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1.
Cell ; 176(6): 1447-1460.e14, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30799039

RESUMEN

The presence of DNA in the cytoplasm is normally a sign of microbial infections and is quickly detected by cyclic GMP-AMP synthase (cGAS) to elicit anti-infection immune responses. However, chronic activation of cGAS by self-DNA leads to severe autoimmune diseases for which no effective treatment is available yet. Here we report that acetylation inhibits cGAS activation and that the enforced acetylation of cGAS by aspirin robustly suppresses self-DNA-induced autoimmunity. We find that cGAS acetylation on either Lys384, Lys394, or Lys414 contributes to keeping cGAS inactive. cGAS is deacetylated in response to DNA challenges. Importantly, we show that aspirin can directly acetylate cGAS and efficiently inhibit cGAS-mediated immune responses. Finally, we demonstrate that aspirin can effectively suppress self-DNA-induced autoimmunity in Aicardi-Goutières syndrome (AGS) patient cells and in an AGS mouse model. Thus, our study reveals that acetylation contributes to cGAS activity regulation and provides a potential therapy for treating DNA-mediated autoimmune diseases.


Asunto(s)
ADN/inmunología , Nucleotidiltransferasas/metabolismo , Autotolerancia/inmunología , Acetilación , Secuencia de Aminoácidos , Animales , Aspirina/farmacología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Autoinmunidad , Línea Celular , ADN/genética , ADN/metabolismo , Modelos Animales de Enfermedad , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutación , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Células THP-1
2.
Nat Immunol ; 9(5): 533-41, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18362886

RESUMEN

Despite rapid progress in elucidating the molecular mechanisms of activation of the kinase IKK, the processes that regulate IKK deactivation are still unknown. Here we demonstrate that CUE domain-containing 2 (CUEDC2) interacted with IKKalpha and IKKbeta and repressed activation of the transcription factor NF-kappaB by decreasing phosphorylation and activation of IKK. Notably, CUEDC2 also interacted with GADD34, a regulatory subunit of protein phosphatase 1 (PP1). We found that IKK, CUEDC2 and PP1 existed in a complex and that IKK was released from the complex in response to inflammatory stimuli such as tumor necrosis factor. CUEDC2 deactivated IKK by recruiting PP1 to the complex. Therefore, CUEDC2 acts as an adaptor protein to target IKK for dephosphorylation and inactivation by recruiting PP1.


Asunto(s)
Proteínas Portadoras/metabolismo , Quinasa I-kappa B/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Proteínas Portadoras/inmunología , Dominio Catalítico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Femenino , Humanos , Quinasa I-kappa B/química , Inflamación/inmunología , Interleucina-6/biosíntesis , Interleucina-6/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteínas Represoras/inmunología , Regulación hacia Arriba
3.
Proc Natl Acad Sci U S A ; 110(27): 11017-22, 2013 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-23776205

RESUMEN

DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G1 arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G1 arrest. The mechanism of UV-induced G1 arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C(Cdh1)), a critical ubiquitin ligase in G1 phase, thereby stabilizing Cyclin A and promoting G1-S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C(Cdh1)-mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G1 arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G1-S block. These results establish CUEDC2 as an APC/C(Cdh1) inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G1 arrest.


Asunto(s)
Proteínas Portadoras/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/fisiología , Proteínas de la Membrana/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular Tumoral , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de la radiación , Células HEK293 , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida , Fosforilación/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Rayos Ultravioleta
4.
J Biol Chem ; 287(1): 382-392, 2012 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-22084247

RESUMEN

Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway.


Asunto(s)
Proteínas Portadoras/metabolismo , Janus Quinasa 1/metabolismo , Proteínas de la Membrana/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Elonguina , Activación Enzimática , Humanos , Fosforilación , Estabilidad Proteica , Proteolisis , Proteína 3 Supresora de la Señalización de Citocinas , Factores de Transcripción/metabolismo , Transcripción Genética
5.
Zhonghua Yi Xue Za Zhi ; 93(14): 1099-103, 2013 Apr 09.
Artículo en Zh | MEDLINE | ID: mdl-23902846

RESUMEN

OBJECTIVE: To establish Gankyrin knocking down 4T1-luc cell model and detect the effects of Gankyrin expression on breast cancer metastasis. METHODS: 4T1-luc cells carrying shGankyrin construct were established by lentivirus infection and antibiotic screening. Western blotting and real-time PCR were used to check the expression levels of Gankyrin. In vivo imaging system was used to monitor the effects of Gankyrin knocked down on cell growth and tumor metastasis after the in situ implantation of Gankyrin knocking down 4T1-luc cells in BALB/c mice. RESULTS: The cell expression decreased at the protein and mRNA levels. Gankyrin mRNA expression in different shGankyrin 4T1-luc cells was respectively 4.9%, 25.1% and 69.8% versus the control cells. ShGankyrin#2 4T1-luc cells were chosen for in situ implantation into BAL/c mice because luminescent intensity was consistent with cell numbers. The photon flux of lung metastatic tumor induced by Gankyrin knocking down 4T1-luc cell was 3.02 × 10(6), while that of lung metastasis induced by control cells was 10.9 × 10(6). The differences between two groups were significant. In pathology, Gankyrin was detected positive in lung metastasis tumors induced by control group. However, Gankyrin was negative in the Gankyrin knockdown group. CONCLUSIONS: Lentivirus infection may be effectively used to establish Gankyrin knocking down 4T1-luc cell model. Because of its involvement in the in vivo pulmonary metastasis of breast cancers, Gankyrin should be a novel target for tumor therapy.


Asunto(s)
Neoplasias Mamarias Experimentales/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas/genética , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia
6.
Proc Natl Acad Sci U S A ; 106(10): 3788-93, 2009 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-19234109

RESUMEN

DNA damage response (DDR) acts as a tumorigenesis barrier, and any defects in the DDR machinery may lead to cancer. SOX4 expression is elevated in many types of tumors; however, its role in DDR is still largely unknown. Here, we show that SOX4, a new DNA damage sensor, is required for the activation of p53 tumor suppressor in response to DNA damage. Notably, SOX4 interacts with and stabilizes p53 protein by blocking Mdm2-mediated p53 ubiquitination and degradation. Furthermore, SOX4 enhances p53 acetylation by interacting with p300/CBP and facilitating p300/CBP/p53 complex formation. In concert with these results, SOX4 promotes cell cycle arrest and apoptosis, and it inhibits tumorigenesis in a p53-dependent manner. Therefore, these findings highlight SOX4 as a potential key factor in regulating DDR-associated cancer.


Asunto(s)
Daño del ADN , Factores de Transcripción SOXC/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Unión Proteica , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ubiquitinación
7.
Cell Res ; 29(4): 265-273, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30814679

RESUMEN

The re-emergence of Zika virus (ZIKV) in the Western Hemisphere has resulted in global public health crisis since 2015. ZIKV preferentially infects and targets human neural progenitor cells (hNPCs) and causes fetal microcephaly upon maternal infection. hNPCs not only play critical roles during fetal brain development, but also persist in adult brain throughout life. Yet the mechanism of innate antiviral immunity in hNPCs remains largely unknown. Here, we show that ZIKV infection triggers the abundant production of virus-derived small interfering RNAs in hNPCs, but not in the more differentiated progenies or somatic cells. Ablation of key RNAi machinery components significantly enhances ZIKV replication in hNPCs. Furthermore, enoxacin, a broad-spectrum antibiotic that is known as an RNAi enhancer, exerts potent anti-ZIKV activity in hNPCs and other RNAi-competent cells. Strikingly, enoxacin treatment completely prevents ZIKV infection and circumvents ZIKV-induced microcephalic phenotypes in brain organoid models that recapitulate human fetal brain development. Our findings highlight the physiological importance of RNAi-mediated antiviral immunity during the early stage of human brain development, uncovering a novel strategy to combat human congenital viral infections through enhancing RNAi.


Asunto(s)
Encéfalo/inmunología , Células-Madre Neurales/inmunología , Organoides/inmunología , ARN Viral/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/genética , Animales , Antivirales/farmacología , Encéfalo/patología , Línea Celular , Enoxacino/farmacología , Humanos , Inmunidad Innata , Células-Madre Neurales/patología , Organoides/patología , Interferencia de ARN , Replicación Viral , Virus Zika/inmunología , Virus Zika/fisiología
8.
Nucleic Acids Res ; 34(19): 5552-66, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17020914

RESUMEN

Progesterone receptor (PR) plays a critical role in cell proliferation and differentiation, and its transcriptional activity is known to be modulated by cofactor proteins. In the present study, we demonstrated that in the presence of progesterone, protein inhibitor of activated STAT-3 (PIAS3) significantly inhibited the PR transcriptional activity and the expression of progesterone-responsive genes. Reduction of endogenous PIAS3 by PIAS3 small-interfering RNA enhanced PR transactivation in a ligand-dependent manner. PIAS3 interacted with PR both in vitro and in vivo and the interaction was enhanced by progesterone. Furthermore, our findings suggested that PIAS3 strongly induced PRB sumoylation at three sites, Lys-7, Lys-388 and Lys-531. In addition, novel roles in PRB nuclear retention and transactivation were identified for these sites. Our data also suggested that PIAS3 was recruited in a largely hormone-dependent manner in response to a progesterone-responsive promoter. Finally, we demonstrated that PIAS3 inhibited the DNA-binding activity of PR and influenced its nuclear export as well as PR transactivation. Taken together, these data strongly suggested that PIAS3 played an important physiological role in PR function.


Asunto(s)
Núcleo Celular/química , Chaperonas Moleculares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Receptores de Progesterona/metabolismo , Activación Transcripcional , Animales , Humanos , Progesterona/antagonistas & inhibidores , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Receptores de Progesterona/análisis , Receptores de Progesterona/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo
9.
Nat Cell Biol ; 13(8): 924-33, 2011 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-21743465

RESUMEN

Aneuploidy and chromosomal instability are major characteristics of human cancer. These abnormalities can result from defects in the spindle assembly checkpoint (SAC), which is a surveillance mechanism for accurate chromosome segregation through restraint of the activity of the anaphase-promoting complex/cyclosome (APC/C). Here, we show that a CUE-domain-containing protein, CUEDC2, is a cell-cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 during mitosis. Depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition. Phosphorylated CUEDC2 binds to Cdc20, an activator of APC/C, and promotes the release of Mad2 from APC/C-Cdc20 and subsequent APC/C activation. CUEDC2 overexpression causes earlier activation of APC/C, leading to chromosome missegregation and aneuploidy. Interestingly, CUEDC2 is highly expressed in many types of tumours. These results suggest that CUEDC2 is a key regulator of mitosis progression, and that CUEDC2 dysregulation might contribute to tumour development by causing chromosomal instability.


Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Portadoras/metabolismo , Inestabilidad Cromosómica/fisiología , Proteínas de la Membrana/metabolismo , Huso Acromático/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Ciclosoma-Complejo Promotor de la Anafase , Aneuploidia , Proteínas de Unión al Calcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Inestabilidad Cromosómica/genética , Células HeLa , Humanos , Proteínas Mad2 , Mitosis/genética , Mitosis/fisiología , Complejos Multiproteicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , Proteínas Represoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo
10.
Nat Med ; 17(6): 708-14, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21572428

RESUMEN

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.


Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/fisiopatología , Proteínas Portadoras/fisiología , Resistencia a Antineoplásicos/fisiología , Proteínas de la Membrana/fisiología , Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Portadoras/biosíntesis , Línea Celular Tumoral , Regulación hacia Abajo , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/fisiología , Proteínas de la Membrana/biosíntesis , Fosforilación , Receptores de Progesterona/metabolismo , Receptores de Progesterona/fisiología , Tamoxifeno/uso terapéutico , Ubiquitinación
11.
J Clin Invest ; 120(8): 2829-41, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20628200

RESUMEN

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil-containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras-induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.


Asunto(s)
Transformación Celular Neoplásica , Genes ras , Neoplasias Pulmonares/etiología , Transducción de Señal , Factores de Transcripción/fisiología , Proteínas de Unión al GTP rho/fisiología , Quinasas Asociadas a rho/fisiología , Animales , Inhibidores de Disociación de Guanina Nucleótido/fisiología , Humanos , Ratones , Células 3T3 NIH , Fosfohidrolasa PTEN/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Inhibidor alfa de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico , Proteína de Unión al GTP rhoA
12.
EMBO J ; 26(7): 1831-42, 2007 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-17347654

RESUMEN

Accumulated evidence indicates that progesterone receptors (PR) are involved in proliferation of breast cancer cells and are implicated in the development of breast cancer. In this paper, a yeast two-hybrid screen for PR led to the identification of CUE domain containing 2 (CUEDC2), whose function is unknown. Our results demonstrate that CUEDC2 interacts with PR and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. The inhibition of endogenous CUEDC2 by siRNA nearly abrogated the progesterone-induced degradation of PR, suggesting that CUEDC2 is involved in progesterone-induced PR ubiquitination and degradation. Moreover, we identify the sumoylation site Lys-388 of PR as the target of CUEDC2-promoted ubiquitination. CUEDC2 decreases the sumoylation while promoting ubiquitination on Lys-388 of PRB. We also show that CUEDC2 represses PR transactivation, inhibits the ability of PR to stimulate rapid MAPK activity, and impairs the effect of progesterone on breast cancer cell growth. Therefore, our results identify a key post-translational mechanism that controls PR protein levels and for the first time provide an important insight into the function of CUEDC2 in breast cancer proliferation.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Progesterona/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Ligandos , Proteínas de la Membrana/genética , Proteínas Mutantes/metabolismo , Progesterona/farmacología , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Receptores de Progesterona/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética
13.
J Proteome Res ; 6(11): 4397-406, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17894482

RESUMEN

A global understanding of ubiquitinated proteins in vivo is key to unraveling the biological significance of ubiquitination. There are, however, a few effective screening methods for rapid analysis of ubiquitinated proteins. In the current study, we designed a cell-based cDNA expression array combined with cell imaging for the rapid identification of polyubiquitinated proteins, which normally accumulate to form the unique "dot" structure following inhibition of ubiquitin proteasomes. The array consisted of 112 cDNAs encoding key components of major cellular pathways and potential targets of polyubiquitination. Among them, 40 proteins formed accumulation dots in response to proteasome inhibitor, MG-132, treatment. More importantly, 24 of those 40 proteins, such as MAPKAPK3, NLK, and RhoGDI2, are previously not known as the targets of ubiquitin. We further validated our findings by examining the endogenous counterparts of some of these proteins and found that those endogenous proteins form a similar "dot" structure. Immunoprecipitation assays confirmed that these accumulated proteins are polyubiquitinated. Our results demonstrate that this large-scale application of cell-based arrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins. Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cell signaling.


Asunto(s)
Proteínas/química , Proteómica/métodos , Ubiquitina/química , Línea Celular Tumoral , ADN Complementario/metabolismo , Electroforesis en Gel Bidimensional , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leupeptinas/farmacología , Inhibidores de Proteasoma , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico
14.
Biochem Biophys Res Commun ; 345(1): 355-61, 2006 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-16678796

RESUMEN

Recent reports have shown that MDM2 may attenuate hypertrophy of cardiac myocytes. However, mechanism of MDM2 involving in this process is unclear. In this study, we identified a novel specific MDM2-binding protein TCAP by the yeast two-hybrid screen. It was validated by GST pull-down and co-immunoprecipitation assays. Confocal analysis showed that MDM2 and TCAP co-localized in the nucleus, and elevated MDM2 expression could alter the subcellular localization of TCAP. Notably, MDM2 downregulated the protein level of TCAP through the proteasomal pathway, and this downregulation was inhibited by p14(ARF). In addition, our results suggested that the degradation of TCAP by MDM2 was through the ubiquitin-independent pathway. Given that TCAP is a key component involving in the cardiac hypertrophy, the degradation of TCAP by MDM2 might be connected with the roles of MDM2 in cardiac hypertrophy. Further investigation will focus on the biological significance of MDM2-TCAP interaction in cardiac hypertrophy.


Asunto(s)
Proteínas Musculares/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Sarcómeros/metabolismo , Fracciones Subcelulares/metabolismo , Conectina , Regulación hacia Abajo , Proteínas Musculares/química , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/química , Sarcómeros/química , Fracciones Subcelulares/química
15.
J Proteome Res ; 5(10): 2815-23, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17022653

RESUMEN

We implemented a proteomics approach to the systematical analysis of the alterations in the proteome of NIH3T3 cells transformed by oncogenic H-RasV12. Forty-four proteins associated with Ras-mediated transformation have been identified, and 28 proteins were not previously reported. RT-PCR analysis showed that approximately 44% of target proteins identified showed concomitant changes in mRNA abundance. A principal finding was the up-regulation of gankyrin, which was the first evidence to show that gankyrin pathway was implicated in Ras-activated transformation.


Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteína Oncogénica p21(ras)/genética , Proteoma/análisis , Proteómica , Animales , Western Blotting , Transformación Celular Neoplásica/genética , Análisis por Conglomerados , Humanos , Ratones , Células 3T3 NIH , Proteoma/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección
16.
J Biol Chem ; 279(47): 49251-8, 2004 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-15371445

RESUMEN

Although eukaryotic translation initiation factor 5A (eIF5A) was originally designated as an "initiation factor," recent data have shown it to be also involved in apoptosis. However, the actual function of eIF5A in apoptosis is still unknown. In this study, we performed yeast two-hybrid screens to identify eIF5A-interacting proteins to help us understand the mechanisms of eIF5A. Our results demonstrated that eIF5A and syntenin could engage in a specific interaction both in vitro and in vivo and functioned collaboratively to regulate p53 activity. Our findings, for the first time, revealed a new biological activity for eIF5A as the regulator of p53. Overexpression of eIF5A or its EFP domain resulted in up-regulation of p53, and silencing eIF5A by small interfering RNA reduced the p53 protein level. Further analysis by reverse transcription PCR showed eIF5A-activated p53 transcription. The effect of eIF5A on p53 transcriptional activity was further demonstrated by the increasing expressions of p21 and Bax, well known target genes of p53. In contrast, a point mutant of eIF5A, hypusination being abolished, was revealed to be functionally defective in p53 up-regulation. Overexpression of eIF5A led to a p53-dependent apoptosis or sensitized cells to induction of apoptosis by chemotherapeutic agents. However, when eIF5A interacted with its novel partner, syntenin, the eIF5A-induced increase in p53 protein level was significantly inhibited. Therefore, eIF5A seems to be a previously unrecognized regulator of p53 that may define a new pathway for p53-dependent apoptosis, and syntenin might regulate p53 by balancing the regulation of eIF5A signaling to p53 for apoptosis.


Asunto(s)
Apoptosis , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Factores de Iniciación de Péptidos/fisiología , Proteínas de Unión al ARN/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Células COS , Línea Celular , Línea Celular Tumoral , ADN Complementario/metabolismo , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Silenciador del Gen , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Mutación , Factores de Iniciación de Péptidos/química , Fosforilación , Plásmidos/metabolismo , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinteninas , Factores de Tiempo , Transfección , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba , Proteína X Asociada a bcl-2 , Factor 5A Eucariótico de Iniciación de Traducción
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