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1.
BMB Rep ; 52(12): 712-717, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31818360

RESUMO

Translocase of outer mitochondrial membrane 20 (TOMM20) plays an essential role as a receptor for proteins targeted to mitochondria. TOMM20 was shown to be overexpressed in various cancers. However, the oncological function and therapeutic potential for TOMM20 in cancer remains largely unexplored. The purpose of this study was to elucidate the underlying molecular mechanism of TOMM20's contribution to tumorigenesis and to explore the possibility of its therapeutic potential using colorectal cancer as a model. The results show that TOMM20 overexpression resulted in an increase in cell proliferation, migration, and invasion of colorectal cancer (CRC) cells, while siRNA-mediated inhibition of TOMM20 resulted in significant decreases in cell proliferation, migration, and invasion. TOMM20 expression directly impacted the mitochondrial function including ATP production and maintenance of membrane potential, which contributed to tumorigenic cellular activities including regulation of S phase cell cycle and apoptosis. TOMM20 was overexpressed in CRC compared to the normal tissues and increased expression of TOMM20 to be associated with malignant characteristics including a higher number of lymph nodes and perineural invasion in CRC. Notably, knockdown of TOMM20 in the xenograft mouse model resulted in a significant reduction of tumor growth. This is the first report demonstrating a relationship between TOMM20 and tumorigenesis in colorectal cancer and providing promising evidence for the potential for TOMM20 to serve as a new therapeutic target of colorectal cancer. [BMB Reports 2019; 52(12): 712-717].


Assuntos
Neoplasias Colorretais/patologia , Receptores de Superfície Celular/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Metástase Neoplásica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Biol Chem ; 294(28): 10900-10912, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31142615

RESUMO

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.


Assuntos
Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico , Proteólise , Via Secretória , Vesículas Secretórias/metabolismo , Ubiquitinação
3.
Nucleic Acids Res ; 47(2): 762-778, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30445466

RESUMO

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.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Humanos , Lisina/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Fatores de Transcrição de Octâmero/química , Proteínas de Ligação a RNA/química , Fase S , Transdução de Sinais , Ubiquitinação , Raios Ultravioleta
4.
Proc Natl Acad Sci U S A ; 115(46): 11766-11771, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30377265

RESUMO

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.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Epigênese Genética , Células HEK293/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Interleucina-6/metabolismo , Janus Quinase 2/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Ativação Transcricional
5.
Biochim Biophys Acta Mol Basis Dis ; 1864(6 Pt A): 2199-2213, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29635000

RESUMO

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.


Assuntos
Distroglicanas/metabolismo , Distrofina/metabolismo , Distrofias Musculares/patologia , Ubiquitina-Proteína Ligases/metabolismo , Utrofina/metabolismo , Animais , Sítios de Ligação , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Camundongos , Distrofias Musculares/genética , Mutação de Sentido Incorreto , Domínios Proteicos/genética , Estabilidade Proteica , Proteólise , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Utrofina/genética
6.
Mol Cell ; 69(5): 866-878.e7, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29499138

RESUMO

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.


Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Células HeLa , Chaperonas de Histonas , Humanos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Ubiquitinas/metabolismo
7.
J Cell Physiol ; 232(8): 2083-2093, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27648923

RESUMO

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.


Assuntos
Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Telomerase/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/enzimologia , Células HeLa , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Knockout , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Proteólise , RNA/genética , RNA/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA , Telomerase/genética , Fatores de Tempo , Transfecção , Tunicamicina/farmacologia , Ubiquitinação
8.
FEBS J ; 283(24): 4583-4599, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27808481

RESUMO

The protease-associated (PA) domain-containing E3 ubiquitin ligases are transmembrane proteins located in intracellular organelles such as the endoplasmic reticulum, endosomes, or lysosomes. The functional roles of these ubiquitin ligases are not well defined. To understand the function of E3 ubiquitin ligases, identification of their substrates is of critical importance. In this study, we describe a newly devised method based on proximity-dependent biotin labeling to identify substrates of ubiquitin ligases. Application of this method to RING finger protein 167 (RNF167), a member of the PA domain-containing E3 family, led to identification of Arl8B as its substrate. We demonstrated that RNF167 ubiquitinates Arl8B at the lysine residue K141 and reduces the level of the Arl8B protein. Overexpression and knockdown of RNF167 revealed its regulatory role in Arl8B-dependent lysosome positioning and endocytic trafficking to lysosomes. Furthermore, we found that the ubiquitination-defective Arl8B K141R mutant counteracts RNF167 in these cellular events. These results indicate that RNF167 plays a crucial role as an E3 ubiquitin ligase targeting Arl8B to regulate lysosome positioning and endocytic trafficking.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Endocitose , Endossomos/metabolismo , Lisossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Ribosilação do ADP/genética , Sítios de Ligação/genética , Células HeLa , Humanos , Immunoblotting , Lisina/genética , Lisina/metabolismo , Microscopia Confocal , Mutação , Transporte Proteico , Proteólise , Interferência de RNA , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
9.
Biochem Biophys Res Commun ; 480(2): 280-287, 2016 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-27769858

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/metabolismo , Proteínas Repressoras/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Transporte Proteico , Proteólise , Proteínas Repressoras/genética , Transdução de Sinais
10.
Neurobiol Learn Mem ; 135: 50-56, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27321162

RESUMO

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.


Assuntos
Amnésia/metabolismo , Tonsila do Cerebelo/metabolismo , Proteína de Ligação a CREB/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Memória/fisiologia , Proteína Quinase C/metabolismo , Amnésia/fisiopatologia , Tonsila do Cerebelo/fisiopatologia , Animais , Comportamento Animal/fisiologia , Células Cultivadas , Embrião de Mamíferos , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Proteína Quinase C/antagonistas & inibidores
11.
PLoS One ; 8(5): e62981, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23667555

RESUMO

The ubiquitin-like modifier (UBL) domain of ubiquitin-like domain proteins (UDPs) interacts specifically with subunits of the 26 S proteasome. A novel UDP, ubiquitin-like domain-containing C-terminal domain phosphatase (UBLCP1), has been identified as an interacting partner of the 26 S proteasome. We determined the high-resolution solution structure of the UBL domain of human UBLCP1 by nuclear magnetic resonance spectroscopy. The UBL domain of hUBLCP1 has a unique ß-strand (ß3) and ß3-α2 loop, instead of the canonical ß4 observed in other UBL domains. The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1. Data from backbone dynamics shows that the ß3-α2 loop is relatively rigid although it might have intrinsic dynamic profile. The positively charged residues of the ß3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Soluções , Fator 2 Associado a Receptor de TNF
12.
J Biol Chem ; 288(10): 6998-7011, 2013 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-23293021

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas Reguladoras de Apoptose , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poliubiquitina/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Ubiquitina/metabolismo , Proteína com Valosina
13.
Mol Cell ; 48(4): 572-86, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23063525

RESUMO

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.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Células MCF-7 , Metilação , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Serina-Treonina Quinases , Especificidade por Substrato
14.
Biochem J ; 447(2): 313-20, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22849407

RESUMO

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.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Benzoquinonas/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Feminino , Células HEK293 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia
15.
Sci Signal ; 5(219): ra30, 2012 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-22494971

RESUMO

Although the guanosine triphosphate/guanosine diphosphate loading switch is a major regulatory mechanism that controls the activity of the guanosine triphosphatase Ras, we report a distinct mechanism for regulating Ras activity through phosphorylation-mediated degradation and describe the role of this second regulatory mechanism in the suppression of cellular transformation and tumors induced by Ras mutations. We found that negative regulators of Wnt/ß-catenin signaling contributed to the polyubiquitin-dependent degradation of Ras after its phosphorylation by glycogen synthase kinase 3ß (GSK3ß) and the subsequent recruitment of ß-TrCP-E3 ligase. We found a positive association between tumorigenesis and Ras stabilization resulting from the aberrant activation of Wnt/ß-catenin signaling in adenomas from two mouse models of colon cancer, human colonic tumors from various stages, and colon polyps of patients with familial adenomatous polyposis. Our results indicated that GSK3ß plays an essential role in Ras degradation and that inhibition of this degradation pathway by aberrant Wnt/ß-catenin signaling may contribute to Ras-induced transformation in colorectal tumorigenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias Intestinais/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas ras/metabolismo , Animais , Proliferação de Células , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Mutação , Fosforilação , Transdução de Sinais
16.
J Biol Chem ; 287(20): 16681-8, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22442153

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/fisiologia , Folículo Piloso/metabolismo , Proteínas de Homeodomínio/biossíntese , Queratinas/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Animais , Proteínas de Homeodomínio/genética , Queratinas/genética , Camundongos , Camundongos Pelados , Fatores de Transcrição/genética
17.
Biol Pharm Bull ; 35(3): 369-75, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22382323

RESUMO

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.


Assuntos
Fígado Gorduroso/metabolismo , Fenretinida/farmacologia , Hipoglicemiantes/farmacologia , Hipolipemiantes/farmacologia , Resistência à Insulina , Obesidade/metabolismo , Adiponectina/sangue , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Dieta Hiperlipídica , Fígado Gorduroso/patologia , Leptina/sangue , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/patologia , Pré-Albumina/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Aumento de Peso/efeitos dos fármacos
18.
J Biol Chem ; 287(15): 11891-8, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22334659

RESUMO

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.


Assuntos
Cromatina/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Processamento de Proteína Pós-Traducional , Origem de Replicação , Sequência de Aminoácidos , Sequência Conservada , Células HeLa , Humanos , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem/química , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Fase S
19.
J Biol Chem ; 287(12): 9269-79, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22275368

RESUMO

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.


Assuntos
Adenosina Trifosfatases/metabolismo , Nucleotídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
20.
Biochem Biophys Res Commun ; 415(4): 720-6, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-22085717

RESUMO

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis without affecting primary tumorigenesis. The regulatory mechanism of BRMS1 at the protein level has not been revealed until recently. Here, we found that cullin 3 (Cul3), a component of E3 ubiquitin ligase, is a new binding partner of BRMS1 and the interaction between BRMS1 and Cul3 is mediated by the SPOP adaptor protein. Intriguingly, BRMS1 turns out to be a potent substrate that is ubiquitinated by the Cul3-SPOP complex. Knockdown of SPOP increases the level of BRMS1 protein and represses the expression of BRMS1 repressive target genes such as OPN and uPA in breast cancer cells. These results suggest that the novel regulatory mechanism of BRMS1 by Cul3-SPOP complex is important for breast cancer progression.


Assuntos
Proteínas Culina/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas Culina/genética , Feminino , Células HEK293 , Humanos , Estabilidade Proteica , Ubiquitinação
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