RESUMEN
Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Proteínas Nucleares/metabolismo , Reparación del ADN por Recombinación/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Proteína BRCA1/genética , Proteínas Portadoras/genética , Proteínas de Unión al ADN , Células HeLa , Chaperonas de Histonas , Humanos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Humanos , Lisina/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Factores de Transcripción de Octámeros/química , Proteínas de Unión al ARN/química , Fase S , Transducción de Señal , Ubiquitinación , Rayos UltravioletaRESUMEN
Janus tyrosine kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway is essential for modulating cellular development, differentiation, and homeostasis. Thus, dysregulation of JAK2-STAT3 signaling pathway is frequently associated with human malignancies. Here, we provide evidence that lysine-specific demethylase 3A (KDM3A) functions as an essential epigenetic enzyme for the activation of JAK2-STAT3 signaling pathway. KDM3A is tyrosine-phosphorylated by JAK2 in the nucleus and functions as a STAT3-dependent transcriptional coactivator. JAK2-KDM3A signaling cascade induced by IL-6 leads to alteration of histone H3K9 methylation as a predominant epigenetic event, thereby providing the functional and mechanistic link between activation of JAK2-STAT3 signaling pathway and its epigenetic control. Together, our findings demonstrate that inhibition of KDM3A phosphorylation could be a potent therapeutic strategy to control oncogenic effect of JAK2-STAT3 signaling pathway.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/metabolismo , Epigénesis Genética , Células HEK293/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Interleucina-6/metabolismo , Janus Quinasa 2/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
The E3 ubiquitin ligase membrane-associated ring-CH-type finger 2 (MARCH2) is known to be involved in intracellular vesicular trafficking, but its role in the early secretory pathway between the endoplasmic reticulum (ER) and Golgi compartments is largely unknown. Human ER-Golgi intermediate compartment protein 2 (ERGIC2) and ERGIC3 are orthologs of Erv41 and Erv46 in yeast, proteins that form a heteromeric complex, cycle between the ER and Golgi, and function as cargo receptors in both anterograde and retrograde protein trafficking. Here, we report that MARCH2 directs ubiquitination and subsequent degradation of ERGIC3 and that MARCH2 depletion increases endogenous ERGIC3 levels. We provide evidence that the lysine residues at positions 6 and 8 of ERGIC3 are the major sites of MARCH2-mediated ubiquitination. Of note, MARCH2 did not significantly decrease the levels of an ERGIC3 variant with lysine-to-arginine substitutions at residues 6 and 8. We also show that ERGIC3 binds to itself or to ERGIC2, whereas ERGIC2 is unable to interact with itself. Our results indicate that α1-antitrypsin and haptoglobin are likely to be cargo proteins of ERGIC3. We further observed that α1-antitrypsin and haptoglobin specifically bind to ERGIC3 and that ERGIC3 depletion decreases their secretion. Moreover, MARCH2 reduced secretion of α1-antitrypsin and haptoglobin, and coexpression of the ubiquitination-resistant ERGIC3 variant largely restored their secretion, suggesting that MARCH2-mediated ERGIC3 ubiquitination is the major cause of the decrease in trafficking of ERGIC3-binding secretory proteins. Our findings provide detailed insights into the regulation of the early secretory pathway by MARCH2 and into ERGIC3 function.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Transporte de Proteínas , Proteolisis , Vías Secretoras , Vesículas Secretoras/metabolismo , UbiquitinaciónRESUMEN
Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing "methyl degron"; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Células MCF-7 , Metilación , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas , Especificidad por SustratoRESUMEN
Dystroglycan is a ubiquitous membrane protein that functions as a mechanical connection between the extracellular matrix and cytoskeleton. In skeletal muscle, dystroglycan plays an indispensable role in regulating muscle regeneration; a malfunction in dystroglycan is associated with muscular dystrophy. The regulation of dystroglycan stability is poorly understood. Here, we report that WWP1, a member of NEDD4 E3 ubiquitin ligase family, promotes ubiquitination and subsequent degradation of ß-dystroglycan. Our results indicate that dystrophin and utrophin protect ß-dystroglycan from WWP1-mediated degradation by competing with WWP1 for the shared binding site at the cytosolic tail of ß-dystroglycan. In addition, we show that a missense mutation (arginine 440 to glutamine) in WWP1-which is known to cause muscular dystrophy in chickens-increases the ubiquitin ligase-mediated ubiquitination of both ß-dystroglycan and WWP1. The R440Q missense mutation in WWP1 decreases HECT domain-mediated intramolecular interactions to relieve autoinhibition of the enzyme. Our results provide new insight into the regulation of ß-dystroglycan degradation by WWP1 and other Nedd4 family members and improves our understanding of dystroglycan-related disorders.
Asunto(s)
Distroglicanos/metabolismo , Distrofina/metabolismo , Distrofias Musculares/patología , Ubiquitina-Proteína Ligasas/metabolismo , Utrofina/metabolismo , Animales , Sitios de Unión , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Ratones , Distrofias Musculares/genética , Mutación Missense , Dominios Proteicos/genética , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Utrofina/genéticaRESUMEN
Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA-dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra-telomeric functions that are independent of its telomere-regulatory function, including tumor progression, and neuronal cell death regulation. In this study, we evaluated these additional hTERT non-telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin-dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. J. Cell. Physiol. 232: 2083-2093, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Telomerasa/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/enzimología , Células HeLa , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones Noqueados , Chaperonas Moleculares/genética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Unión Proteica , Proteolisis , ARN/genética , ARN/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN , Telomerasa/genética , Factores de Tiempo , Transfección , Tunicamicina/farmacología , UbiquitinaciónRESUMEN
Signaling through many transmembrane receptors is terminated by their sorting to the intraluminal vesicles (ILVs) of multivescular bodies (MVBs) and subsequent lysosomal degradation. ILV formation requires the endosomal sorting complex required for transport (ESCRT) machinery. CC2D1A and CC2D1B interact with the CHMP4 family of proteins, the major subunit of the ESCRT-III complex, however, their roles in receptor degradation and signaling are poorly defined. Here, we report that CC2D1A binds to CHMP4B polymers formed on endosomes to regulate the endosomal sorting pathway. We show that depletion of CC2D1A and B accelerates degradation of EGFR and elicits rapid termination of its downstream signaling through ERK1 and 2. Depletion of CC2D1A and B promotes sorting of EGFR to ILV leading to its rapid lysosomal degradation. In addition, we show that knockdown of CC2D1A and B has similar effects on degradation and downstream signaling of another membrane receptor, TLR4. Thus, these findings suggest that CC2D1A and B may have broad effects on transmembrane receptors by preventing premature ILV sorting and termination of signaling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores ErbB/metabolismo , Proteínas Represoras/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Transporte de Proteínas , Proteolisis , Proteínas Represoras/genética , Transducción de SeñalRESUMEN
Recently, protein kinase M ζ (PKMζ) has emerged as an important player for maintaining memory. It has been reported that PKMζ regulates the trafficking of GluA2 in postsynaptic membranes to maintain memory. However, there has been no study on PKMζ outside the synaptic region regarding memory maintenance. Here, we found that PKMζ is transported to the nucleus in a neural activity-dependent manner. Moreover, we found that PKMζ phosphorylates CREB-binding protein (CBP) at serine residues and that PKMζ inhibition reduces the acetylation of histone H2B and H3. Finally, we showed that the amnesic effect of PKMζ inhibition can be rescued by enhancing histone acetylation level. These results suggest the possibility that nuclear PKMζ has a crucial role in memory maintenance.
Asunto(s)
Amnesia/metabolismo , Amígdala del Cerebelo/metabolismo , Proteína de Unión a CREB/metabolismo , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Memoria/fisiología , Proteína Quinasa C/metabolismo , Amnesia/fisiopatología , Amígdala del Cerebelo/fisiopatología , Animales , Conducta Animal/fisiología , Células Cultivadas , Embrión de Mamíferos , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas , Proteína Quinasa C/antagonistas & inhibidoresRESUMEN
Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys(48)-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Proteínas Reguladoras de la Apoptosis , Sitios de Unión/genética , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poliubiquitina/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Ubiquitina/metabolismo , Proteína que Contiene ValosinaRESUMEN
Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Although Pontin is frequently overexpressed in human cancers of various types and implicated in oncogenic functions, the upstream signaling network leading to the regulation of Pontin that in turn affects transcription of downstream target genes has not been extensively studied. Here, we identify Pontin is methylated by G9a/GLP methyltransferases in hypoxic condition and potentiates HIF-1α-mediated activation by increasing the recruitment of p300 coactivator to a subset of HIF-1α target promoters. Intriguingly, Pontin methylation results in the increased invasive and migratory properties by activating downstream target gene, Ets1. In contrast, inhibition of Pontin methylation results in the suppression of tumorigenic and metastatic properties. Together, our data provide new approaches by targeting Pontin methylation and its downstream targets for the development of therapeutic agents for human cancers.
Asunto(s)
Proteínas Portadoras/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , ATPasas Asociadas con Actividades Celulares Diversas , Hipoxia de la Célula , Línea Celular Tumoral , Cromatina/genética , Epigenómica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Metilación , Metiltransferasas/metabolismo , Proteínas de Neoplasias/metabolismo , Transcripción GenéticaRESUMEN
The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Nucleótidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
During the late M to the G(1) phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2-5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2-5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.
Asunto(s)
Cromatina/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Procesamiento Proteico-Postraduccional , Origen de Réplica , Secuencia de Aminoácidos , Secuencia Conservada , Células HeLa , Humanos , Datos de Secuencia Molecular , Complejo de Reconocimiento del Origen/química , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Fase SRESUMEN
The Hairless (Hr), a transcription factor, is expressed in the suprabasal cell layer of the interfollicular epidermis and the lower portion of the hair follicle epithelium, where its expression is dependent on the hair cycle. Recently, we reported a new Hr mutant mouse, Hr(Hp), in which the hairless protein (HR) was overexpressed. In this study, we documented abnormal formation of inner root sheath (IRS), suppressed expression of Dlx3, and IRS keratins in the Hr(Hp)/Hr(Hp) skin. We also found that HR down-regulated Dlx3 mRNA expression through suppression of Dlx3 promoter activity. In addition, we showed that Dlx3 regulated the expression of IRS keratins. Our results demonstrate that regulation of Dlx3 by HR affects the IRS keratin expression, thus modulating the formation of IRS of hair follicle.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Folículo Piloso/metabolismo , Proteínas de Homeodominio/biosíntesis , Queratinas/biosíntesis , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Animales , Proteínas de Homeodominio/genética , Queratinas/genética , Ratones , Ratones Pelados , Factores de Transcripción/genéticaRESUMEN
PTK6 [protein tyrosine kinase 6; also known as Brk (breast tumour kinase)] is a non-receptor tyrosine kinase, closely related to Src, but evolutionarily distinct, that is up-regulated in various cancers, including breast cancer. Hsp90 (heat-shock protein 90) was identified as a PTK6-interacting protein in HEK (human embryonic kidney)-293 cells overexpressing PTK6. Hsp90 interacted with the PTK6 tyrosine kinase catalytic domain, but catalytic activity was not required for the interaction. Geldanamycin, an Hsp90 inhibitor, significantly decreased the PTK6 protein level through proteasome-dependent degradation, but did not affect the level of Src. Geldanamycin treatment also decreased phosphorylation of PTK6 substrates due to reduced amounts of PTK6. Moreover, overexpression of CHIP [C-terminus of Hsc70 (heat-shock cognate 70)-interacting protein], a chaperone-dependent E3 ligase, enhanced proteosomal degradation of PTK6. Geldanamycin increased the interaction of PTK6 with CHIP, but decreased the interaction of PTK6 with Hsp90. We also found that endogenous PTK6 associated with Hsp90 and geldanamycin decreased expression of endogenous PTK6 in breast carcinoma cells. Finally, we report that silencing endogenous CHIP expression in breast carcinoma cells inhibited geldanamycin-induced PTK6 reduction. These results demonstrate that Hsp90 plays an essential role in regulating PTK6 stability and suggest that Hsp90 inhibitors may be useful as therapeutic drugs for PTK6-positive cancers, including breast cancer.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Benzoquinonas/farmacología , Neoplasias de la Mama , Línea Celular Tumoral , Femenino , Células HEK293 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Lactamas Macrocíclicas/farmacologíaRESUMEN
Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant hair disorder. Through the study of a mouse model, we identified a mutation in the 5'-untranslated region of the hairless (HR) gene in patients with MUHH in a Caucasian family. The corresponding mutation, named 'hairpoor', was found in mutant mice that were generated through N-ethyl-N-nitrosourea mutagenesis. Hairpoor mouse mutants display partial hair loss at an early age and progress to near alopecia, which resembles the MUHH phenotype. This mutation conferred overexpression of HR through translational derepression and, in turn, decreased the expression of Sfrp2, an inhibitor of the Wnt signaling pathway. This study indicates that the gain in function of HR also results in alopecia, as seen with the loss of function of HR, via abnormal upregulation of the Wnt signaling pathway.
Asunto(s)
Expresión Génica , Hipotricosis/congénito , Hipotricosis/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Proteínas Wnt/metabolismo , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Hipotricosis/genética , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Linaje , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proteínas Wnt/genéticaRESUMEN
Fenretinide (FEN), a ligand of retinol binding protein 4 (RBP4), has been suggested as a measure to reduce insulin resistance and its associated disorders such as obesity, and fatty liver by reducing serum RBP4. We investigated whether there is another possible mechanism by which fenretinide reduces insulin resistance and fatty liver in genetically obese (ob/ob) mice. Male obese mice fed a high-fat diet (45% of calories from fat) were divided into two groups (n=13 each). One (FEN) received fenretinide (20 mg/kg body weight, intraperitoneally) and the other (O) received vehicle three times weekly for 24 d. C57BL/6J mice fed a normal-fat diet (16% of calories from fat) were used as a control (C; n=13). No changes in fat weight and serum leptin level could be observed in FEN mice. Lower plasma RBP4 was observed in FEN mice compared with O mice. Fenretinide improved whole-body insulin sensitivity based on glucose and insulin tolerance tests and the homeostasis model assessment of insulin resistance. Fenretinide decreased the plasma lipid (triglyceride, cholesterol, and free-fatty acid) levels, hepatic TG level, and histological steatosis score. The mechanism by which fenretinide prevents fatty liver may be explained by an increased plasma adiponectin level, increased activation of hepatic AMP-activated protein kinase, and the expression of peroxisome proliferator-activated protein-α and peroxisomal acyl-CoA oxidase, which promote fat oxidation. FEN alleviated insulin resistance and fatty liver in obese mice and thus may act as an anti-lipidemic and anti-diabetic drug.
Asunto(s)
Hígado Graso/metabolismo , Fenretinida/farmacología , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Resistencia a la Insulina , Obesidad/metabolismo , Adiponectina/sangre , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Dieta Alta en Grasa , Hígado Graso/patología , Leptina/sangre , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/patología , Prealbúmina/metabolismo , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
Parkinson's disease (PD) is a common neurodegenerative disease that involves the deterioration of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD remains poorly understood, recent genetic, postmortem, and experimental evidence shows that abnormal protein accumulation and subsequent aggregate formation are prominent features of both sporadic and familial PD. While proteasome dysfunction is observed in PD, diverse mutations in the parkin gene are linked to early-onset autosomal-recessive forms of familial PD. We demonstrate that parkin, an E3 ubiquitin ligase, activates the 26S proteasome in an E3 ligase activity-independent manner. Furthermore, an N-terminal ubiquitin-like domain within parkin is critical for the activation of the 26S proteasome through enhancing the interaction between 19S proteasomal subunits, whereas the PD-linked R42P mutant abolishes this action. The current findings point to a novel role for parkin for 26S proteasome assembly and suggest that parkin mutations contribute to the proteasomal dysfunction in PD.
Asunto(s)
Enfermedad de Parkinson/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Animales Modificados Genéticamente , Drosophila/genética , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Activación Enzimática/genética , Genes Recesivos , Células HeLa , Humanos , Ratones , Ratones Noqueados , Mutación , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2.
Asunto(s)
Adenosina Trifosfato/química , Ósmosis , Inhibidores de Proteasoma , Ubiquitina/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Sitios de Unión , Células HeLa , Hexosiltransferasas , Humanos , Sistema de Señalización de MAP Quinasas , Espectrometría de Masas/métodos , Péptidos/química , Fosfopéptidos/química , Fosforilación , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión ProteicaRESUMEN
The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.