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1.
EMBO J ; 28(6): 652-62, 2009 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-19197236

RESUMO

The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.


Assuntos
Infecções por Adenoviridae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Hidrolases Anidrido Ácido , Adenoviridae/fisiologia , Proteínas E4 de Adenovirus/química , Proteínas E4 de Adenovirus/metabolismo , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Humanos , Proteína Homóloga a MRE11 , Dados de Sequência Molecular , Fosforilação , Transporte Proteico , Proteínas Supressoras de Tumor/metabolismo , Replicação Viral
2.
Curr Biol ; 16(5): 480-5, 2006 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-16527742

RESUMO

APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.


Assuntos
Citidina Desaminase/fisiologia , Dependovirus/fisiologia , Proteínas Nucleares/fisiologia , Proteínas/fisiologia , Retroelementos/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Dependovirus/patogenicidade , Humanos , Macrófagos/enzimologia , Monócitos/enzimologia , RNA Mensageiro/metabolismo , Replicação Viral/fisiologia
3.
Sci Rep ; 8(1): 4241, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523796

RESUMO

Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid profile improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using a combination of N-glycan engineering for enhanced protease resistance and improved solubility, Fc fusion for further half-life extension, and a single point mutation for improving manufacturability in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability profile that is compatible with subcutaneous (SC) administration. In particular, it showed a low systemic clearance (0.243 mL/hr/kg) and long terminal half-life (~200 hours for intact protein) in cynomolgus monkeys that approaches those of monoclonal antibodies. Furthermore, the superior PK properties translated into robust improvement in glucose tolerance and the effects lasted 14 days post single SC dose in ob/ob mice. PF-06645849 also caused greater body weight loss in DIO mice at lower and less frequent SC doses, compared to previous FGF21 analogue PF-05231023. In summary, the overall PK/PD and pharmaceutical profile of PF-06645849 offers great potential for development as weekly to twice-monthly SC administered therapeutic for chronic treatment of metabolic diseases.


Assuntos
Fatores de Crescimento de Fibroblastos/farmacocinética , Animais , Células CHO , Cricetinae , Cricetulus , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/química , Glicosilação , Células HEK293 , Humanos , Injeções Subcutâneas , Macaca fascicularis , Taxa de Depuração Metabólica , Camundongos , Estabilidade Proteica , Proteólise , Distribuição Tecidual
4.
PLoS One ; 9(11): e111767, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25365322

RESUMO

Fibroblast growth factor 21 (FGF21) has evolved as a major metabolic regulator, the pharmacological administration of which causes weight loss, insulin sensitivity and glucose control in rodents and humans. To understand the molecular mechanisms by which FGF21 exerts its metabolic effects, we developed a human in vitro model of adipocytes to examine crosstalk between FGF21 and insulin signaling. Human adipose stem cell-derived (hASC) adipocytes were acutely treated with FGF21 alone, insulin alone, or in combination. Insulin signaling under these conditions was assessed by measuring tyrosine phosphorylation of insulin receptor (InsR), insulin receptor substrate-1 (IRS-1), and serine 473 phosphorylation of Akt, followed by a functional assay using 14C-2-deoxyglucose [14C]-2DG to measure glucose uptake in these cells. FGF21 alone caused a modest increase of glucose uptake, but treatment with FGF21 in combination with insulin had a synergistic effect on glucose uptake in these cells. The presence of FGF21 also effectively lowered the insulin concentration required to achieve the same level of glucose uptake compared to the absence of FGF21 by 10-fold. This acute effect of FGF21 on insulin signaling was not due to IR, IGF-1R, or IRS-1 activation. Moreover, we observed a substantial increase in basal S473-Akt phosphorylation by FGF21 alone, in contrast to the minimal shift in basal glucose uptake. Taken together, our data demonstrate that acute co-treatment of hASC-adipocytes with FGF21 and insulin can result in a synergistic improvement in glucose uptake. These effects were shown to occur at or downstream of Akt, or separate from the canonical insulin signaling pathway.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Resistência à Insulina , Insulina/farmacologia , Células-Tronco/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Fatores de Crescimento de Fibroblastos/agonistas , Glucose/metabolismo , Humanos , Insulina/agonistas , Proteínas Substratos do Receptor de Insulina/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
J Virol ; 79(17): 11382-91, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16103189

RESUMO

Virus infections have dramatic effects on structural and morphological characteristics of the host cell. The gene product of open reading frame 3 in the early region 4 (E4orf3) of adenovirus serotype 5 (Ad5) is involved in efficient replication and late protein synthesis. During infection with adenovirus mutants lacking the E4 region, the viral genomic DNA is joined into concatemers by cellular DNA repair factors, and this requires the Mre11/Rad50/Nbs1 complex. Concatemer formation can be prevented by the E4orf3 protein, which causes the cellular redistribution of the Mre11 complex. Here we show that E4orf3 colocalizes with components of the Mre11 complex in nuclear tracks and also in large cytoplasmic accumulations. Rearrangement of Mre11 and Rad50 by Ad5 E4orf3 is not dependent on interactions with Nbs1 or promyelocytic leukemia protein nuclear bodies. Late in infection the cytoplasmic inclusions appear as a distinct juxtanuclear accumulation at the centrosome and this requires an intact microtubule cytoskeleton. The large cytoplasmic accumulations meet the criteria defined for aggresomes, including gamma-tubulin colocalization and formation of a surrounding vimentin cage. E4orf3 also appears to alter the solubility of the cellular Mre11 complex. These data suggest that E4orf3 can target the Mre11 complex to an aggresome and may explain how the cellular repair complex is inactivated during adenovirus infection.


Assuntos
Proteínas E4 de Adenovirus/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hidrolases Anidrido Ácido , Adenovírus Humanos/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Humanos , Proteína Homóloga a MRE11 , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Solubilidade , Tubulina (Proteína)/metabolismo
6.
J Virol ; 79(11): 6664-73, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15890904

RESUMO

The early transcriptional region 4 (E4) of adenovirus type 5 (Ad5) encodes gene products that modulate splicing, apoptosis, transcription, DNA replication, and repair pathways. Viruses lacking both E4orf3 and E4orf6 have a severe replication defect, partially characterized by the formation of genome concatemers. Concatemer formation is dependent upon the cellular Mre11 complex and is prevented by both the E4orf3 and E4orf6 proteins. The Mre11/Rad50/Nbs1 proteins are targeted for proteasome-mediated degradation by the Ad5 viral E1b55K/E4orf6 complex. The expression of Ad5 E4orf3 causes a redistribution of Mre11 complex members and results in their exclusion from viral replication centers. For this study, we further analyzed the interactions of E4 proteins from different adenovirus serotypes with the Mre11 complex. Analyses of infections with serotypes Ad4 and Ad12 demonstrated that the degradation of Mre11/Rad50/Nbs1 proteins is a conserved feature of the E1b55K/E4orf6 complex. Surprisingly, Nbs1 and Rad50 were localized to the replication centers of both Ad4 and Ad12 viruses prior to Mre11 complex degradation. The transfection of expression vectors for the E4orf3 proteins of Ad4 and Ad12 did not alter the localization of Mre11 complex members. The E4orf3 proteins of Ad4 and Ad12 also failed to complement defects in both concatemer formation and late protein production of a virus with a deletion of E4. These results reveal surprising differences among the highly conserved E4orf3 proteins from different serotypes in the ability to disrupt the Mre11 complex.


Assuntos
Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/classificação , Adenovírus Humanos/patogenicidade , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas E4 de Adenovirus/genética , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , DNA Viral/genética , Teste de Complementação Genética , Células HeLa , Humanos , Proteína Homóloga a MRE11 , Complexos Multiproteicos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Filogenia , Proteína da Leucemia Promielocítica , Sorotipagem , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor , Replicação Viral
7.
EMBO J ; 22(24): 6610-20, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-14657032

RESUMO

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.


Assuntos
Adenovírus Humanos/fisiologia , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Vírus Defeituosos/genética , Proteínas Serina-Treonina Quinases/genética , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Fase G2 , Células HeLa , Humanos , Proteína Homóloga a MRE11 , Mitose , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor , Replicação Viral
8.
Mol Ther ; 10(3): 604-8, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15336660

RESUMO

Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.


Assuntos
Dependovirus/genética , Miocárdio/metabolismo , Transdução Genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Dependovirus/classificação , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/metabolismo , Especificidade da Espécie
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