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1.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802742

RESUMO

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.


Assuntos
Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Endorribonucleases/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Pulmão/patologia , Terapia de Alvo Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Células Cultivadas , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citocinas/biossíntese , Endorribonucleases/genética , Epitélio/efeitos dos fármacos , Epitélio/patologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Inflamação/genética , Modelos Biológicos , Naftalenos/química , Naftalenos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Pirazinas/química , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína 1 de Ligação a X-Box/metabolismo
2.
J Biol Chem ; 290(41): 25174-87, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26330556

RESUMO

X-linked primate-specific melanoma antigen-A11 (MAGE-A11) is a human androgen receptor (AR) coactivator and proto-oncogene expressed at low levels in normal human reproductive tract tissues and at higher levels in castration-resistant prostate cancer where it is required for androgen-dependent cell growth. In this report, we show that MAGE-A11 is targeted for degradation by human p14-ARF, a tumor suppressor expressed from an alternative reading frame of the p16 cyclin-dependent kinase inhibitor INK4a/ARF gene. MAGE-A11 degradation by the proteasome was mediated by an interaction with p14-ARF and was independent of lysine ubiquitination. A dose-dependent inverse relationship between MAGE-A11 and p14-ARF correlated with p14-ARF inhibition of the MAGE-A11-induced increase in androgen-dependent AR transcriptional activity and constitutive activity of a splice variant-like AR. Reciprocal stabilization between MAGE-A11 and AR did not protect against degradation promoted by p14-ARF. p14-ARF prevented MAGE-A11 interaction with the E2F1 oncoprotein and inhibited the MAGE-A11-induced increase in E2F1 transcriptional activity. Post-translational down-regulation of MAGE-A11 promoted by p14-ARF was independent of HDM2, the human homologue of mouse double minute 2, an E3 ubiquitin ligase inhibited by p14-ARF. However, MAGE-A11 had a stabilizing effect on HDM2 in the absence or presence of p14-ARF and cooperated with HDM2 to increase E2F1 transcriptional activity in the absence of p14-ARF. We conclude that degradation of MAGE-A11 promoted by the human p14-ARF tumor suppressor contributes to low levels of MAGE-A11 in nontransformed cells and that higher levels of MAGE-A11 associated with low p14-ARF increase AR and E2F1 transcriptional activity and promote the development of castration-resistant prostate cancer.


Assuntos
Antígenos de Neoplasias/metabolismo , Regulação para Baixo , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Fator de Transcrição E2F1/metabolismo , Humanos , Masculino , Fenótipo , Neoplasias de Próstata Resistentes à Castração/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proto-Oncogene Mas , Transcrição Gênica
3.
J Biol Chem ; 288(34): 24809-24, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23853093

RESUMO

Melanoma antigen-A11 (MAGE-A11) is a low-abundance, primate-specific steroid receptor coregulator in normal tissues of the human reproductive tract that is expressed at higher levels in prostate cancer. Increased expression of MAGE-A11 enhances androgen receptor transcriptional activity and promotes prostate cancer cell growth. Further investigation into the mechanisms of MAGE-A11 function in prostate cancer demonstrated interactions with the retinoblastoma-related protein p107 and Rb tumor suppressor but no interaction with p130 of the Rb family. MAGE-A11 interaction with p107 was associated with transcriptional repression in cells with low MAGE-A11 and transcriptional activation in cells with higher MAGE-A11. Selective interaction of MAGE-A11 with retinoblastoma family members suggested the regulation of E2F transcription factors. MAGE-A11 stabilized p107 by inhibition of ubiquitination and linked p107 to hypophosphorylated E2F1 in association with the stabilization and activation of E2F1. The androgen receptor and MAGE-A11 modulated endogenous expression of the E2F1-regulated cyclin-dependent kinase inhibitor p27(Kip1). The ability of MAGE-A11 to increase E2F1 transcriptional activity was similar to the activity of adenovirus early oncoprotein E1A and depended on MAGE-A11 interactions with p107 and p300. The immunoreactivity of p107 and MAGE-A11 was greater in advanced prostate cancer than in benign prostate, and knockdown with small inhibitory RNA showed that p107 is a transcriptional activator in prostate cancer cells. These results suggest that MAGE-A11 is a proto-oncogene whose increased expression in prostate cancer reverses retinoblastoma-related protein p107 from a transcriptional repressor to a transcriptional activator of the androgen receptor and E2F1.


Assuntos
Antígenos de Neoplasias/metabolismo , Fator de Transcrição E2F1/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Transcrição Gênica , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animais , Antígenos de Neoplasias/genética , Células COS , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Fator de Transcrição E2F1/genética , Células HeLa , Humanos , Masculino , Proteínas de Neoplasias/genética , Fosforilação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Estabilidade Proteica , Proto-Oncogene Mas , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Proteína p107 Retinoblastoma-Like/genética
4.
J Biol Chem ; 288(3): 1939-52, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23172223

RESUMO

Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.


Assuntos
Antígenos de Neoplasias/genética , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Ativação Transcricional , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Neoplasias/metabolismo , Plasmídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Multimerização Proteica , Receptores Androgênicos/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas
5.
J Biol Chem ; 287(14): 10905-15, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22334658

RESUMO

Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.


Assuntos
Síndrome de Resistência a Andrógenos/genética , Antígenos de Neoplasias/metabolismo , Éxons/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Ativação Transcricional/genética , Motivos de Aminoácidos , Síndrome de Resistência a Andrógenos/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína p300 Associada a E1A/metabolismo , Feminino , Humanos , Masculino , Fenótipo , Fosforilação/genética , Receptores Androgênicos/genética
6.
J Biol Chem ; 287(41): 34809-24, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22891251

RESUMO

Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH(2)-terminal (110)LLXXVLXXLL(119) motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.


Assuntos
Antígenos de Neoplasias/metabolismo , Endométrio/metabolismo , Proteínas de Neoplasias/metabolismo , Gravidez/fisiologia , Receptores de Progesterona/metabolismo , Proteínas de Ligação a Tacrolimo/biossíntese , Ativação Transcricional/fisiologia , Adolescente , Adulto , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação para Baixo/fisiologia , Implantação do Embrião/fisiologia , Endométrio/citologia , Feminino , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína , Receptores de Progesterona/genética , Elementos de Resposta/fisiologia , Proteínas de Ligação a Tacrolimo/genética , Regulação para Cima/fisiologia , Proteínas ras/biossíntese , Proteínas ras/genética
7.
Cells ; 12(22)2023 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-37998353

RESUMO

People with cystic fibrosis (pwCF) suffer from chronic and recurring bacterial lung infections that begin very early in life and contribute to progressive lung failure. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ion channel important for maintaining the proper hydration of pulmonary surfaces. When CFTR function is ablated or impaired, airways develop thickened, adherent mucus that contributes to a vicious cycle of infection and inflammation. Therapeutics for pwCF, called CFTR modulators, target the CFTR defect directly, restoring airway surface hydration and mucociliary clearance. However, even with CFTR modulator therapy, bacterial infections persist. To develop a relevant model of diseased airway epithelium, we established a primary human airway epithelium culture system with persistent Pseudomonas aeruginosa infection. We used this model to examine the effects of CFTR modulators on CFTR maturation, CFTR function, and bacterial persistence. We found that the presence of P. aeruginosa increased CFTR mRNA, protein, and function. We also found that CFTR modulators caused a decrease in P. aeruginosa burden. These results demonstrate the importance of including live bacteria to accurately model the CF lung, and that understanding the effects of infection on CFTR rescue by CFTR modulators is critical to evaluating and optimizing drug therapies for all pwCF.


Assuntos
Fibrose Cística , Infecções por Pseudomonas , Humanos , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Técnicas de Cocultura , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pulmão/metabolismo
8.
J Biol Chem ; 286(34): 29951-63, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21730049

RESUMO

Male sex development and growth occur in response to high affinity androgen binding to the androgen receptor (AR). In contrast to complete amino acid sequence conservation in the AR DNA and ligand binding domains among mammals, a primate-specific difference in the AR NH(2)-terminal region that regulates the NH(2)- and carboxyl-terminal (N/C) interaction enables direct binding to melanoma antigen-A11 (MAGE-11), an AR coregulator that is also primate-specific. Human, mouse, and rat AR share the same NH(2)-terminal (23)FQNLF(27) sequence that mediates the androgen-dependent N/C interaction. However, the mouse and rat AR FXXLF motif is flanked by Ala(33) that evolved to Val(33) in primates. Human AR Val(33) was required to interact directly with MAGE-11 and for the inhibitory effect of the AR N/C interaction on activation function 2 that was relieved by MAGE-11. The functional importance of MAGE-11 was indicated by decreased human AR regulation of an androgen-dependent endogenous gene using lentivirus short hairpin RNAs and by the greater transcriptional strength of human compared with mouse AR. MAGE-11 increased progesterone and glucocorticoid receptor activity independently of binding an FXXLF motif by interacting with p300 and p160 coactivators. We conclude that the coevolution of the AR NH(2)-terminal sequence and MAGE-11 expression among primates provides increased regulatory control over activation domain dominance. Primate-specific expression of MAGE-11 results in greater steroid receptor transcriptional activity through direct interactions with the human AR FXXLF motif region and indirectly through steroid receptor-associated p300 and p160 coactivators.


Assuntos
Antígenos de Neoplasias/metabolismo , Evolução Molecular , Regulação da Expressão Gênica/fisiologia , Proteínas de Neoplasias/metabolismo , Receptores Androgênicos/metabolismo , Transcrição Gênica/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/genética , Estrutura Terciária de Proteína , Ratos , Receptores Androgênicos/genética , Especificidade da Espécie , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
9.
J Clin Invest ; 132(18)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35900863

RESUMO

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors' clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Fatores de Terminação de Peptídeos/metabolismo , Animais , Linhagem Celular , Códon sem Sentido , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Transporte de Íons/genética , Camundongos , Mutação
10.
Front Pharmacol ; 12: 628722, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859562

RESUMO

In cystic fibrosis (CF), defective biogenesis and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to airway dehydration and impaired mucociliary clearance, resulting in chronic airway infection and inflammation. The most common CFTR mutation, F508del, results in a processing defect in which the protein is retained in the endoplasmic reticulum and does not reach the apical surface. CFTR corrector compounds address this processing defect to promote mutant CFTR transfer to the apical membrane. When coupled with potentiators to increase CFTR channel activity, these drugs yield significant clinical benefits in CF patients carrying the F508del mutation. However, processing of CFTR and other proteins can be influenced by environmental factors such as inflammation, and the impact of airway inflammation on pharmacological activity of CFTR correctors is not established. The present study evaluated CFTR-rescuing therapies in inflamed CF airway epithelial cultures, utilizing models that mimic the inflammatory environment of CF airways. Primary bronchial epithelial cultures from F508del/F508del CF patients were inflamed by mucosal exposure to one of two inflammatory stimuli: 1) supernatant from mucopurulent material from CF airways with advanced lung disease, or 2) bronchoalveolar lavage fluid from pediatric CF patients. Cultures inflamed with either stimulus exhibited augmented F508del responses following therapy with correctors VX-809 or VX-661, and overcame the detrimental effects of chronic exposure to the CFTR potentiator VX-770. Remarkably, even the improved CFTR rescue responses resulting from a clinically effective triple therapy (VX-659/VX-661/VX-770) were enhanced by epithelial inflammation. Thus, the airway inflammatory milieu from late- and early-stage CF lung disease improves the efficacy of CFTR modulators, regardless of the combination therapy used. Our findings suggest that pre-clinical evaluation of CFTR corrector therapies should be performed under conditions mimicking the native inflammatory status of CF airways, and altering the inflammatory status of CF airways may change the efficacy of CFTR modulator therapies.

11.
J Biol Chem ; 284(50): 34793-808, 2009 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19828458

RESUMO

Androgen-dependent transcriptional activity by the androgen receptor (AR) and its coregulators is required for male reproductive development and function. In humans and other primates, melanoma antigen gene protein-A11 (MAGE-11) is an AR selective coregulator that increases AR transcriptional activity. Here we show that the interaction between AR and MAGE-11 is mediated by AR NH(2)-terminal FXXLF motif binding to a highly conserved MAGE-11 F-box in the MAGE homology domain, and is modulated by serum stimulation of mitogen-activated protein kinase phosphorylation of MAGE-11 Ser-174. The MAGE-11-dependent increase in AR transcriptional activity is mediated by a direct interaction between MAGE-11 and transcriptional intermediary factor 2 (TIF2) through the NH(2)-terminal region of TIF2, and by a MAGE-11 FXXIF motif interaction with an F-box-like region in activation domain 1 of TIF2. The results suggest that MAGE-11 functions as a bridging factor to recruit AR coactivators through a novel FXX(L/I)F motif-F-box interaction paradigm.


Assuntos
Antígenos de Neoplasias/metabolismo , Motivos F-Box , Proteínas de Neoplasias/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Receptores Androgênicos/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Células COS , Chlorocebus aethiops , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Coativador 2 de Receptor Nuclear/genética , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Androgênicos/genética , Alinhamento de Sequência
12.
Anal Methods ; 10(1): 46-50, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29568335

RESUMO

The rapid release of new tobacco products requires high-throughput quantitative methods to support tobacco research. Sample preparation for LC-MS and GC-MS is time consuming and limits throughput. Paper spray tandem mass spectrometry (PS-MS/MS) is proposed and validated as a simple and rapid method for quantification of nicotine and cotinine in complex matrices to support tobacco-related research. Air liquid interface (ALI) human tracheobronchial epithelial cell (HTBEC) cultures were exposed to tobacco smoke using a Vitrocell VC-10 smoking machine. Apical culture washes (phosphate buffered saline, PBS) and basolateral media were analyzed with the PS-MS/MS method. GC-MS/MS was used as a comparative quantitative technique. The PS-MS/MS approach allowed for direct spotting of samples on the paper substrate, whereas the GC-MS/MS method required additional sample preparation in the form of solvent-solvent extraction. Limits of quantitation (LOQs) were higher with the PS-MS/MS approach than GC-MS/MS, but still below the relevant concentrations found in HTBEC smoke exposure experiments as well as most clinical applications. PS-MS/MS is readily achieved on mass spectrometers that include atmospheric pressure inlets, and allows for convenient quantification from complex matrices that would otherwise require additional sample preparation and chromatographic separation.

13.
Mol Cell Endocrinol ; 439: 1-9, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27720894

RESUMO

Melanoma antigen-A11 (MAGE-A11) is a proto-oncogene involved in androgen receptor signaling and androgen-dependent cell growth. In this report we provide evidence that MAGE-A11 interacts with Skp2 (S phase kinase-associated protein), the substrate recognition protein of the Skp1-Cullin1-F-box E3 ubiquitin ligase, and with Skp2 binding protein, cyclin A. A similar cyclin A binding motif in MAGE-A11 and Skp2 was consistent with a competitive relationship between MAGE-A11 and Skp2 in binding cyclin A. Skp2 inhibited MAGE-A11 interaction with cyclin A. Differential effects of MAGE-A11 on Skp2-mediated protein degradation were also revealed. MAGE-A11 increased Skp2-mediated degradation of cyclin A and retinoblastoma-related protein p130. In contrast, MAGE-A11 decreased Skp2-mediated degradation of E2F1 and Skp2 self-ubiquitination. Stabilization of E2F1 by MAGE-A11 was associated with sequestration and inactivation of Skp2 through the formation of an E2F1-MAGE-A11-Skp2 complex. We conclude that direct interactions of MAGE-A11 with Skp2 and cyclin A regulate the substrate-specificity of Skp2-mediated protein degradation.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/metabolismo , Proteólise , Proteínas Quinases Associadas a Fase S/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Sítios de Ligação , Células COS , Chlorocebus aethiops , Ciclina A/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator de Transcrição E2F1/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lentivirus/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/química , Ligação Proteica , Proto-Oncogene Mas , RNA Interferente Pequeno/metabolismo , Receptores Androgênicos/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Proteínas Quinases Associadas a Fase S/química , Especificidade por Substrato , Ubiquitinação
14.
Mol Cell Endocrinol ; 443: 42-51, 2017 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-28042025

RESUMO

Androgen receptor (AR) transcriptional activity depends on interactions between the AR NH2-terminal region and transcriptional coregulators. A yeast two-hybrid screen of a human testis library using predicted α-helical NH2-terminal fragment AR-(370-420) as bait identified suppressor of variegation 3-9 homolog 2 (SUV39H2) histone methyltransferase as an AR interacting protein. SUV39H2 interaction with AR and the AR coregulator, melanoma antigen-A11 (MAGE-A11), was verified in two-hybrid, in vitro glutathione S-transferase affinity matrix and coimmunoprecipitation assays. Fluorescent immunocytochemistry colocalized SUV39H2 and AR in the cytoplasm without androgen, in the nucleus with androgen, and with MAGE-A11 in the nucleus independent of androgen. Chromatin immunoprecipitation using antibodies raised against SUV39H2 demonstrated androgen-dependent recruitment of AR and SUV39H2 to the androgen-responsive upstream enhancer of the prostate-specific antigen gene. SUV39H2 functioned cooperatively with MAGE-A11 to increase androgen-dependent AR transcriptional activity. SUV39H2 histone methyltransferase is an AR coactivator that increases androgen-dependent transcriptional activity through interactions with AR and MAGE-A11.


Assuntos
Antígenos de Neoplasias/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Androgênicos/metabolismo , Sequência de Aminoácidos , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Receptores Androgênicos/química , Receptores Androgênicos/genética , Transcrição Gênica
15.
Mol Cell Endocrinol ; 348(2): 403-10, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-21664945

RESUMO

Human androgen receptor (AR) transcriptional activity involves interdomain and coactivator interactions with the agonist-bound AR ligand binding domain (LBD). Structural determinants of the AR NH(2)- and carboxyl-terminal interaction between the AR NH(2)-terminal FXXLF motif and activation function 2 (AF2) in the LBD were shown previously by crystallography. In this report, we provide evidence for a region in AR LBD helix 12 outside the AF2 binding cleft that facilitates interactions with the FXXLF and LXXLL motifs. Mutagenesis of glutamine 902 to alanine in AR LBD helix 12 (Q902A) disrupted AR FXXLF motif binding to AF2, but enhanced coactivator LXXLL motif binding. Functional compensation for defective FXXLF motif binding by AR-Q902A was suggested by the slower dissociation rate of bound androgen. Functional importance of glutamine 902 was indicated by the charged residue germline mutation Q902R that caused partial androgen insensitivity, and a similar somatic mutation Q902K reported in prostate cancer, both of which increased the androgen dissociation rate and decreased AR transcriptional activity. High affinity equilibrium androgen binding was retained by alanine substitution mutations at Tyr-739 in AR LBD helix 5 or Lys-905 in helix 12 structurally adjacent to AF2, whereas transcriptional activity decreased and the androgen dissociation increased. Deleterious effects of these loss of function mutations were rescued by the helix stabilizing AR prostate cancer somatic mutation H874Y. Sequence NH(2)-terminal to the AR FXXLF motif contributed to the AR NH(2)- and carboxyl-terminal interaction based on greater AR-2-30 FXXLF motif peptide binding to the agonist-bound AR LBD than a shorter AR-20-30 FXXLF motif peptide. We conclude that helix 12 residues outside the AF2 binding cleft modulate AR transcriptional activity by providing flexibility to accommodate FXXLF or LXXLL motif binding.


Assuntos
Receptores Androgênicos/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Androgênios/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Di-Hidrotestosterona/química , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Coativador 2 de Receptor Nuclear/metabolismo , Estrutura Terciária de Proteína , Receptores Androgênicos/química , Receptores Androgênicos/genética , Propriedades de Superfície , Ativação Transcricional
16.
J Biol Chem ; 281(10): 6648-63, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16365032

RESUMO

The androgen receptor (AR) is a ligand-activated transcription factor required for male sex development and virilization and contributes to prostate cancer initiation and progression. High affinity androgen binding triggers conformational changes required for AR transactivation. Here we characterized naturally occurring AR gene mutations in the region of activation function 2 (AF2) that decrease or increase AR transcriptional activity by altering the region bounded by AF2 and the ligand binding pocket without affecting equilibrium androgen binding affinity. In the androgen insensitivity syndrome, germ line AR mutations increase the androgen dissociation rate and reduce AR FXXLF motif binding and the recruitment of steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs. In prostate cancer, somatic AR mutations in AF2 or near the bound ligand slow androgen dissociation and increase AR stabilization and coactivator recruitment. Crystal structures of the AR ligand binding domain bound to R1881 and FXXLF or LXXLL motif peptide indicate the mutations are proximal to the AF2 bound peptide, adjacent to the ligand pocket, or in a putative ligand gateway. The results suggest a bidirectional structural relay between bound ligand and coactivator that establishes AR functional potency in vivo.


Assuntos
Sítio Alostérico/fisiologia , Síndrome de Resistência a Andrógenos/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Sítio Alostérico/genética , Motivos de Aminoácidos , Síndrome de Resistência a Andrógenos/genética , Ligação Competitiva , Feminino , Células HeLa , Humanos , Ligantes , Masculino , Mutação Puntual , Neoplasias da Próstata/genética , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Relação Estrutura-Atividade
17.
J Biol Chem ; 277(12): 10226-35, 2002 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-11779876

RESUMO

The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH(2) terminus. Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection in yeast two-hybrid screens. Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathione S-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain. Mutagenesis of residues within activation function 2 indicates distinct but overlapping binding sites where specificity depends on sequences within and flanking the FXXLF motif. Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription assays. The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins.


Assuntos
Receptores Androgênicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Immunoblotting , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
18.
J Biol Chem ; 277(28): 25631-9, 2002 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-12000757

RESUMO

The agonist-induced androgen receptor NH(2)- and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH(2)-terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH(2)-terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence (433)WHTLF(437) contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation.


Assuntos
Receptores Androgênicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Elementos Facilitadores Genéticos , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Receptores Androgênicos/química , Receptores Androgênicos/genética , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido
19.
J Biol Chem ; 279(29): 30643-53, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15107424

RESUMO

The NH2-terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH2-terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH2-terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH2-terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH2-terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH2-terminal domains and other intrinsically unstructured transcriptional regulatory proteins.


Assuntos
Receptores Androgênicos/química , Ubiquitina-Proteína Ligases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Citoplasma/metabolismo , Evolução Molecular , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico HSP90/química , Células HeLa , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Próstata/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Transcrição Gênica , Transfecção , Transgenes , Técnicas do Sistema de Duplo-Híbrido
20.
Mol Cell ; 16(3): 425-38, 2004 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-15525515

RESUMO

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.


Assuntos
Mutação/genética , Fragmentos de Peptídeos/química , Receptores Androgênicos/química , Receptores Citoplasmáticos e Nucleares/química , Ativação Transcricional , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Masculino , Mutagênese , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coativador 2 de Receptor Nuclear , Fragmentos de Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Receptores Androgênicos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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