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1.
Cell ; 148(5): 988-1000, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22385963

RESUMO

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.


Assuntos
Apoptose , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Esfingolipídeos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Feminino , Células HeLa , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Esfingomielina Fosfodiesterase/metabolismo
2.
EMBO J ; 40(16): e107901, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34169542

RESUMO

How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4+ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca2+ buffering in NKT2 cells. In addition, the low Ca2+ extrusion rate may also contribute to the higher Ca2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.


Assuntos
Cálcio/metabolismo , Diferenciação Celular , Células T Matadoras Naturais/metabolismo , Timo/citologia , Animais , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo
3.
J Biol Chem ; 298(4): 101835, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35304098

RESUMO

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed "minority MOMP." In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.


Assuntos
Apoptose , Caspases , Sobrevivência Celular , Mitocôndrias , Apoptose/fisiologia , Carcinogênese/genética , Carcinogênese/metabolismo , Caspases/metabolismo , Sobrevivência Celular/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo
4.
PLoS Biol ; 17(6): e2004413, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181072

RESUMO

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) ("intrabodies") and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Membranas Mitocondriais/fisiologia , Piruvato Quinase/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Isoformas de Proteínas/metabolismo , Piruvato Quinase/fisiologia , Transdução de Sinais , Anticorpos de Cadeia Única
5.
J Biol Chem ; 295(6): 1623-1636, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31901077

RESUMO

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.


Assuntos
Apoptose , Bicamadas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Humanos , Camundongos , Mitocôndrias/genética , Permeabilidade , Mutação Puntual , Multimerização Proteica , Proteína X Associada a bcl-2/análise , Proteína X Associada a bcl-2/genética
6.
Mol Cell ; 31(4): 557-569, 2008 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-18691924

RESUMO

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Citocromos c/metabolismo , Humanos , Leupeptinas/farmacologia , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/ultraestrutura , Proteínas Mutantes/metabolismo , Peptídeos/farmacologia , Permeabilidade/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/química
7.
PLoS Biol ; 10(9): e1001394, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23049480

RESUMO

Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for "intrinsic" apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the "hit-and-run" hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.


Assuntos
Biocatálise , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Biocatálise/efeitos dos fármacos , Dinamina I/metabolismo , Humanos , Cinética , Lipossomos/metabolismo , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Modelos Biológicos , Permeabilidade/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Quinazolinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Termodinâmica
8.
J Cell Biochem ; 115(4): 632-40, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24453042

RESUMO

Mitochondria play a critical role in apoptosis, or programmed cell death, by releasing apoptogenic factors from the intermembrane space. This process, known as mitochondrial outer membrane permeabilization (MOMP), is tightly regulated by the Bcl-2 family proteins. Pro-apoptotic Bcl-2 family members, Bax and Bak, change their conformation when activated by BH3 domain-only proteins in the family and permeabilize the MOM, whereas pro-survival members inhibit permeabilization. The precise nature of the apoptotic pore in the MOM is unknown, but is probably lipidic. Furthermore, it has been realized that there is another layer of MOMP regulation by a protein factor termed the catalyst in the MOM in order for Bax/Bak to achieve efficient and complete membrane permeabilization. Mitochondrial dynamics do not affect MOMP directly, but seem closely coordinated with MOMP for swift protein efflux from mitochondria. This review will present current views on the molecular mechanisms and regulation of MOMP and conclude with recent developments in clinical applications based on the knowledge gleaned from the investigation.


Assuntos
Apoptose/fisiologia , Membranas Mitocondriais/metabolismo , Terapia de Alvo Molecular/métodos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Humanos , Permeabilidade , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
9.
Dev Cell ; 14(2): 193-204, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18267088

RESUMO

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.


Assuntos
Dinaminas/antagonistas & inibidores , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Quinazolinonas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Dinaminas/ultraestrutura , Citometria de Fluxo , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Permeabilidade/efeitos dos fármacos , Quinazolinonas/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Relação Estrutura-Atividade
10.
J Biol Chem ; 286(1): 491-501, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21041309

RESUMO

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína 11 Semelhante a Bcl-2 , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Citocromos c/metabolismo , Células HEK293 , Humanos , Lipossomos/metabolismo , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína X Associada a bcl-2/química , Proteína bcl-X/metabolismo
11.
Curr Opin Cell Biol ; 15(6): 691-9, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14644193

RESUMO

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.


Assuntos
Apoptose , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Cálcio/química , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Potenciais da Membrana , Mitocôndrias/patologia , Permeabilidade , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
12.
Proc Natl Acad Sci U S A ; 105(51): 20327-32, 2008 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-19074266

RESUMO

Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity, as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 effector molecules, BAX and BAK. Sustainable cellular stress induces proteins (e.g., BID, BIM, and cytosolic p53) capable of directly activating BAX and/or BAK, but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g., BCL-2, BCL-xL, and MCL-1). In the event of accumulated or marked cellular stress, a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator protein-mediated MOMP. We examined the effect of ABT-737, a BCL-2 antagonist, and PUMA, a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire, with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis, which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data show that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires "covert" levels of direct activators of BAX and BAK at the OMM.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Células Cultivadas , Camundongos , Membranas Mitocondriais , Permeabilidade
13.
Methods Mol Biol ; 1877: 247-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536011

RESUMO

During apoptosis, mitochondria permeabilize the outer membranes to release apoptogenic proteins from the intermembrane space. This process termed mitochondrial outer membrane permeabilization (MOMP) is regulated by Bcl-2 family proteins. Bax is an effector proapoptotic molecule that permeabilizes the lipid membranes when it is activated by activator BH3-only proteins. We investigated this critical event by developing simple but faithful vesicle systems-outer membrane vesicles (OMVs) and liposomes-to visualize the pores in the membrane by using cryo-electron microscopy (cryo-EM). We have revealed the morphology of the pore, determined the localization of Bax labeled with nanogold and have performed image analysis to help understand the mechanisms of pore formation induced by Bax.


Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Microscopia Crioeletrônica/métodos , Humanos , Lipídeos de Membrana/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
14.
Sci Rep ; 6: 27299, 2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27255832

RESUMO

How the pro-apoptotic Bax protein permeabilizes the mitochondrial outer membrane is not fully understood. Previously, using cryo-electron microscopy (cryo-EM), we showed that activated Bax forms large, growing pores. Whether formed in liposomes or in mitochondrial outer membranes, Bax-induced pores exhibit the same morphology, with negative curvature flanking the edges and with no visible protein structure protruding from the membranes. Here we used cryo-EM to show that gold-labeled Bax molecules, after activation by Bid, became localized strictly at pore edges. This argues that Bax acts at short range to deform the membrane. Also, Bax molecules populated the walls of both small and large pores at the same density, implying that Bax is continuously recruited to the pores as they widen. Moreover, because all Bax molecules became oligomerized after membrane insertion, we infer that Bax oligomers are present at pore edges. We suggest that oligomerization may promote pore enlargement.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/fisiologia , Permeabilidade , Proteína X Associada a bcl-2/metabolismo , Microscopia Crioeletrônica
15.
Mol Biol Cell ; 26(2): 339-49, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25411335

RESUMO

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.


Assuntos
Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Proteínas de Xenopus/farmacologia , Proteína X Associada a bcl-2/farmacologia , Animais , Cromatografia em Gel , Microscopia Crioeletrônica , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Dextranos/química , Dextranos/metabolismo , Feminino , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Oócitos/metabolismo , Permeabilidade/efeitos dos fármacos , Porosidade , Proteínas Recombinantes/farmacologia , Xenopus , Proteínas de Xenopus/genética , Proteína X Associada a bcl-2/genética
16.
Clin Cancer Res ; 16(16): 4217-25, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20601444

RESUMO

PURPOSE: Inhibition of the antiapoptotic BCL2 family is one of the most promising areas of anticancer drug development. However, ABT-737, a specific BCL2 inhibitor, is neither orally bioavailable nor metabolically stable. To overcome these problems, the structurally related molecule ABT-263 was synthesized and recently entered clinical trials in hematologic malignancies, including chronic lymphocytic leukemia (CLL). Almost all laboratory studies have been carried out with ABT-737 rather than ABT-263, the drug being used in clinical trials. Currently there are no published data on the comparative effects of these inhibitors. To gain insight into the potential value or limitations of ABT-263 in the clinic, we assessed its ability to induce apoptosis in clinically relevant cellular models of CLL. EXPERIMENTAL DESIGN: The susceptibility of freshly isolated primary CLL cells to these inhibitors was compared in standard culture conditions and in conditions that more closely mimic in vivo conditions in a whole blood assay system. RESULTS: ABT-737 was more potent than ABT-263 at inducing apoptosis in CLL cells. In whole blood, approximately 100-fold higher concentrations of both drugs were required to induce apoptosis. We found that ABT-263 was highly bound by albumin and that an increased albumin binding of ABT-263 as compared with ABT-737 accounted for the differential sensitivity of CLL cells. CONCLUSIONS: Our data indicate that the exquisite in vitro sensitivity of CLL cells to BCL2 inhibitors may be lost in vivo due to high cell densities and the albumin binding of ABT-263. Modification of ABT-263 may yield a BCL2 inhibitor with greater bioavailability and more favorable pharmacokinetics.


Assuntos
Albuminas/metabolismo , Compostos de Anilina/farmacocinética , Antineoplásicos/farmacocinética , Compostos de Bifenilo/farmacocinética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Nitrofenóis/farmacocinética , Sulfonamidas/farmacocinética , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Humanos , Imunoprecipitação , Leucemia Linfocítica Crônica de Células B/metabolismo , Nitrofenóis/metabolismo , Nitrofenóis/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacocinética , Piperazinas/farmacologia , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia
17.
Mol Biol Cell ; 20(8): 2276-85, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19244344

RESUMO

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the "pores" and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25-100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/farmacologia , Lipídeos/biossíntese , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/farmacologia , Animais , Cardiolipinas/farmacologia , Microscopia Crioeletrônica , Lipídeos/isolamento & purificação , Membranas Mitocondriais/ultraestrutura , Permeabilidade/efeitos dos fármacos , Proteolipídeos/metabolismo , Lipossomas Unilamelares , Xenopus
18.
J Biol Chem ; 282(4): 2144-55, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17102131

RESUMO

Cytokine deprivation has been classically used to study molecular processes of apoptosis. Following interleukin (IL)-3 withdrawal in FL5.12 cells, Bax undergoes a conformational change that results in its mitochondria targeting, cytochrome c release, activation of caspase-9, and apoptosis. Cells overexpressing Casp9DN (dominant negative caspase-9) or treated with the caspase inhibitor Q-VD-OPh increased viability but failed to increase clonogenic survival. We find that caspase-inhibited cells had a significant fraction of viable cells (herein termed "rescued" cells) that failed to initiate cell division after IL-3 add back. The "rescued" cells had reduced mitochondrial potential, stained for active Bax, and had reduced staining with dihydroethidium, an agent sensitive to superoxide levels. Readdition of IL-3 after deprivation demonstrated that Bax activation was reversed, whereas altered 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and dihydroethidium staining persisted for days. Furthermore, the "rescued" cells were resistant to rotenone, an inhibitor of mitochondrial respiration. The cells were highly sensitive to 2-deoxyglucose, an inhibitor of glycolysis and proposed anti-cancer agent. We conclude that the inhibition of caspase-9 allows cells to retain viability, but cells have prolonged mitochondrial dysfunction and enter a unique nondividing state that shares some properties with malignant cells.


Assuntos
Apoptose , Linfócitos B/fisiologia , Inibidores de Caspase , Ciclo Celular , Interleucina-3 , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Grupo dos Citocromos c/fisiologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hematopoese , Sistema Hematopoético/fisiologia , Humanos , Interleucina-3/deficiência , Interleucina-3/farmacologia , Camundongos , Quinolinas/farmacologia , Proteína X Associada a bcl-2/fisiologia
19.
Science ; 309(5741): 1732-5, 2005 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-16151013

RESUMO

The Trp53 tumor suppressor gene product (p53) functions in the nucleus to regulate proapoptotic genes, whereas cytoplasmic p53 directly activates proapoptotic Bcl-2 proteins to permeabilize mitochondria and initiate apoptosis. Here, we demonstrate that a tripartite nexus between Bcl-xL, cytoplasmic p53, and PUMA coordinates these distinct p53 functions. After genotoxic stress, Bcl-xL sequestered cytoplasmic p53. Nuclear p53 caused expression of PUMA, which then displaced p53 from Bcl-xL, allowing p53 to induce mitochondrial permeabilization. Mutant Bcl-xL that bound p53, but not PUMA, rendered cells resistant to p53-induced apoptosis irrespective of PUMA expression. Thus, PUMA couples the nuclear and cytoplasmic proapoptotic functions of p53.


Assuntos
Apoptose , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Biológicos , Permeabilidade , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/química , Raios Ultravioleta , Proteína X Associada a bcl-2 , Proteína bcl-X
20.
Mol Cell ; 17(4): 525-35, 2005 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-15721256

RESUMO

Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.


Assuntos
Apoptose , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteína 11 Semelhante a Bcl-2 , Proteínas de Transporte/metabolismo , Citocromos c/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Permeabilidade , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Homologia de Sequência de Aminoácidos , Proteína X Associada a bcl-2 , Proteína bcl-X
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