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1.
PLoS Pathog ; 13(7): e1006518, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28742148

RESUMO

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.


Assuntos
Produtos do Gene tax/metabolismo , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Linfócitos T/virologia , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tax/genética , Infecções por HTLV-I/enzimologia , Infecções por HTLV-I/genética , Infecções por HTLV-I/metabolismo , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Transcrição Gênica , beta-N-Acetil-Hexosaminidases/genética
2.
FASEB J ; 28(2): 1010-21, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24174424

RESUMO

O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic ß-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 ß cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Glucose/farmacologia , Imunoprecipitação , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Ratos
3.
Structure ; 17(4): 547-58, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19368888

RESUMO

UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms. These structures, coupled with mutational analyses and comparison with the crystal structure of class I UvrA from Bacillus stearothermophilus, suggest a previously unsuspected role for the identified insertion domains of UvrAs in both DNA binding and damage recognition. Taken together, the available information suggests a model for how UvrA interacts with DNA and thus sheds new light on the molecular mechanisms underlying the role of UvrA in the early steps of NER.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Dano ao DNA , DNA Bacteriano/metabolismo , Deinococcus/enzimologia , Adenosina Trifosfatases/classificação , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalização , Análise Mutacional de DNA , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Geobacillus stearothermophilus/enzimologia , Hidrólise , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Tempo
4.
DNA Repair (Amst) ; 64: 34-44, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29482102

RESUMO

ERCC1/XPF endonuclease plays an important role in multiple DNA repair pathways and stands as a potential prognostic and predictive biomarker for cisplatin-based chemotherapy. Four distinct ERCC1 isoforms arising from alternative splicing have been described (201, 202, 203 and 204) but only the 202 isoform is functional in DNA excision repair, when interacting with its obligate partner XPF. Currently, there is no tool to assess specifically the expression of ERCC1-202 due to high sequence homology between the four isoforms. Here, we generated monoclonal antibodies directed against the heterodimer of ERCC1 and its obligate interacting partner XPF by genetic immunization. We obtained three monoclonal antibodies (2C11, 7C3 and 10D10) recognizing specifically the heterodimer ERCC1-202/XPF as well as the ERCC1-204/XPF with no affinity to ERCC1 or XPF monomers. By combining one of these three heterodimer-specific antibodies with a commercial anti-ERCC1 antibody (clone 4F9) unable to recognize the 204 isoform in a proximity ligation assay (PLA), we managed to specifically detect the functional ERCC1-202 isoform. This methodological breakthrough can constitute a basis for the development of clinical tests to evaluate ERCC1 functional proficiency.


Assuntos
Anticorpos Monoclonais , Especificidade de Anticorpos , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Endonucleases/análise , Endonucleases/metabolismo , Imunoensaio/métodos , Processamento Alternativo , Sequência de Aminoácidos , Linhagem Celular , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Endonucleases/genética , Humanos , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Alinhamento de Sequência
5.
J Clin Invest ; 128(4): 1671-1687, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29447131

RESUMO

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non-small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house-generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro - ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells - and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Reparo do DNA , Neoplasias Pulmonares/metabolismo , NAD/biossíntese , Neoplasias Experimentais/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Nus , NAD/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo
6.
ESMO Open ; 3(1): e000257, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29344407

RESUMO

BACKGROUND: Antiprogrammed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) therapies have demonstrated promising activity in advanced head and neck squamous cell carcinoma (HNSCC), with overall response rates of approximately 20% in unselected populations and survival benefit. Whether induction docetaxel, platinum and fluorouracil (TPF) modifies PD-L1 expression or tumour immune infiltrates is unknown. PATIENTS AND METHODS: Patients with locally advanced HNSCC treated at Gustave Roussy (Villejuif, France) between 2006 and 2013 by induction TPF followed by surgery were retrospectively considered. Patients with paired samples (pre-TPF and post-TPF) were kept for further analysis. PD-L1 expression was quantified by immunohistochemistry according to a validated protocol. The objective of the study was to compare PD-L1 expression on tumour cells (TC) and immune cells (IC) (positivity threshold of ≥5%) before and after TPF. CD8+ and Foxp3+ lymphocytes densities before and after TPF were also quantified. RESULTS: Out of 313 patients receiving induction TPF, 86 underwent surgery; paired samples were available for 21 of them. Baseline PD-L1 expression was ≥5% in two and five samples for TC and IC, respectively. A significant increase of PD-L1 expression was observed after TPF, with 15 samples (71%) presenting a positive staining in IC after induction chemotherapy (P=0.003; Wilcoxon rank-sum test) and eight samples (38%) in TC (P=0.005; Wilcoxon rank-sum test). Tumour-infiltrating CD8+ mean densities also significantly increased post-TPF (P=0.01). There was no significant difference in Foxp3+ expression, CD8/Foxp3 ratio or correlation with outcome. CONCLUSION: TPF induction chemotherapy in advanced HNSCC increases PD-L1 positivity on tumour-infiltrating ICs, as well as CD8+ lymphocytes density. These results warrant independent validation on larger datasets and might help therapeutic strategy in advanced HNSCC.

7.
Clin Cancer Res ; 22(24): 5983-5991, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27401242

RESUMO

BACKGROUND: The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far. METHODS: A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations. RESULTS: Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations. CONCLUSIONS: Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Aminopiridinas , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Crizotinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Lactamas , Lactamas Macrocíclicas/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Mutação/genética , Proteínas de Fusão Oncogênica/genética , Pirimidinas/uso terapêutico , Sulfonas/uso terapêutico
8.
PLoS One ; 9(3): e92737, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24647478

RESUMO

Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Fosfatos de Fosfatidilinositol/análise , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Humanos
9.
Microbiology (Reading) ; 150(Pt 2): 437-446, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14766922

RESUMO

Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB-lacZ was used to assay for beta-galactosidase activity and thus follow RcsA stability. The expression of cpsB-lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB-lacZ expression. Additionally, a P(BAD)-rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB-lacZ expression. The effect of RcsA on cpsB-lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA(Red)-lacZ translational fusion construct showed higher activity of RcsA(Red)-LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased beta-galactosidase levels of RcsA(Red)-LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.


Assuntos
Endopeptidase Clp , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Protease La , Serina Endopeptidases/genética , Proteases Dependentes de ATP , Sequência de Bases , Primers do DNA , Regulação Enzimológica da Expressão Gênica/genética , Genótipo , Proteínas de Choque Térmico/metabolismo , Cinética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismo
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