RESUMO
Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.
Assuntos
Proteínas Reguladoras de Apoptose , Neoplasias , Humanos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/químicaRESUMO
Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/fisiologia , Humanos , Bicamadas Lipídicas/metabolismoRESUMO
Small-molecule mediated modulation of protein interactions of Bcl-2 (B-cell lymphoma-2) family proteins was clinically validated in 2015 when Venetoclax, a selective inhibitor of the antiapoptotic protein BCL-2, achieved breakthrough status designation by the FDA for treatment of lymphoid malignancies. Since then, substantial progress has been made in identifying inhibitors of other interactions of antiapoptosis proteins. However, targeting their pro-apoptotic counterparts, the "executioners" BAX, BAK, and BOK that both initiate and commit the cell to dying, has lagged behind. However, recent publications demonstrate that these proteins can be positively or negatively regulated using small molecule tool compounds. The results obtained with these molecules suggest that pharmaceutical regulation of apoptosis will have broad implications that extend beyond activating cell death in cancer. We review recent advances in identifying compounds and their utility in the exogenous control of life and death by regulating executioner proteins, with emphasis on the prototype BAX.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Éteres Fenílicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismoRESUMO
Axonal degeneration and neuronal cell death are fundamental processes in development and contribute to the pathology of neurological disease in adults. Both processes are regulated by BCL-2 family proteins which orchestrate the permeabilization of the mitochondrial outer membrane (MOM). MOM permeabilization (MOMP) results in the activation of pro-apoptotic molecules that commit neurons to either die or degenerate. With the success of small-molecule inhibitors targeting anti-apoptotic BCL-2 proteins for the treatment of lymphoma, we can now envision the use of inhibitors of apoptosis with exquisite selectivity for BCL-2 family protein regulation of neuronal apoptosis in the treatment of nervous system disease. Critical to this development is deciphering which subset of proteins is required for neuronal apoptosis and axon degeneration, and how these two different outcomes are separately regulated. Moreover, noncanonical BCL-2 family protein functions unrelated to the regulation of MOMP, including impacting necroptosis and other modes of cell death may reveal additional potential targets and/or confounders. This review highlights our current understanding of BCL-2 family mediated neuronal cell death and axon degeneration, while identifying future research questions to be resolved to enable regulating neuronal survival pharmacologically.
Assuntos
Apoptose , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Humanos , Membranas Mitocondriais/metabolismoRESUMO
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
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
Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases.
Assuntos
Apoptose/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Feminino , Ácido Glutâmico/toxicidade , Células HCT116 , Humanos , Lipossomos/metabolismo , Masculino , Camundongos , Membranas Mitocondriais/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Permeabilidade/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genéticaRESUMO
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.