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1.
J Cell Sci ; 128(9): 1707-17, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25770104

RESUMO

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Diferenciação Celular , Desenvolvimento Muscular , Animais , Apoptose , Hidrolases de Éster Carboxílico/deficiência , Caspase 3/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fibras Musculares Esqueléticas/patologia , Mioblastos/enzimologia , Mioblastos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/metabolismo , Transfecção
2.
Oncotarget ; 5(16): 6964-75, 2014 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-25026282

RESUMO

Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.


Assuntos
Clatrina/metabolismo , Endopeptidases/metabolismo , Receptores ErbB/metabolismo , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Endocitose , Endopeptidases/genética , Células HeLa , Humanos , Transfecção
3.
Biochem J ; 457(2): 289-300, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24134311

RESUMO

Processing of the 'CaaX' motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these 'CaaX' proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper 'CaaX' processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.


Assuntos
Membrana Celular/metabolismo , Endopeptidases/metabolismo , Endopeptidases/fisiologia , Genes ras/fisiologia , Membrana Celular/química , Células HEK293 , Células HeLa , Humanos , Isoformas de Proteínas/metabolismo , Proto-Oncogene Mas
4.
Small GTPases ; 2(4): 192-201, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22145091

RESUMO

The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.

5.
Nat Commun ; 2: 259, 2011 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-21448158

RESUMO

Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.


Assuntos
Movimento Celular/fisiologia , Endopeptidases/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Interleucina-8/metabolismo , Transporte Proteico , Proteínas rho de Ligação ao GTP/análise
6.
Cancer Res ; 70(8): 3329-39, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20388806

RESUMO

Ubiquitination is a reversible posttranslational modification that is essential for cell cycle control, and it is becoming increasingly clear that the removal of ubiquitin from proteins by deubiquitinating enzymes (DUB) is equally important. In this study, we have identified high levels of the DUB USP17 in several tumor-derived cell lines and primary lung, colon, esophagus, and cervix tumor biopsies. We also report that USP17 is tightly regulated during the cell cycle in all the cells examined, being abundantly evident in G(1) and absent in S phase. Moreover, regulated USP17 expression was necessary for cell cycle progression because its depletion significantly impaired G(1)-S transition and blocked cell proliferation. Previously, we have shown that USP17 regulates the intracellular translocation and activation of the GTPase Ras by controlling Ras-converting enzyme 1 (RCE1) activation. RCE1 also regulates the processing of other proteins with a CAAX motif, including Rho family GTPases. We now show that USP17 depletion blocks Ras and RhoA localization and activation. Moreover, our results confirm that USP17-depleted cells have constitutively elevated levels of the cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1), known downstream targets of Ras and RhoA signaling. These observations clearly show that USP17 is tightly regulated during cell division and that its expression is necessary to coordinate cell cycle progression, and thus, it may be considered a promising novel cancer therapeutic target.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Motivos de Aminoácidos , Biópsia , Ciclo Celular , Linhagem Celular Tumoral , Fase G1 , GTP Fosfo-Hidrolases/metabolismo , Inativação Gênica , Células HeLa , Humanos , Transporte Proteico , Fase S , Proteases Específicas de Ubiquitina
7.
J Biol Chem ; 285(16): 12028-36, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20147298

RESUMO

The proto-oncogenic Ras isoforms (H, N, and K) have a C-terminal CAAX motif and undergo the same post-translational processing steps, although they traffic to the plasma membrane through different routes. Previously, we have shown that overexpression of the deubiquitinating enzyme USP17 inhibits H-Ras localization to the plasma membrane. Now we report that whereas H-Ras and N-Ras were unable to localize to the plasma membrane in the presence of USP17, K-Ras4b localization was unaffected. EGF stimulation was unable to induce N-Ras membrane localization in USP17-expressing cells. In addition, N-Ras activity and downstream signaling through the MAPK MEK/ERK and PI3K/JNK pathways were blunted. However, we still detected abundant N-Ras localization at the ER and Golgi in USP17-expressing cells. Collectively, our data showed that the deubiquitinating enzyme USP17 blocks EGF-induced N-Ras membrane trafficking and activation, but left K-Ras unaffected.


Assuntos
Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Brefeldina A/farmacologia , Membrana Celular/metabolismo , Endopeptidases/genética , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Proteases Específicas de Ubiquitina
8.
J Biol Chem ; 284(14): 9587-95, 2009 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-19188362

RESUMO

The proto-oncogene Ras undergoes a series of post-translational modifications at its carboxyl-terminal CAAX motif that are essential for its proper membrane localization and function. One step in this process is the cleavage of the CAAX motif by the enzyme Ras-converting enzyme 1 (RCE1). Here we show that the deubiquitinating enzyme USP17 negatively regulates the activity of RCE1. We demonstrate that USP17 expression blocks Ras membrane localization and activation, thereby inhibiting phosphorylation of the downstream kinases MEK and ERK. Furthermore, we show that this effect is caused by the loss of RCE1 catalytic activity as a result of its deubiquitination by USP17. We also show that USP17 and RCE1 co-localize at the endoplasmic reticulum and that USP17 cannot block proliferation or Ras membrane localization in RCE1 null cells. These studies demonstrate that USP17 modulates Ras processing and activation, at least in part, by regulating RCE1 activity.


Assuntos
Endopeptidases/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Endopeptidases/deficiência , Endopeptidases/genética , Retículo Endoplasmático/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proto-Oncogene Mas , Proteases Específicas de Ubiquitina , Ubiquitinação , Proteínas ras/genética
9.
J Clin Invest ; 117(5): 1440-9, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17431503

RESUMO

The Fanconi anemia (FA) pathway maintains genomic stability in replicating cells. Some sporadic breast, ovarian, pancreatic, and hematological tumors are deficient in FA pathway function, resulting in sensitivity to DNA-damaging agents. FA pathway dysfunction in these tumors may result in hyperdependence on alternative DNA repair pathways that could be targeted as a treatment strategy. We used a high-throughput siRNA screening approach that identified ataxia telangiectasia mutated (ATM) as a critical kinase for FA pathway-deficient human fibroblasts. Human fibroblasts and murine embryonic fibroblasts deficient for the FA pathway were observed to have constitutive ATM activation and Fancg(-/-)Atm(-/-) mice were found to be nonviable. Abrogation of ATM function in FA pathway-deficient cells resulted in DNA breakage, cell cycle arrest, and apoptotic cell death. Moreover, Fanconi anemia complementation group G- (FANCG-) and FANCC-deficient pancreatic tumor lines were more sensitive to the ATM inhibitor KU-55933 than isogenic corrected lines. These data suggest that ATM and FA genes function in parallel and compensatory roles to maintain genomic integrity and cell viability. Pharmaceutical inhibition of ATM may have a role in the treatment of FA pathway-deficient human cancers.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/fisiologia , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/fisiologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Supressoras de Tumor/deficiência
10.
Mol Cancer Ther ; 5(4): 952-61, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16648566

RESUMO

Cisplatin resistance occurs, at least in part, through the function of the Fanconi anemia (FA)/BRCA pathway, a DNA-damage response pathway required for repair of cisplatin cross-links. In the current study, we designed a cell-based screening strategy to identify small-molecule inhibitors of the FA/BRCA pathway with the hypothesis that such molecules could restore sensitivity to platinum agents. We identified four inhibitors, including three protein kinase inhibitors (wortmannin, H-9, and alsterpaullone) and one natural compound (curcumin) that inhibit the FA/BRCA pathway. We show that curcumin, a compound that is generally regarded as safe, inhibits the monoubiquitination of the FANCD2 protein as predicted by the screen and consequently sensitizes ovarian and breast tumor cell lines to cisplatin through apoptotic cell death. We believe that this study shows an efficient, high-throughput method for identifying new compounds that may sensitize cancer cells to DNA-damaging chemotherapy.


Assuntos
Proteína BRCA1/fisiologia , Cisplatino/farmacologia , Curcumina/farmacologia , Androstadienos/farmacologia , Proteína BRCA1/efeitos dos fármacos , Benzazepinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Anemia de Fanconi/genética , Células HeLa , Humanos , Indóis/farmacologia , Isoquinolinas/farmacologia , Sulfonamidas/farmacologia , Wortmanina
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