RESUMO
Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
Assuntos
Antimitóticos/química , Morte Celular , Microtúbulos/efeitos dos fármacos , Mitose , Estilbenos/química , Animais , Antimitóticos/toxicidade , Linhagem Celular Tumoral , Citoesqueleto/química , Humanos , Luz , Camundongos , Polimerização , Estilbenos/toxicidadeRESUMO
Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5'UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.
Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Hepacivirus/genética , Hepatite C/genética , Sítios Internos de Entrada Ribossomal/genética , Animais , Linhagem Celular , Hepacivirus/patogenicidade , Hepatite C/patologia , Hepatite C/virologia , Humanos , Ligação Proteica/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Viral/química , RNA Viral/genética , Coelhos , Ribossomos/química , Ribossomos/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.
Assuntos
Proliferação de Células , Transformação Celular Viral , Modelos Animais de Doenças , Produtos do Gene tax/metabolismo , Infecções por HTLV-I/patologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Linfócitos T/patologia , Animais , Feminino , Perfilação da Expressão Gênica , Produtos do Gene tax/genética , Células HEK293 , Infecções por HTLV-I/metabolismo , Infecções por HTLV-I/virologia , Humanos , Ativação Linfocitária , Masculino , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Domínios PDZ , Ligação Proteica , Linfócitos T/metabolismoRESUMO
The INT6/EIF3E protein has been implicated in mouse and human breast carcinogenesis. This subunit of the eIF3 translation initiation factor that includes a PCI domain exhibits specific features such as presence in the nucleus and ability to interact with other important cellular protein complexes like the 26S proteasome and the COP9 signalosome. It has been previously shown that INT6 was not essential for bulk translation, and this protein is considered to regulate expression of specific mRNAs. Based on the results of a two-hybrid screen performed with INT6 as bait, we characterize in this article the MIF4GD/SLIP1 protein as an interactor of this eIF3 subunit. MIF4GD was previously shown to associate with SLBP, which binds the stem-loop located at the 3' end of the histone mRNAs, and to be necessary for efficient translation of these cell cycle-regulated mRNAs that lack a poly(A) tail. In line with the interaction of both proteins, we show using the RNA interference approach that INT6 is also essential to S-phase histone mRNA translation. This was observed by analyzing expression of endogenous histones and by testing heterologous constructs placing the luciferase reporter gene under the control of the stem-loop element of various histone genes. With such a reporter plasmid, silencing and overexpression of INT6 exerted opposite effects. In agreement with these results, INT6 and MIF4GD were observed to colocalize in cytoplasmic foci. We conclude from these data that INT6, by establishing interactions with MIF4GD and SLBP, plays an important role in translation of poly(A) minus histone mRNAs.
Assuntos
Proteínas de Transporte/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Histonas/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Transporte/química , Células Cultivadas , Fator de Iniciação 3 em Eucariotos/química , Humanos , Linfócitos , Ligação Proteica , Proteínas de Ligação a RNARESUMO
Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteína p300 Associada a E1A/metabolismo , Regulação Neoplásica da Expressão Gênica , Leucemia de Células T/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Telomerase/genética , Proteínas Virais/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Proteína p300 Associada a E1A/genética , Células HeLa , Humanos , Imunoprecipitação , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas dos Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genéticaRESUMO
In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células HEK293 , Humanos , Células Jurkat , RNA Helicases , Proteínas dos Retroviridae , Linfócitos T/metabolismo , Linfócitos T/virologia , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
In eukaryotic cells, the spatial distribution between cytoplasm and nucleus is essential for cell homeostasis. This dynamic distribution is selectively regulated by the nuclear pore complex (NPC), which allows the passive or energy-dependent transport of proteins between these two compartments. Viruses possess many strategies to hijack nucleocytoplasmic shuttling for the benefit of their viral replication. Here, we review how viruses interfere with the karyopherin CRM1 that controls the nuclear export of protein cargoes. We analyze the fact that the viral hijacking of CRM1 provokes are-localization of numerous cellular factors in a suitable place for specific steps of viral replication. While CRM1 emerges as a critical partner for viruses, it also takes part in antiviral and inflammatory response regulation. This review also addresses how CRM1 hijacking affects it and the benefits of CRM1 inhibitors as antiviral treatments.
Assuntos
Carioferinas , Vírus , Transporte Ativo do Núcleo Celular , Carioferinas/metabolismo , Vírus/genética , Citoplasma/metabolismo , Antivirais/metabolismo , Cromossomos/metabolismo , Núcleo Celular/metabolismoRESUMO
BACKGROUND & AIMS: We previously reported the frequent overexpression of the FZD7 membrane receptor in hepatocellular carcinoma (HCC) and its role for controlling cancer phenotype. Herein, this study aimed at assessing the anticancer properties of compounds inhibiting FZD7 activity by disrupting its binding with the cytosolic Dishevelled (DVL) adaptator. METHODS: We have designed small interfering peptides (RHPDs) that are able to enter within cells and to competitively antagonize the binding of FZD7 to the PDZ domain of DVL. Their anti-neoplastic properties were assessed in vitro on a panel of human HCC cell lines and in vivo on the SV40-TAg transgenic mouse model of HCC. RESULTS: We have shown that RHPDs decrease cell viability via apoptosis depending on their affinity for PDZ, with a therapeutic index between cancerous and non-cancerous cells. RHPD properties were linked to ß-catenin degradation and PKCδ activation. In transgenic mice, intra-tumor injection of RHPDs inhibited HCC progression. CONCLUSIONS: We have completed a proof-of-concept showing that in vitro and in vivo the pharmacological inhibition of FZD7 displays anti-cancerous properties against HCC. The mechanisms can involve ß-catenin and PKCδ modulations. Further studies are warranted to design protocols showing the compatibility with systemic in vivo applications.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Receptores Frizzled/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Antígenos Transformantes de Poliomavirus/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas Desgrenhadas , Humanos , Camundongos , Camundongos Transgênicos , Fosfoproteínas/química , Proteína Quinase C-delta/fisiologia , Proteína Supressora de Tumor p53/fisiologia , beta Catenina/metabolismoRESUMO
T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays important roles in differentiation of hematopoietic and endothelial cells and is deregulated in a high percentage of T-cell acute lymphoblastic leukemia (T-ALL). In this report we show that the intracellular concentration of TAL1 is regulated by transforming growth factor beta (TGF-beta), which triggers its polyubiquitylation and degradation by the proteasome. This effect is mediated by AKT1, which phosphorylates TAL1 at threonine 90. Immunoprecipitation experiments showed that this event increases association of TAL1 with the E3 ubiquitin ligase CHIP. The E47 heterodimerization partner of TAL1 hinders this association. Our observations indicate that activation of the TGF-beta and phosphatidylinositol 3-kinase/AKT pathways might reverse overexpression of TAL1 in leukemic cells by inducing proteolysis of this important oncogene.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Ubiquitina/metabolismo , Substituição de Aminoácidos , Androstadienos/farmacologia , Dimerização , Células HeLa/metabolismo , Humanos , Células Jurkat/metabolismo , Leupeptinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Fosfotreonina/metabolismo , Inibidores de Proteassoma , Mapeamento de Interação de Proteínas , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição TCF/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição , Ubiquitina-Proteína Ligases/metabolismo , WortmaninaRESUMO
The stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs.
Assuntos
Bioensaio , Estabilidade Proteica , Proteínas , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas/genéticaRESUMO
Altered expression of the translation factor eIF3e is associated with breast cancer occurrence. We have previously shown that eIF3e deficiency leads to an impaired DNA damage response with a marked decrease in DNA repair by homologous recombination. Here, we explored the possibility to exploit this DNA repair defect in targeted cancer therapy using PARP inhibitors. Surprisingly, eIF3e-deficient breast cancer cells are resistant to these drugs, in contrast to BRCA1-deficient cells. Studying this, we found that eIF3e-depleted cells synthesize lowered amounts of PARP1 protein, due to a weakened translation of the corresponding mRNA, associated with a strong decrease in cellular poly(ADP-ribosyl)ation. Additionally, we discovered that the mTORC1 signaling pathway is aberrantly activated in response to eIF3e suppression. Together, these PARP1 and mTORC1 dysfunctions upon eIF3e depletion are causally linked to induction of cellular senescence associated with a pro-inflammatory secretory phenotype. This study provides mechanistic insights into how eIF3e protects against breast cancer, with potential novel cancer therapeutic opportunities. While PARP inhibitors appear as inappropriate drugs for eIF3e-deficient breast tumors, our findings suggest that such cancers may benefit from senolytic drugs or mTORC1 inhibitors.
RESUMO
Telomeric repeat-binding factor 2 (TRF2) is a subunit of the shelterin protein complex, which binds to and protects telomeres from unwanted DNA damage response (DDR) activation. TRF2 expression plays a pivotal role in aging and cancer, being downregulated during cellular senescence and overexpressed during oncogenesis. Cancers overexpressing TRF2 often exhibit a poor prognosis. In cancer cells, TRF2 plays multiple functions, including telomere protection and non-cell autonomous roles, promoting neo-angiogenesis and immunosuppression. We present here an original screening strategy, which enables identification of small molecules that decrease or increase TRF2 expression. By screening a small library of Food and Drug Agency (FDA)-approved drugs, we identified two molecules (AR-A014418 and alexidine·2HCl) that impaired tumor growth, neo-angiogenesis and immunosuppression by downregulating TRF2 expression in a mouse xenograft model. These results support the chemotherapeutic strategy of downregulating TRF2 expression to treat aggressive human tumors and validate this cell-based assay capable of screening for potential anti-cancer and anti-aging molecules by modulating TRF2 expression levels.
RESUMO
Before the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated.
RESUMO
The molecular identification of proviruses from ancient tissues (and particularly from bones) remains a contentious issue. It can be expected that the copy number of proviruses will be low, which magnifies the risk of contamination with retroviruses from exogenous sources. To assess the feasibility of paleoretrovirological studies, we attempted to identify proviruses from early 20th century bones of museum specimens while following a strict ancient DNA methodology. Simian T-cell leukemia virus type 1 sequences were successfully obtained and authenticated from a Chlorocebus pygerythrus specimen. This represents the first clear evidence that it will be possible to use museum specimens to better characterize simian and human T-tropic retrovirus genetic diversity and analyze their origin and evolution, in greater detail.
Assuntos
Cercopithecinae/virologia , DNA Viral/isolamento & purificação , Evolução Molecular , Variação Genética , Provírus/isolamento & purificação , Vírus Linfotrópico T Tipo 1 de Símios/isolamento & purificação , Animais , DNA Viral/genética , DNA Viral/história , História do Século XX , Filogenia , Provírus/classificação , Provírus/genética , Vírus Linfotrópico T Tipo 1 de Símios/classificação , Vírus Linfotrópico T Tipo 1 de Símios/genética , Sequências Repetidas TerminaisRESUMO
The human T-cell leukemia virus type 1 (HTLV-1) Tax transactivator is known to induce or repress various cellular genes, several of them encoding transcription factors. As Tax is known to deregulate various basic bHLH factors, we looked more specifically at its effect on TAL1 (T-cell acute lymphoblastic leukemia 1), also known as SCL (stem cell leukemia). Indeed, TAL1 is deregulated in a high percentage of T-cell acute lymphoblastic leukemia cells, and its oncogenic properties are well-established. Here we show that Tax induces transcription of this proto-oncogene by stimulating the activity of the TAL1 gene promoter 1b, through both the CREB and NF-kappaB pathways. It was also observed that TAL1 upregulates HTLV-1 promoter activity, in either the presence or the absence of Tax. The viral promoter is inhibited in trans by expression of the E2A protein E47, and TAL1 is able to abrogate this inhibition. These data show the existence of a positive feedback loop between Tax and TAL1 expression and support the notion that this proto-oncogene participates in generation of adult T-cell leukemia/lymphoma by increasing the amount of the Tax oncoprotein but also possibly by its own transforming activities.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T do Adulto/virologia , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Linhagem Celular , Retroalimentação Fisiológica , Células HeLa , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Timo/citologiaRESUMO
BACKGROUND: We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form. RESULTS: Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway. CONCLUSION: Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.
Assuntos
Produtos do Gene tat/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/metabolismo , Transdução Genética/métodos , Ubiquitina/metabolismo , Produtos do Gene tat/genética , Humanos , Células Jurkat , Peptídeos/genética , Estrutura Terciária de Proteína , Ubiquitina/genéticaRESUMO
A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.
Assuntos
Metabolismo Energético/fisiologia , Biossíntese de Proteínas , Reticulócitos/metabolismo , Animais , Sistema Livre de Células , Cicloeximida/farmacologia , Glucose/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Fosfocreatina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Coelhos , Reticulócitos/citologia , Ribossomos/metabolismoRESUMO
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.
Assuntos
Produtos do Gene tax/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Transativadores/metabolismo , Trifosfato de Adenosina/metabolismo , Produtos do Gene tax/genética , Células HeLa/virologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Mutação , Domínios Proteicos , Transporte Proteico , RNA Helicases/genética , Transativadores/genéticaRESUMO
The Int-6 protein has been originally identified as the product of a mouse gene being a frequent integration site of the mouse mammary tumour virus. Here, we show that reducing Int-6 expression by RNA interference in HeLa cells markedly alters mitosis progression. Defects in spindle formation, chromosome segregation and cytokinesis were observed. These abnormalities of mitosis completion are correlated with an inhibition of cyclin B-Cdk1 kinase activity, due to a prolonged inhibitory phosphorylated state of Cdk1. In line with this observation, the Wee1 tyrosine kinase that negatively controls Cdk1 was less efficiently inactivated during G2 in Int-6-depleted cells. These findings support the notion that the oncogenic properties associated with alteration of Int-6 originate from chromosomal instability.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Ciclina B/antagonistas & inibidores , Fator de Iniciação 3 em Eucariotos/genética , Mitose/genética , Interferência de RNA , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/genética , Citocinese/genética , Fator de Iniciação 3 em Eucariotos/análise , Células HeLa , Humanos , Neoplasias/etiologia , Neoplasias/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/genética , Fuso Acromático/imunologia , Fuso Acromático/metabolismoRESUMO
The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.