RESUMO
As antimicrobial resistance threatens our ability to treat common bacterial infections, new antibiotics with limited cross-resistance are urgently needed. In this regard, natural products that target the bacterial ribosome have the potential to be developed into potent drugs through structure-guided design, provided their mechanisms of action are well understood. Here we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X primarily inhibits peptide bond formation between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3' adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of tetracenomycin X on the bacterial ribosome and suggests a path forward for the development of novel aromatic polyketide antibiotics.
Assuntos
Antibacterianos , Policetídeos , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeos/farmacologia , Peptídeos/química , Policetídeos/farmacologia , Biossíntese de ProteínasRESUMO
Mitochondrial division is essential for mitosis and metazoan development, but a mechanistic role in cancer biology remains unknown. Here, we examine the direct effects of oncogenic RAS(G12V)-mediated cellular transformation on the mitochondrial dynamics machinery and observe a positive selection for dynamin-related protein 1 (DRP1), a protein required for mitochondrial network division. Loss of DRP1 prevents RAS(G12V)-induced mitochondrial dysfunction and renders cells resistant to transformation. Conversely, in human tumor cell lines with activating MAPK mutations, inhibition of these signals leads to robust mitochondrial network reprogramming initiated by DRP1 loss resulting in mitochondrial hyper-fusion and increased mitochondrial metabolism. These phenotypes are mechanistically linked by ERK1/2 phosphorylation of DRP1 serine 616; DRP1(S616) phosphorylation is sufficient to phenocopy transformation-induced mitochondrial dysfunction, and DRP1(S616) phosphorylation status dichotomizes BRAF(WT) from BRAF(V600E)-positive lesions. These findings implicate mitochondrial division and DRP1 as crucial regulators of transformation with leverage in chemotherapeutic success.
Assuntos
Transformação Celular Neoplásica/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Células HT29 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Serina/metabolismo , Proteínas ras/genéticaRESUMO
Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.
Assuntos
GTP Fosfo-Hidrolases/genética , Mitocôndrias Hepáticas/genética , Membranas Mitocondriais/metabolismo , Forma das Organelas/genética , Proteína X Associada a bcl-2/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Dinâmica Mitocondrial/genética , Membranas Mitocondriais/ultraestrutura , Permeabilidade , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The remarkably complex architecture and organization of bacterial nanomachines originally raised the enigma to how they are assembled in a coordinated manner. Over the years, the assembly processes of the flagellum and evolutionary-related injectisome complexes have been deciphered and were shown to rely on a conserved protein secretion machine: the type-III secretion system. In this book chapter, we demonstrate how individually evolved mechanisms cooperate in highly versatile and robust secretion machinery to export and assemble the building blocks of those nanomachines.
Assuntos
Metabolismo Energético , Sistemas de Secreção Tipo III/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias , Flagelos , Transporte ProteicoRESUMO
mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5' or 3' end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3'-to-5' exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3'-to-5' exoRNase for RNA turnover in S. pyogenes In particular, PNPase is responsible for the degradation of regulatory elements from 5' untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3'-to-5' exoRNases.
Assuntos
Exorribonucleases/metabolismo , Estabilidade de RNA/fisiologia , Streptococcus pyogenes/genética , Exorribonucleases/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/metabolismoRESUMO
Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Proteínas de Membrana/metabolismo , Sistemas de Secreção Tipo III , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , FilogeniaRESUMO
During assembly of the bacterial flagellum, protein subunits that form the exterior structures are exported through a specialized secretion apparatus energized by the proton gradient. This category of protein transport, together with the similar process that occurs in the injectisomes of gram-negative pathogens, is termed type-III secretion. The membrane-embedded part of the flagellar export apparatus contains five essential proteins: FlhA, FlhB, FliP, FliQ and FliR. Here, we have undertaken a variety of experiments that together support the proposal that the protein-conducting conduit is formed primarily, and possibly entirely, by FliP. Chemical modification experiments demonstrate that positions near the center of certain FliP trans-membrane (TM) segments are accessible to polar reagents. FliP expression sensitizes cells to a number of chemical agents, and mutations at predicted channel-facing positions modulate this effect. Multiple assays are used to show that FliP suffices to form a channel that can conduct a variety of medium-sized, polar molecules. Conductance properties are strongly modulated by mutations in a methionine-rich loop that is predicted to lie at the inner mouth of the channel, which might form a gasket around cargo molecules undergoing export. The results are discussed in the framework of an hypothesis for the architecture and action of the cargo-conducting part of the type-III secretion apparatus.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Transporte Proteico/genética , Salmonella enterica/genética , Salmonella enterica/metabolismoRESUMO
The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteína X Associada a bcl-2/fisiologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína 11 Semelhante a Bcl-2 , Benzamidas/química , Benzamidas/farmacologia , Benzopiranos/química , Benzopiranos/farmacologia , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Células HeLa , Humanos , Indóis , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Mimetismo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Nitrilas/química , Nitrilas/farmacologia , Nitrofenóis/química , Nitrofenóis/farmacologia , Permeabilidade , Piperazinas/química , Piperazinas/farmacologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Pirróis/química , Pirróis/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonas/química , Sulfonas/farmacologia , Lipossomas Unilamelares/química , Proteína X Associada a bcl-2/química , Proteína bcl-X/química , Proteína bcl-X/fisiologiaRESUMO
The mitochondrial pathway of apoptosis proceeds when the outer mitochondrial membrane (OMM) is compromised by the pro-apoptotic BCL-2 family members, BAK and BAX. Once activated, BAK and BAX form proteolipid pores in the OMM leading to mitochondrial outer membrane permeabilization (MOMP), and the release of inner membrane space proteins, such as cytochrome c, which promotes caspase activation. The use of isolated mitochondria has been instrumental to understanding the key interactions necessary to engage BAK and BAX activation, MOMP, and apoptosis. Furthermore, it is possible to biochemically define the relationships between BCL-2 family function and mitochondrial physiology using isolated systems. Our laboratory uses freshly isolated mitochondria from numerous sources to better understand BCL-2 family function and requirements for BAK and BAX activation. Here, we will discuss commonly used in vitro techniques to perform MOMP and cytochrome c release assays; and provide several key methodologies to implicate BAK and BAX activity in these processes.
Assuntos
Apoptose , Citocromos c/metabolismo , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Bioensaio , Caspases/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Camundongos , Mitocôndrias Hepáticas/patologia , Membranas Mitocondriais/patologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Sensing small molecules is crucial for microorganisms to adapt their genetic programs to changes in their environment. Arrest peptides encoded by short regulatory open reading frames program the ribosomes that translate them to undergo translational arrest in response to specific metabolites. Ribosome stalling in turn controls the expression of downstream genes on the same messenger RNA by translational or transcriptional means. In this review, we present our current understanding of the mechanisms by which ribosomes translating arrest peptides sense different metabolites, such as antibiotics or amino acids, to control gene expression.
Assuntos
Biossíntese de Proteínas , Ribossomos , Ribossomos/genética , Ribossomos/metabolismo , Peptídeos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismoRESUMO
The flagellum is the most complex macromolecular structure known in bacteria and comprised of around two dozen distinct proteins. The main building block of the long, external flagellar filament, flagellin, is secreted through the flagellar type-III secretion system at a remarkable rate of several tens of thousands amino acids per second, significantly surpassing the rates achieved by other pore-based protein secretion systems. The evolutionary implications and potential benefits of this high secretion rate for flagellum assembly and function, however, have remained elusive. In this study, we provide both experimental and theoretical evidence that the flagellar secretion rate has been evolutionarily optimized to facilitate rapid and efficient construction of a functional flagellum. By synchronizing flagellar assembly, we found that a minimal filament length of 2.5 µm was required for swimming motility. Biophysical modelling revealed that this minimal filament length threshold resulted from an elasto-hydrodynamic instability of the whole swimming cell, dependent on the filament length. Furthermore, we developed a stepwise filament labeling method combined with electron microscopy visualization to validate predicted flagellin secretion rates of up to 10,000 amino acids per second. A biophysical model of flagellum growth demonstrates that the observed high flagellin secretion rate efficiently balances filament elongation and energy consumption, thereby enabling motility in the shortest amount of time. Taken together, these insights underscore the evolutionary pressures that have shaped the development and optimization of the flagellum and type-III secretion system, illuminating the intricate interplay between functionality and efficiency in assembly of large macromolecular structures. Significance statement: Our study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.
RESUMO
Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Flagelos/metabolismo , Salmonella typhimurium/genéticaRESUMO
RNA degradation is an essential process that allows bacteria to control gene expression and adapt to various environmental conditions. It is usually initiated by endoribonucleases (endoRNases), which produce intermediate fragments that are subsequently degraded by exoribonucleases (exoRNases). However, global studies of the coordinated action of these enzymes are lacking. Here, we compare the targetome of endoRNase Y with the targetomes of 3'-to-5' exoRNases from Streptococcus pyogenes, namely, PNPase, YhaM, and RNase R. We observe that RNase Y preferentially cleaves after guanosine, generating substrate RNAs for the 3'-to-5' exoRNases. We demonstrate that RNase Y processing is followed by trimming of the newly generated 3' ends by PNPase and YhaM. Conversely, the RNA 5' ends produced by RNase Y are rarely further trimmed. Our strategy enables the identification of processing events that are otherwise undetectable. Importantly, this approach allows investigation of the intricate interplay between endo- and exoRNases on a genome-wide scale.
Assuntos
Exorribonucleases/metabolismo , RNA-Seq , Transcriptoma/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Guanosina/metabolismo , Óperon/genética , Estabilidade de RNA/genética , Streptococcus pyogenes/genéticaRESUMO
Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells.
Assuntos
Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Compostos de Bifenilo/farmacologia , Humanos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Neoplasias/genética , Neoplasias/patologia , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sulfonamidas/farmacologia , Proteína X Associada a bcl-2/genética , Proteína bcl-X/genéticaRESUMO
Many bacteria swim through liquids or crawl on surfaces by rotating long appendages called flagella. Flagellar filaments are assembled from thousands of subunits that are exported through a narrow secretion channel and polymerize beneath a capping scaffold at the tip of the growing filament. The assembly of a flagellum uses a significant proportion of the biosynthetic capacities of the cell with each filament constituting ~1% of the total cell protein. Here, we addressed a significant question whether a flagellar filament can form a new cap and resume growth after breakage. Re-growth of broken filaments was visualized using sequential 3-color fluorescent labeling of filaments after mechanical shearing. Differential electron microscopy revealed the formation of new cap structures on broken filaments that re-grew. Flagellar filaments are therefore able to re-grow if broken by mechanical shearing forces, which are expected to occur frequently in nature. In contrast, no re-growth was observed on filaments that had been broken using ultrashort laser pulses, a technique allowing for very local damage to individual filaments. We thus conclude that assembly of a new cap at the tip of a broken filament depends on how the filament was broken.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Flagelos/ultraestrutura , Flagelina/genética , Flagelina/metabolismo , Microscopia Eletrônica , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestruturaRESUMO
The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially â¼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.
Assuntos
Flagelos/metabolismo , Flagelina/metabolismo , Biogênese de Organelas , Salmonella enterica/metabolismo , Modelos Teóricos , Força Próton-MotrizRESUMO
The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.
RESUMO
The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015).
RESUMO
Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated.
Assuntos
Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Substituição de Aminoácidos , Apoptose , Citosol/enzimologia , Citosol/metabolismo , Deleção de Genes , Haploidia , Humanos , Mitocôndrias/enzimologia , Mutação , Fosforilação , Fosfosserina/análogos & derivados , Fosfosserina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMO
Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release.