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1.
J Ovarian Res ; 12(1): 45, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092272

RESUMO

BACKGROUND: While tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8). RESULTS: Our mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect. CONCLUSIONS: The results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.


Assuntos
Mitocôndrias/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular , Linhagem Celular Tumoral , Feminino , Humanos , Mutação , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Domínios Proteicos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
2.
Environ Sci Pollut Res Int ; 26(15): 15209-15217, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30924043

RESUMO

Breast cancer is a global public health problem where it is the second most prevalent cancer. Historical cancer treatment with graviola has been reported. This study aimed to investigate the protective effects of graviola on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast cancer. Fifty female Wistar rats were allocated into four groups: control group (gastro-gavaged by sesame oil), DMBA-treated group (gastro-gavaged a single dose of DMBA [50 mg/kg body mass, diluted in 1 ml sesame oil]) at the age 57 days, DMBA+G37-treated group (gastro-gavaged a single dose of DMBA [50 mg/kg body mass, diluted in 1 ml sesame oil]) at the age of 57 days plus graviola (200 mg/kg body mass) two times weekly (p.o.) at the age of 37 days till the end of the experiment, and DMBA+G57-treated group (received a single dose of DMBA [50 mg/kg body mass, diluted in 1 ml sesame oil]) plus graviola (200 mg/kg body mass) two times weekly at the age of 57 days until the end of the experiment. After the 30-week experimental period, blood samples were collected. Then, animals were sacrificed to determine the apoptotic indices, antioxidant status, and mammary gland tumor marker (CA 15-3). The DMBA upregulated the expression of one of the main anti-apoptotic genes: B-cell lymphoma protein 2 (BCL2) and estrogen receptor alpha (ER-α) gene. Moreover, it significantly increased breast lipid peroxidation and serum CA 15-3 but decreased breast antioxidant enzymatic activities (glutathione peroxidase, glutathione S-transferase, catalase, and superoxide dismutase). Nevertheless, administration of DMBA and graviola especially DMBA+G37 induced apoptosis through at least 1.5-fold in gene expression levels of pro-apoptotic genes: BCL2-associated X protein (BAX), tumor suppressor gene (P53), and cysteinyl-aspartic acid-protease-3 (caspase-3). A critical role of P53 in the regulation of the BCL2 and BAX has been reported. These proteins can determine if the cell undergoes apoptosis or cancels the process. Once the BAX gene activates caspase-3, there is no irreversible way toward cell death. Also, graviola ameliorated the DMBA effects on antioxidant enzymatic activities and tumor marker CA 15-3. This study concludes that graviola ameliorated DMBA-induced breast cancer potentially through upregulating apoptotic genes, downregulating the ER-α gene, increasing antioxidants, and decreasing lipid peroxidation levels.


Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/induzido quimicamente , Caspase 3/metabolismo , Catalase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Receptores Estrogênicos/metabolismo , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/genética , 9,10-Dimetil-1,2-benzantraceno/química , 9,10-Dimetil-1,2-benzantraceno/metabolismo , Animais , Antioxidantes/química , Caspase 3/química , Catalase/química , Feminino , Glutationa Peroxidase/química , Peroxidação de Lipídeos , Ratos , Ratos Wistar , Superóxido Dismutase/química , Proteína X Associada a bcl-2/química
3.
Methods Mol Biol ; 1886: 191-202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30374868

RESUMO

Atomic force microscopy (AFM) is a form of contact microscopy that uses a very sharp tip to scan the surface of a sample. It provides a 3D image of the surface structure and in the force mode it can also be used to test the mechanical properties of the sample. AFM has been successfully applied to study the molecular mechanism of pore-forming proteins on model membranes. It gives information about both the structural reorganization of the membrane surface and the changes in the force required for membrane piercing upon incubation with this special type of proteins. Here we describe robust protocols to investigate the effect of pore-forming proteins in supported lipid bilayers .


Assuntos
Microscopia de Força Atômica , Porinas , Bicamadas Lipídicas/química , Microscopia de Força Atômica/métodos , Porinas/química , Porinas/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Proteína X Associada a bcl-2/química
4.
Cell Death Differ ; 25(10): 1717-1731, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30185826

RESUMO

Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.


Assuntos
Proteína X Associada a bcl-2/metabolismo , Dimerização , Humanos , Membranas Mitocondriais/metabolismo , Conformação Proteica em alfa-Hélice , Estrutura Terciária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
5.
Structure ; 26(10): 1346-1359.e5, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30122452

RESUMO

BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.


Assuntos
Mutação , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Regulação Alostérica , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citosol/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Humanos , Ictaluridae/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Proteína X Associada a bcl-2/genética
6.
Protein J ; 37(5): 428-443, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30128635

RESUMO

The B cell lymphoma 2 (BCL2) proteins are a family of evolutionarily related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Overexpression of anti-apoptotic BCL2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes and pathogenic cell survival. Consequently, anti-apoptotic BCL2 proteins have attracted considerable interest as therapeutic targets. We recently reported the development of a novel class of synthetic protein based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic BCL2 protein Bax. These studies showed that the number and position of native disulfide linkages contained within the ScTx-Bax structure significantly influences the ability for these constructs to target anti-apoptotic BCL2 proteins in vitro. The goal of the present study is to investigate the contribution of two disulfide linkages in the folding and biological activity of ScTx-Bax proteins. Here, we report the full chemical synthesis of three ScTx-Bax sequence variants, each presenting two native disulfide linkages at different positions within the folded structure. It was observed that two disulfide linkages were sufficient to fold ScTx-Bax proteins into native-like architectures reminiscent of wild-type ScTx. Furthermore, we show that select (bis)disulfide ScTx-Bax variants can target Bcl-2 (proper) in vitro and that the position of the disulfide bonds significantly influences binding affinity. Despite exhibiting only modest binding to Bcl-2, the successful synthesis of ScTx-Bax proteins containing two disulfide linkages represents a viable route to ScTx-based BH3 domain mimetics that preserve native-like conformations. Finally, structural models of ScTx-Bax proteins in complex with Bcl-2 indicate that these helical mimetics bind in similar configurations as wild-type Bax BH3 domains. Taken together, these results suggest that ScTx-Bax proteins may serve as potent lead compounds that expand the repertoire of "druggable" protein-protein interactions.


Assuntos
Dissulfetos/química , Proteínas Recombinantes de Fusão , Venenos de Escorpião , Proteína X Associada a bcl-2 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Venenos de Escorpião/biossíntese , Venenos de Escorpião/química , Venenos de Escorpião/genética , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
7.
Cell Physiol Biochem ; 48(2): 692-704, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30025410

RESUMO

BACKGROUND/AIMS: Myocardial apoptosis plays an important role in doxorubicin (Dox) cardiotoxicity. MicroRNA-29 (miR-29) is suggested to function as an anti-fibrotic factor with potential therapeutic effects on cardiac fibrosis. However, it has not been shown whether there is an association between miR-29b and myocardial apoptosis. METHODS: Male Wistar rats were transfected with miR-29b agomir by local delivery to the myocardium prior to Dox treatment. Rat cardiomyocytes were pretreated with miR-29b mimics or inhibitor followed by Dox incubation in vitro. Cardiac function and underlying mechanisms were evaluated by echocardiography, immunofluorescence, flow cytometry, real-time PCR, and western blotting. RESULTS: Our results revealed that miR-29b is the only member of the miR-29 family that was significantly downregulated in myocardium from Dox-treated rats. Delivery of miR-29b agomir to myocardium resulted in a marked improvement of cardiac function. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining showed that rescue of miR-29b expression inhibited Dox-induced myocardial apoptosis, concomitantly with increased Bcl-2 expression and decreased Bax expression and caspase-3 activity. In vitro, miR-29b overexpression mitigated, whereas inhibition of miR-29b promoted, Dox-induced cardiomyocyte apoptosis. Mechanistically, miR-29b negatively regulated Bax expression by directly targeting the 3' untranslated region of Bax. In Dox-treated cardiomyocytes, upregulation of miR-29b resulted in a significant decrease in Bax expression, with an increase in Bcl-2 expression, accompanied by inhibition of mitochondrial membrane depolarization, cytochrome c release, and caspase activation. However, inhibition of miR-29b produced the opposite effects by further augmenting the effects of Dox. CONCLUSIONS: These data demonstrate that miR-29b prevents Dox-induced myocardial apoptosis through inhibition of the mitochondria-dependent pathway by directly targeting Bax, suggesting that miR-29b is a potential novel therapeutic target for the treatment of Dox cardiotoxicity.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Doxorrubicina/toxicidade , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Proteína X Associada a bcl-2/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Sequência de Bases , Caspase 3/metabolismo , Caspase 9/metabolismo , Coração/efeitos dos fármacos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Mitocôndrias/efeitos dos fármacos , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Alinhamento de Sequência , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
8.
Biochim Biophys Acta Mol Cell Res ; 1865(5): 684-694, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29470995

RESUMO

MOAP-1 is a pro-apoptotic tumor suppressor molecule with a growing set of known interacting partners. We have demonstrated that during death receptor-dependent apoptosis, MOAP-1 is recruited to TNF-R1 or TRAIL-R1, followed by RASSF1A and Bax association. MOAP-1/Bax association promotes Bax conformational change resulting in the translocation of Bax into the mitochondrial membrane, mitochondrial membrane insertion and dysregulation resulting in several hallmark events that execute apoptosis. Although a role in apoptosis is established, it is currently unknown how MOAP-1 is regulated and how it links to Bax to promote apoptosis. In this study, we demonstrate robust association with RACK1, a versatile scaffolding protein that responds to activation of protein kinase C. Furthermore, we can demonstrate that RACK1 functions to bring the E3 ligase, TRAF2, to MOAP-1 in order to undergo a K63-dependent ubiquitination. Furthermore, RACK1 associates with MOAP-1 via electrostatic associations similar to those observed between MOAP-1/RASSF1A and MOAP-1/TNF-R1. These events illustrate the complex nature of MOAP-1 regulation and characterizes the important role of the scaffolding protein, RACK1, in influencing MOAP-1 biology.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteínas de Neoplasias/genética , Receptores de Quinase C Ativada/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Reguladoras de Apoptose/química , Humanos , Células Jurkat , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Morte Celular/química , Receptores de Morte Celular/genética , Receptores Tipo I de Fatores de Necrose Tumoral/química , Eletricidade Estática , Fator 2 Associado a Receptor de TNF/química , Fator 2 Associado a Receptor de TNF/genética , Proteínas Supressoras de Tumor/química , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
9.
J Biomed Opt ; 23(1): 1-10, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29313324

RESUMO

Based on our recently developed quantitative fluorescence resonance energy transfer (FRET) measurement method using simultaneous spectral unmixing of excitation and emission spectra (ExEm-spFRET), we here set up an improved spectrometer-microscope (SM) for implementing modified ExEm-spFRET (mExEm-spFRET), in which a system correction factor (fsc) is introduced. Our SM system is very stable for at least six months. Implementation of mExEm-spFRET with four or two excitation wavelengths on SM for single living cells expressing different FRET constructs obtained consistent FRET efficiency (E) and acceptor-donor concentration ratio (Rc) values. We also performed mExEm-spFRET measurement for single living cells coexpressing cyan fluorescent protein (CFP)-Bax and yellow fluorescent protein (YFP)-Bax and found that the E values between CFP-Bax and YFP-Bax were very low (2.2%) and independent of Rc for control cells, indicating that Bax did not exist as homooligomer in healthy cells, but positively proportional to Rc in the case of Rc<1 and kept constant value (25%) when Rc>1 for staurosporine (STS)-treated cells, demonstrating that all Bax formed homooligomer after STS treatment for 6 h.


Assuntos
Transferência Ressonante de Energia de Fluorescência/instrumentação , Microscopia de Fluorescência/instrumentação , Análise de Célula Única/instrumentação , Desenho de Equipamento , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Fotodegradação , Multimerização Proteica , Proteína X Associada a bcl-2/análise , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo
10.
Int J Biochem Cell Biol ; 95: 35-42, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29233735

RESUMO

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25-/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting.


Assuntos
Apoptose , Citosol/metabolismo , Mitocôndrias/metabolismo , Acetiltransferase N-Terminal B/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína X Associada a bcl-2/metabolismo , Acetilação , Animais , Células Cultivadas , Cruzamentos Genéticos , Citosol/enzimologia , Embrião de Mamíferos/citologia , Deleção de Genes , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/enzimologia , Acetiltransferase N-Terminal B/genética , Conformação Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Especificidade por Substrato , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
11.
FEBS J ; 285(3): 416-431, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28755482

RESUMO

Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Animais , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/química , Dimerização , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Mobilização Lipídica , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Porosidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/agonistas , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo
12.
J Biomol Struct Dyn ; 36(6): 1637-1648, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28511583

RESUMO

B-cell lymphoma 2 (Bcl-2) family proteins are the central regulators of apoptosis, functioning via mitochondrial outer membrane permeabilization. The family members are involved in several stages of apoptosis regulation. The overexpression of the anti-apoptotic proteins leads to several cancer pathological conditions. This overexpression is modulated or inhibited by heterodimerization of pro-apoptotic BH3 domain or BH3-only peptides to the hydrophobic groove present at the surface of anti-apoptotic proteins. Additionally, the heterodimerization displayed differences in binding affinity profile among the pro-apoptotic peptides binding to anti-apoptotic proteins. In light of discovering the novel peptide/drug molecules that contain the potential to inhibit specific anti-apoptotic protein, it is necessary to understand the molecular basis of recognition between the protein and its binding partner (peptide or ligand) along with its binding energies. Therefore, the present work focused on deciphering the molecular basis of recognition between pro-apoptotic Bak peptide binding to different anti-apoptotic (Bcl-xL, Bfl-1, Bcl-W, Mcl-1, and Bcl-2) proteins using advanced Molecular Dynamics (MD) approach such as Molecular Mechanics-Generalized Born Solvent Accessible. The results from our investigation revealed that the predicted binding free energies showed excellent correlation with the experimental values (r2 = .95). The electrostatic (ΔGele) contributions are the major component that drives the interaction between Bak peptides and different anti-apoptotic peptides. Additionally, van der Waals (ΔGvdw) energies also play an indispensible role in determining the binding free energy. Furthermore, the decomposition analysis highlighted the comprehensive information about the energy contributions of hotspot residues involved in stabilizing the interaction between Bak peptide and different anti-apoptotic proteins.


Assuntos
Proteínas Reguladoras de Apoptose/química , Apoptose/fisiologia , Peptídeos/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína X Associada a bcl-2/química
13.
Proc Natl Acad Sci U S A ; 114(51): 13453-13458, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29208709

RESUMO

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.


Assuntos
Sequência Conservada , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Evolução Molecular , Humanos , Lisina/genética , Lisina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Ligação Proteica , Serina/genética , Serina/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
14.
Cancer Cell ; 32(4): 490-505.e10, 2017 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-29017059

RESUMO

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.


Assuntos
Apoptose/efeitos dos fármacos , Hidrazonas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Tiazóis/farmacologia , Proteína X Associada a bcl-2/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Conformação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/química
15.
J Cell Sci ; 130(17): 2903-2913, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28760928

RESUMO

The pro-apoptotic BCL-2 protein BAX commits human cells to apoptosis by permeabilizing the outer mitochondrial membrane. BAX activation has been suggested to require the separation of helix α5 from α6 - the 'latch' from the 'core' domain - among other conformational changes. Here, we show that conformational changes in this region impair BAX translocation to the mitochondria and retrotranslocation back into the cytosol, and therefore BAX inhibition, but not activation. Redirecting misregulated BAX to the mitochondria revealed an alternative mechanism of BAX inhibition. The E3 ligase parkin, which is known to trigger mitochondria-specific autophagy, ubiquitylates BAX K128 and targets the pro-apoptotic BCL-2 protein for proteasomal degradation. Retrotranslocation-deficient BAX is completely degraded in a parkin-dependent manner. Although only a minor pool of endogenous BAX escapes retrotranslocation into the cytosol, parkin-dependent targeting of misregulated BAX on the mitochondria provides substantial protection against BAX apoptotic activity.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Citoproteção , Células HCT116 , Humanos , Lisina/metabolismo , Mitocôndrias/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Ubiquitinação , Proteína X Associada a bcl-2/química
16.
Exp Cell Res ; 359(2): 342-355, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28807790

RESUMO

Bax∆2 is a functional pro-apoptotic Bax isoform having alterations in its N-terminus, but sharing the rest of its sequence with Baxα. Bax∆2 is unable to target mitochondria due to the loss of helix α1. Instead, it forms cytosolic aggregates and activates caspase 8. However, the functional domain(s) responsible for BaxΔ2 behavior have remained elusive. Here we show that disruption of helix α1 makes Baxα mimic the behavior of Bax∆2. However, the other alterations in the Bax∆2 N-terminus have no significant impact on aggregation or cell death. We found that the hallmark BH3 domain is necessary but not sufficient for aggregation-mediated cell death. We also noted that the core region shared by Baxα and Bax∆2 is required for the formation of large aggregates, which is essential for BaxΔ2 cytotoxicity. However, aggregation by itself is unable to trigger cell death without the C-terminus. Interestingly, the C-terminal helical conformation, not its primary sequence, appears to be critical for caspase 8 recruitment and activation. As Bax∆2 shares core and C-terminal sequences with most Bax isoforms, our results not only reveal a structural basis for Bax∆2-induced cell death, but also imply an intrinsic potential for aggregate-mediated caspase 8-dependent cell death in other Bax family members.


Assuntos
Sequência de Aminoácidos , Caspase 8/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Deleção de Sequência , Proteína X Associada a bcl-2/química , Sítios de Ligação , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular , Clonagem Molecular , Expressão Gênica , Células HCT116 , Humanos , Modelos Moleculares , Agregados Proteicos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
17.
Nat Commun ; 8(1): 73, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28706229

RESUMO

The Bcl-2 proteins form a complex interaction network that controls mitochondrial permeabilization and apoptosis. The relative importance of different Bcl-2 complexes and their spatio-temporal regulation is debated. Using fluorescence cross-correlation spectroscopy to quantify the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, we show that membrane insertion drastically alters the pattern of Bcl-2 complexes, and that the C-terminal helix of Bcl-xL determines its binding preferences. At physiological temperature, Bax can spontaneously activate in a self-amplifying process. Strikingly, Bax also recruits Bcl-xL to membranes, which is sufficient to retrotranslocate Bax back into solution to secure membrane integrity. Our study disentangles the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.The permeabilization of the mitochondrial outer membrane to induce apoptosis is regulated by complex interactions between Bcl-2 family members. Here the authors develop a quantitative interactome of a membrane Bcl-2 network and identify a hierarchy of protein complexes in apoptosis induction.


Assuntos
Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose , Membrana Celular , Humanos , Camundongos , Modelos Químicos , Ligação Proteica , Lipossomas Unilamelares/química
18.
Nat Chem Biol ; 13(9): 961-967, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28692068

RESUMO

BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.


Assuntos
Éteres Fenílicos/farmacologia , Proteína X Associada a bcl-2/química , Apoptose , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Éteres Fenílicos/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2
19.
Structure ; 25(8): 1310-1316.e3, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28712810

RESUMO

Bax is known for its pro-apoptotic role within the mitochondrial pathway of apoptosis. However, the mechanism for transitioning Bax from cytosolic to membrane-bound oligomer remains elusive. Previous nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) studies defined monomeric Bax as conformationally homogeneous. Yet it has recently been proposed that monomeric Bax exists in equilibrium with a minor state that is distinctly different from its NMR structure. Here, we revisited the structural analysis of Bax using methods uniquely suited for unveiling "invisible" states of proteins, namely, NMR paramagnetic relaxation enhancements and EPR double electron-electron resonance (DEER). Additionally we examined the effect of glycerol, the co-solvent of choice in DEER studies, on the structure of Bax using NMR chemical-shift perturbations and residual dipolar couplings. Based on our combined NMR and EPR results, Bax is a conformationally homogeneous protein prior to its activation.


Assuntos
Proteína X Associada a bcl-2/química , Glicerol/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica
20.
Sci Rep ; 7(1): 2635, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28572603

RESUMO

How cytochrome C is released from the mitochondria to the cytosol via Bax oligomeric pores, a process which is required for apoptosis, is still a mystery. Based on experimentally measured residue-residue distances, we recently solved the first atomic model for Bax oligomeric pores at the membranes using computational approaches. Here, we investigate the mechanism at the microsecond time- and nanometer space- scale using MD simulations. Our free energy landscape depicts a low barrier for the permeation of cytochrome C into the Bax C-terminal mouth, with the pathway proceeding to the inner cavity and exiting via the N-terminal mouth. Release is guided by organized charged/hydrophilic surfaces. The hydrophilicity and negative charge of the pore surface gradually increase along the release pathway from the pore entry to the exit opening. Rather than inert passing of the cytochrome C through a rigid pore, the flexible pore may selectively aid the cytochrome C passage. Once the Bax pore is formed in the membrane, with a low energy barrier, the release of cytochrome C may be readily achieved through energy fluctuations. Collectively, our work provides mechanistic insight in atomic detail into the release of cytochrome C through Bax oligomeric pores.


Assuntos
Citocromos c/química , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Apoptose , Modelos Biológicos , Simulação de Dinâmica Molecular , Propriedades de Superfície
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