RESUMO
The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Motivos de Aminoácidos , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/química , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Mutação , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The human adenovirus E4orf4 protein is toxic in both human tumor cells and Saccharomyces cerevisiae. Previous studies indicated that most of this toxicity is dependent on an interaction of E4orf4 protein with the B55 class of regulatory subunits of protein phosphatase 2A (PP2A) and in yeast with the B55 homolog Cdc55. We have found previously that E4orf4 inhibits PP2A activity against at least some substrates. In an attempt to understand the mechanism of this inhibition, we used a genetic approach to identify residues in the seven-bladed ß-propeller proteins B55α and Cdc55 required for E4orf4 binding. In both cases, amino-terminal polypeptides composed only of blade 1 and at least part of blade 2 were found to bind E4orf4 and overexpression blocked E4orf4 toxicity in yeast. Furthermore, certain amino acid substitutions in blades 1 and 2 within full-length B55α and Cdc55 resulted in loss of E4orf4 binding. Recent mutational analysis has suggested that segments of blades 1 and 2 present on the top face of B55α form part of the "substrate-binding groove." Additionally, these segments are in close proximity to the catalytic C subunit of the PP2A holoenzyme. Thus, our results are consistent with the hypothesis that E4orf4 binding could affect the access of substrates, resulting in the failure to dephosphorylate some PP2A substrates.
Assuntos
Proteínas de Ciclo Celular/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Virais/metabolismo , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genéticaRESUMO
SELEX was used to create an RNA aptamer targeted to protein tyrosine phosphatase 1B (PTP1B), an enzyme implicated in type 2 diabetes, breast cancer and obesity. We found an aptamer that strongly inhibits PTP1B in vitro with a Ki of less than 600 pM. This slow-binding, high-affinity inhibitor is also highly selective, with no detectable effect on most other tested phosphatases and approximately 300:1 selectivity over the closely related TC-PTP. Through controlled synthesis of truncated variants of the aptamer, we isolated shorter forms that inhibit PTP1B. We also investigated various single-nucleotide modifications to probe their effects on the aptamer's secondary structure and inhibition properties. This family of aptamers represents an exciting option for the development of lead nucleotide-based compounds in combating several human cancers and metabolic diseases.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/uso terapêutico , Humanos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirróis/farmacologia , Animais , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Indóis , Melanoma/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.
Assuntos
Apoptose , Cálcio/metabolismo , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Adenoviridae/genética , Animais , Células CHO , Linhagem Celular , Cricetinae , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citosol/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP20 , Células HeLa , Humanos , Proteínas de Membrana/genética , Modelos Biológicos , Proteínas Musculares/metabolismo , Ratos , Transdução de Sinais , Células Tumorais Cultivadas , Utrofina , Receptor fas/metabolismoRESUMO
Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens.
Assuntos
Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Descoberta de Drogas , Feminino , Humanos , Camundongos , Morfogênese , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Organogênese , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidoresRESUMO
A DNA microarray analysis identified the BH3-only BCL-2 family member, BIK/NBK, as a transcript that is upregulated during induction of apoptosis by oncogenic E1A. E1A depended on wild-type p53 to induce BIK and activate the death program. Further, p53 independently induced BIK RNA and protein, and BIK alone stimulated cell death in p53-null cells, dependent on the activation of caspases. BIK function, however, was abrogated by a disabling point mutation within the BH3 domain. Collectively, these results argue that BIK is a downstream apoptotic effector of p53 in response to a physiological p53-mediated death stimulus provided by E1A. Elevated BCL-2 functioned downstream of p53 and BIK induction to inhibit the E1A death pathway, with the ratio of anti-apoptotic BCL-2 and pro-apoptotic BIK determining cell death or survival in E1A-expressing cells. Cells expressing BCL-2 or treated with the pan caspase inhibitor, zVAD-fmk, allowed accumulation of high levels of cytotoxic BIK compared to control cells. Of note, a significant fraction of either ectopic or endogenous BIK was found associated with the endoplasmic reticulum, suggesting that this organelle, in addition to mitochondria, may be a target of BIK function.
Assuntos
Proteínas E1A de Adenovirus/farmacologia , Apoptose , Retículo Endoplasmático/química , Proteínas de Membrana , Biossíntese de Proteínas , Proteínas/análise , Proteína Supressora de Tumor p53/fisiologia , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Caspases/fisiologia , Linhagem Celular , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Vetores Genéticos , Humanos , Proteínas Mitocondriais , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/análise , Alinhamento de Sequência , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
The "BH3-only" proapoptotic BCL-2 family members are sentinels of intracellular damage. Here, we demonstrated that the BH3-only BID protein partially localizes to the nucleus in healthy cells, is important for apoptosis induced by DNA damage, and is phosphorylated following induction of double-strand breaks in DNA. We also found that BID phosphorylation is mediated by the ATM kinase and occurs in mouse BID on two ATM consensus sites. Interestingly, BID-/- cells failed to accumulate in the S phase of the cell cycle following treatment with the topoisomerase II poison etoposide; reintroducing wild-type BID restored accumulation. In contrast, introducing a nonphosphorylatable BID mutant did not restore accumulation in the S phase and resulted in an increase in cellular sensitivity to etoposide-induced apoptosis. These results implicate BID as an ATM effector and raise the possibility that proapoptotic BID may also play a prosurvival role important for S phase arrest.
Assuntos
Apoptose/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Sítios de Ligação/fisiologia , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Etoposídeo/farmacologia , Fibroblastos/metabolismo , Genes cdc/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/fisiologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fase S/efeitos dos fármacos , Fase S/fisiologia , Inibidores da Topoisomerase II , Proteínas Supressoras de Tumor/genéticaRESUMO
The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, expression of non-apoptotic levels of either ncBID or wtBID in Bid -/- MEFs induced a similar and significant enhancement in apoptosis in response to a variety of death signals, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.