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1.
Genes Dev ; 31(17): 1754-1769, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28982759

RESUMO

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.


Assuntos
Apoptose/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Dineínas/metabolismo , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Proteína 11 Semelhante a Bcl-2/genética , Células CACO-2 , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HeLa , Humanos , Células MCF-7 , Camundongos , Ligação Proteica , Multimerização Proteica/genética , Estabilidade Proteica , Interferência de RNA , Proteína X Associada a bcl-2/genética
2.
J Cell Sci ; 132(4)2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29361534

RESUMO

Dynamin-related protein 1 (Drp1), an 80 kDa mechanochemical GTPase of the dynamin superfamily, is required for mitochondrial division in mammals. Despite the role of Drp1 dysfunction in human disease, its molecular mechanism remains poorly understood. Here, we examined the effect of Drp1 on membrane curvature using tubes pulled from giant unilamellar vesicles (GUVs). We found that GTP promoted rapid rearrangement of Drp1 from a uniform distribution to discrete foci, in line with the assembly of Drp1 scaffolds at multiple nucleation sites around the lipid tube. Polymerized Drp1 preserved the membrane tube below the protein coat, also in the absence of pulling forces, but did not induce spontaneous membrane fission. Strikingly, Drp1 polymers stabilized membrane curvatures similar to those of constricted mitochondria against pressure changes. Our findings support a new model for mitochondrial division whereby Drp1 mainly acts as a scaffold for membrane curvature stabilization, which sets it apart from other dynamin homologs.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Cardiolipinas/metabolismo , Citocinese/fisiologia , Dinaminas , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Polimerização , Multimerização Proteica/fisiologia
3.
Cell Death Differ ; 27(2): 434-450, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31189926

RESUMO

The BH3-only class of Bcl-2 family proteins triggers mitochondrial apoptosis. Several mechanisms are used to restrain the pro-apoptotic activity of these proteins. Dynein light chain (DYNLL) 1 and 2 has been proposed to negatively regulate the activity of Bim and Bmf, respectively, and the Bim-DYNLL1 interaction leads to the formation of large protein complexes on mitochondria. Here we found that Bim and Bmf interact with both isoforms of DYNLL (DYNLL1 and DYNLL2). DYNLL1/2 not only induced homo-dimerization of Bim and Bmf but also led to the formation of ternary complexes (Bim-DYNLL-Bmf), both in cell-free and in cellular systems. DYNLL-induced oligomerization stabilized Bmf in cultured cells and inhibited its degradation by the ubiquitin-independent 20S proteasome in a cell-free system. Surprisingly, overexpression of wild-type Bmf but not of a DYNLL-binding-deficient mutant induced degradation of endogenous Bim in different cell lines, but both variants sensitized to apoptosis. Mutant Bmf incapable of interacting with anti-apoptotic Bcl-2 proteins and of inducing apoptosis still caused Bim degradation. These results suggest that Bmf overexpression-induced Bim degradation is not due to the displacement of Bim from anti-apoptotic Bcl-2 proteins but a direct consequence of the modulation of Bim-DYNLL association. A peptide derived from the DYNLL-binding domain of Bim also led to the degradation of Bim as well as of its preferred binding partner Mcl-1. Thus DYNLL regulates the mitochondrial pathway of apoptosis by determining the stability of Bmf, Bim, and Mcl-1 proteins.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Dineínas do Citoplasma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sítios de Ligação , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
4.
ACS Chem Biol ; 12(4): 989-1000, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28170214

RESUMO

The prosurvival Bcl-2 proteins exhibit a specific pattern of interactions with BH3-only proteins that determines the cellular dependence on apoptotic stress. This specificity is crucial for the development of BH3 mimetics, a class of anticancer molecules based on the BH3 domain with promising activity in clinical trials. Although complex formation mainly takes place in the mitochondrial outer membrane, most studies so far addressed the interaction between BH3 peptides and truncated Bcl-2 proteins in solution. As a consequence, quantitative understanding of the sequence specificity determinants of BH3 peptides in the membrane environment is missing. Here, we tackle this issue by systematically quantifying the ability of BH3 peptides to compete for the complexes between cBid and Bcl-xL in giant unilamellar vesicles and compare it with solution and mitochondria. We show that the BH3 peptides derived from Hrk, Bim, Bid, and Bad are the most efficient in disrupting cBid/Bcl-xL complexes in the membrane, which correlates with their activity in mitochondria. Our findings support the targeting to the membrane of small molecules that bind Bcl-2 proteins as a strategy to improve their efficiency.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína bcl-X/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Sítios de Ligação , Membrana Celular/metabolismo , Humanos , Simulação de Acoplamento Molecular , Mimetismo Molecular , Peptídeos/metabolismo , Ligação Proteica
5.
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
6.
FEBS J ; 284(5): 711-724, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28064468

RESUMO

The BCL-2 family members are key regulators of the intrinsic apoptotic pathway, which is defined by permeabilization of the mitochondrial outer membrane by members of the BAX-like subfamily. BOK is classified as a BAX-like protein; however, its (patho-)physiological role remains largely unclear. We therefore assessed the membrane permeabilization potential of C-terminally truncated recombinant BOK, BOK∆C . We show that BOK∆C can permeabilize liposomes mimicking the composition of mitochondrial outer membrane, but not of endoplasmic reticulum, forming large and stable pores over time. Importantly, pore formation was enhanced by the presence of cBID and refractory to the addition of antiapoptotic BCL-XL . However, isolated mitochondria from Bax-/- Bak-/- cells were resistant to BOK-induced cytochrome c release, even in the presence of cBID. Taken together, we show that BOK∆C can permeabilize liposomes, and cooperate with cBID, but its role in directly mediating mitochondrial permeabilization is unclear and may underlie a yet to be determined negative regulation.


Assuntos
Apoptose/genética , Permeabilidade da Membrana Celular/genética , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Citocromos c/metabolismo , Retículo Endoplasmático/genética , Técnicas de Inativação de Genes , Lipossomos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteína bcl-X/genética
7.
FEBS Open Bio ; 6(2): 126-34, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27239434

RESUMO

Characterization of amorphous protein aggregates may offer insights into the process of aggregation. Eleven single amino acid mutants of lipase (LipA of Bacillus subtilis) were subjected to temperature-induced aggregation, and the resultant aggregates were characterized for recovery of activity in the presence of guanidinium chloride (GdmCl). Based on activity recovery profiles of the aggregates, the mutants could be broadly assigned into four groups. By including at least one mutation from each group, a mutant was generated that showed an increase of ~ 10 °C in melting temperature (T m) compared to the wild-type and did not aggregate even at 75 °C. This method explores characterization of amorphous protein aggregates in the presence of GdmCl and helps in identifying mutations involved in protein aggregation.

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