX1-PREP1 proteins was confirmed by gel shift experiments. Blocking PBX1 protein synthesis resulted in a significant decrease in viral transcription. Collectively, our results represent the first work demonstrating that the homeodomain proteins PBX1 and PREP1 are cellular factors involved in Moloney MLV transcription regulation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Vírus da Leucemia Murina de Moloney/genética , Proteínas Proto-Oncogênicas/fisiologia , Células 3T3 , Animais , Sequência de Bases , Linhagem Celular , Sequência Consenso , Quinases Ciclina-Dependentes/antagonistas & inibidores , DNA Viral/genética , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Camundongos , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/fisiologia , Piperidinas/farmacologia , Fator de Transcrição 1 de Leucemia de Células Pré-B , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Role of epigenetic regulation in the control of gene expression is well established. The impact of several epigenetic mechanisms, such as DNA methylation and histone acetylation, on recombinant protein production in mammalian cells has been investigated recently. Here we investigate the correlation between the selected epigenetic markers and five trastuzumab biosimilar-producing Chinese hamster ovary (CHO) cell lines in which the expression of trastuzumab is driven by human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. We chose the producing clones in which transcription was the determinative step for the production of recombinant trastuzumab. We found that the abundance of trimethylation of histone 3 at lysine 4 (H3K4Me3) on the enhancer of HCMV MIE promoter correlated well with the relative titers of recombinant trastuzumab among the clones. Such close correlation was not observed between the recombinant protein and other epigenetic markers examined in our study. Our results demonstrate that the HCMV MIE enhancer-bound H3K4Me3 epigenetic marker may be used as the epigenetic indicator to predict the relative production of recombinant proteins between the producing CHO cell lines.
Assuntos
Citomegalovirus/genética , Elementos Facilitadores Genéticos , Genes Precoces , Histonas/metabolismo , Lisina/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Trastuzumab/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Metilação de DNA , Dosagem de Genes , Humanos , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trastuzumab/biossíntese , Trastuzumab/genéticaRESUMO
Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells.
Assuntos
Anticorpos Monoclonais/metabolismo , Furina/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Bevacizumab , Células CHO , Cricetinae , Cricetulus , Furina/química , Furina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transfecção , TrastuzumabRESUMO
This study describes the use of DNA functionalized gold nanoparticles (AuNPs) to enhance the synthesis of proteins in cell lysate and examines the mechanisms behind the enhanced mRNA translation. With an appropriate DNA oligomer sequence that hybridizes to the 3'-untranslated region of two mRNA of interest, insulin and green fluorescent protein (GFP), we found that these DNA conjugated AuNPs (AuNP-DNA) introduced into HeLa cell lysate enhanced the synthesis of insulin and GFP by up to 2.18 and 1.80-fold, respectively, over baseline production with just the mRNA present. The insulin synthesis was markedly reduced with non-DNA citrate-capped AuNP (1.25-fold) and AuNP-DNA with a nonspecific poly(T) sequence (1.25-fold). We showed that both nonspecific adsorption of ribosomes and translation factors to form a lysate protein corona on AuNP-DNA and weak hybridization between DNA oligomers and mRNA of interest were important factors that brought translation factors, ribosomes, and mRNA into close proximity of each other. This could reduce the recycling time of ribosomes during mRNA translation, thereby increasing the efficiency of protein synthesis. The outcome of this work shows that with rational DNA design, it could be possible to modulate intracellular biological processes with AuNP-DNA and increase their production of proteins for various biomedical applications.
Assuntos
Coroa de Proteína/química , DNA , Ouro , Células HeLa , Humanos , Nanopartículas Metálicas , Biossíntese de ProteínasRESUMO
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II transcription. Several pharmacological CDK inhibitors (PCIs) are currently in clinical trials as potential cancer therapeutics since CDK hyperactivation is detected in the majority of neoplasias. Within the last few years, the anti-viral effects of PCIs have also been observed against various viruses, including human immunodeficiency virus (HIV), herpes simplex virus, and murine leukemia virus. Through the inhibition of CDK2 and 9, the cellular co-factors for HIV-1 Tat transactivation, HIV-1 replication is blocked by two specific PCIs, CYC202 and flavopiridol, respectively. In this article, we will review the inhibitory mechanisms of flavopiridol and CYC202 and discuss their possible usage in AIDS treatment.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Infecções por HIV/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Flavonoides/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Roscovitina , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb) and is recently identified as a novel positive regulator of p53. We previously showed the basic region (BR) of HEXIM1 mediates the binding of HEXIM1 to a nucleolar protein, nucleophosmin (NPM), and can be ubiquitinated by human double minute 2 protein. Here we identify a cytotoxic peptide derived from the BR of HEXIM1. When fused with a cell-penetrating peptide, the HEXIM1 BR peptide triggers rapid cytotoxic effect independent of p53. Similarly, when the BR peptide is linked with a breast cancer cell targeting peptide, LTV, the LTV-BR fusion peptide exhibits specific killing of breast cancer cells, which is not observed with the commonly used cytotoxic peptide, KLA. Importantly, the BR peptide fails to enter cells by itself and does not induce any cytotoxic effects when it is not guided by any cell-penetrating or cancer targeting peptides. We showed that HEXIM1 BR peptide depolarizes mitochondrial membrane potential in a p53-dependent manner and its cell-killing activity is not suppressed by caspase inhibition. Furthermore, we observed an accumulation of the internalized BR peptide in the nucleoli of treated cells and an altered localization of NPM. These results illustrate a novel mechanism which the BR peptide induces cell death and can potentially be used as a novel therapeutic strategy against breast cancer.