RESUMO
Current models of apoptosis regulation by the Bcl-2 family of proteins postulate that heterodimeric interactions between family members determine whether Bax and Bak are activated to trigger cell death. Thus, the relative abundance and binding affinities between pro- and anti-apoptotic proteins determines the outcome of these interactions. Examination of these interactions using purified mitochondria and liposomes with full-length recombinant proteins revealed that Bcl-xL inhibits apoptosis as a higher-order complex that binds multiple BH3 proteins. Allosteric regulation of this complex by the BH3 sensitizer Bad confers switch-like activity to the indirect activation of Bax. The BH3 activator cBid sequestered by Bcl-xL complexes changes from an inactive to an active form while bound to a Bcl-xL complex only when Bad is also bound. Bcl-xL complexes enable Bad to function as a non-competitive inhibitor of Bcl-xL and allosterically activate cBid, dramatically enhancing the pro-apoptotic potency of Bad.
Assuntos
Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Regulação Alostérica , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , Humanos , Camundongos , Membranas Mitocondriais/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/químicaRESUMO
Apoptosis is a highly regulated form of cell death that controls normal homeostasis as well as the antitumor activity of many chemotherapeutic agents. Commitment to death via the mitochondrial apoptotic pathway requires activation of the mitochondrial pore-forming proteins BAK or BAX. Activation can be effected by the activator BH3-only proteins BID or BIM, which have been considered to be functionally redundant in this role. Herein, we show that significant activation preferences exist between these proteins: BID preferentially activates BAK while BIM preferentially activates BAX. Furthermore, we find that cells lacking BAK are relatively resistant to agents that require BID activation for maximal induction of apoptosis, including topoisomerase inhibitors and TRAIL. Consequently, patients with tumors that harbor a loss of BAK1 exhibit an inferior response to topoisomerase inhibitor treatment in the clinic. Therefore, BID and BIM have nonoverlapping roles in the induction of apoptosis via BAK and BAX, affecting chemotherapy response.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas/genética , Inibidores da Topoisomerase/administração & dosagem , Ativação Transcricional/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
After exposure to stressful stimuli, apoptotic signals can be relayed to mitochondria by pro-apoptotic activator proteins, tBid (truncated Bid/p15) and Bim (Bcl-2 interacting mediator), which activate Bax (Bcl-2 associated X protein) and or Bak (Bcl-2 antagonist/killer) to induce mitochondrial outer membrane (MOM) permeabilization (MOMP). These protein-protein and protein-membrane interactions are critical for apoptosis regulation, since MOMP irreversibly leads to cell death. Whereas the distinct roles of tBid and Bim as sensors of different types of stress are well recognized, it is not known whether the molecular mechanisms whereby they initiate MOMP are the same. In the present study, we compare membrane permeabilization by Bax activated by either cBid [cleaved Bid (p7 and p15)] or Bim and we examine the role of membrane lipids in the recruitment and activation of these three Bcl-2 (B-cell lymphoma 2) pro-apoptotic proteins. We employ fluorescently-labelled proteins and liposomes to quantify the effects of specific lipids on each of the well-characterized steps in Bax-mediated membrane permeabilization. We show that high levels of cholesterol in the membrane inhibit permeabilization by categorically identifying the recruitment of Bax by the activators and Bax insertion in the membrane as the steps being hindered by cholesterol. Furthermore, we show that binding of both cBid and Bim to membranes is facilitated by electrostatic interactions with anionic phospholipids. However, whereas Bim does not require any particular anionic lipids, the conformational change in tBid depends on cardiolipin (CL). This suggests that CL can activate tBid in a similar manner to Mtch2 (mitochondrial carrier homologue 2). Thus, lipids modify multiple aspects of Bax-mediated membrane permeabilization.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Cardiolipinas/metabolismo , Colesterol/metabolismo , Humanos , Lipossomos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Permeabilidade , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Bcl-2 family proteins are central regulators of apoptosis. Various family members are located in the cytoplasm, endoplasmic reticulum, and mitochondrial outer membrane in healthy cells. However during apoptosis most of the interactions between family members that determine the fate of the cell occur at the membranes of intracellular organelles. It has become evident that interactions with membranes play an active role in the regulation of Bcl-2 family protein interactions. Here we provide an overview of various models proposed to explain how the Bcl-2 family regulates apoptosis and discuss how membrane binding affects the structure and function of each of the three categories of Bcl-2 proteins (pro-apoptotic, pore-forming, and anti-apoptotic). We also examine how the Bcl-2 family regulates other aspects of mitochondrial and ER physiology relevant to cell death.
Assuntos
Apoptose , Membrana Celular/metabolismo , Animais , Humanos , Dinâmica Mitocondrial , Modelos Biológicos , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by activating the pore-forming proteins Bax and Bak and by inhibiting the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) triggered apoptosis that correlated with inhibition of anti-apoptotic proteins being sufficient to permeabilize mitochondria isolated from the same cells. Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited Bcl-XL but did not activate Bax. Examination of additional point mutants unexpectedly revealed that the CTS of Bim directly interacts with Bax, is required for physiological concentrations of Bim to activate Bax and that different residues in the CTS enable Bax activation and binding to membranes.
Assuntos
Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Proteína X Associada a bcl-2 , Animais , Proteína 11 Semelhante a Bcl-2/química , Proteína 11 Semelhante a Bcl-2/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Células HCT116 , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Domínios Proteicos , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismoRESUMO
Tumor initiation, progression and resistance to chemotherapy rely on cancer cells bypassing programmed cell death by apoptosis. We report that unlike other pro-apoptotic proteins, Bim contains two distinct binding sites for the anti-apoptotic proteins Bcl-XL and Bcl-2. These include the BH3 sequence shared with other pro-apoptotic proteins and an unexpected sequence located near the Bim carboxyl-terminus (residues 181-192). Using automated Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer (FLIM-FRET) we show that the two binding interfaces enable Bim to double-bolt lock Bcl-XL and Bcl-2 in complexes resistant to displacement by BH3-mimetic drugs currently in use or being evaluated for cancer therapy. Quantifying in live cells the contributions of individual amino acids revealed that residue L185 previously thought involved in binding Bim to membranes, instead contributes to binding to anti-apoptotic proteins. This double-bolt lock mechanism has profound implications for the utility of BH3-mimetics as drugs. â.
Assuntos
Antineoplásicos/farmacologia , Proteína 11 Semelhante a Bcl-2/química , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína bcl-X/química , Apoptose , Proteínas Reguladoras de Apoptose/química , Linhagem Celular Tumoral , Progressão da Doença , Transferência Ressonante de Energia de Fluorescência , Humanos , Processamento de Imagem Assistida por Computador , Células MCF-7 , Domínios ProteicosRESUMO
The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Apoptose , Citocromos c/metabolismo , Humanos , Camundongos , Mapas de Interação de ProteínasRESUMO
Translationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members' structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11-31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP(+/-) mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL.
Assuntos
Biomarcadores Tumorais/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína bcl-X/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Biomarcadores Tumorais/química , Permeabilidade da Membrana Celular , Camundongos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteína Tumoral 1 Controlada por Tradução , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/químicaRESUMO
The Bcl-2 family proteins control apoptosis by regulation of outer mitochondrial membrane permeabilization. Studying the Bcl-2 family is particularly difficult because the functional interactions that regulate apoptosis occur at or within intracellular membranes. Compared to other biophysical methods, fluorescence spectroscopy is well suited to study membrane-bound proteins as experiments can be performed with intact membranes and at protein concentrations similar to those found in cells. For these reasons, fluorescence spectroscopy has been particularly useful in studying the regulation of membrane permeabilization by Bcl-2 family proteins. Here, we discuss four fluorescence-based assays used to study protein dynamics at membranes, with a focus on how these techniques can be used to study the Bcl-2 family proteins.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/análise , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Humanos , Lipossomos/química , Lipossomos/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Conformação Proteica , Proteína X Associada a bcl-2/análise , Proteína X Associada a bcl-2/metabolismoRESUMO
Nanog was identified by its ability to sustain the LIF-independent self-renewal of mouse embryonic stem (ES) cells and has recently been shown to play a role in reprogramming adult fibroblasts into pluripotent stem cells. However, little is known about the structural basis of these remarkable activities of Nanog. We have previously identified an unusually strong transactivator named CD2 at its C terminus. Here we demonstrate that CD2 is required for Nanog to mediate ES cell self-renewal. Furthermore, deletion and point mutation analysis revealed that CD2 relies on at least seven aromatic amino acid residues to generate its potent transactivating activity. A mutant Nanog bearing alanine substitutions for these seven residues fails to confer LIF-independent self-renewal in mouse ES cells. Substitution of CD2 by the viral transactivator VP16 gave rise to Nanog-VP16, which is 10 times more active than wild-type Nanog in ES cells. Surprisingly, the expression of Nanog-VP16 in mouse ES cells induces differentiation and is thus unable to sustain LIF-independent self-renewal for mouse ES cells. Taken together, our results demonstrate that the CD2 domain of Nanog is a unique transactivator that utilizes aromatic residues to confer specific activity absolutely required for ES self-renewal.