RESUMEN
The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."
Asunto(s)
Apoptosis/fisiología , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Liposomas/química , Liposomas/metabolismo , Ratones , Mitocondrias/química , Mitocondrias/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Permeabilidad , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
In Alzheimer's disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aß) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aß of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aß oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aß oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aß oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/farmacología , Apoptosis/efectos de los fármacos , Citocromos c/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Multimerización de Proteína , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular , Ratones , Fragmentos de Péptidos/metabolismo , Porosidad , Estructura Secundaria de Proteína , alfa-Sinucleína/farmacología , Proteínas tau/farmacologíaRESUMEN
During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni(2+)-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the alpha5-alpha6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.
Asunto(s)
Multimerización de Proteína , Proteína Destructora del Antagonista Homólogo bcl-2/química , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Humanos , Ratones , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Nanoporous SiO2 particles which have different pore size and volume were prepared from a colloidal mixture of nano-sized silica particles by a spray heating method. The prepared nanoporous SiO2 particles were employed as a drug carrier to investigate the release behaviors of methylene blue (MB) as a model drug for a selected period of time. The concentration of released MB from the porous particles was measured by a UV-Vis spectroscopy with respect to time. The release of MB from the porous particles was maintained for 400 hours and the maximum amount of the released MB was 0.8 mg at 1.56 cm3/g of pore volume. As pore volume of the nanoporous particles increased, the release rate of MB increased.
Asunto(s)
Portadores de Fármacos , Nanopartículas , Dióxido de Silicio/química , Electroquímica , Microscopía Electrónica de Rastreo , Espectrofotometría UltravioletaRESUMEN
The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter's two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5'-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.
Asunto(s)
Proteínas Portadoras/química , Proteínas Inmovilizadas/metabolismo , Proteínas Mitocondriales/química , Modelos Moleculares , Conformación Proteica , Derivados del Benceno/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Ácido Cítrico/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Liposomas , Proteínas Mitocondriales/metabolismo , Fosfato de Piridoxal/metabolismo , Marcadores de Spin , Ácidos Tricarboxílicos/metabolismo , LevadurasRESUMEN
The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.
Asunto(s)
Apoptosis/fisiología , Membrana Dobles de Lípidos/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/química , Animales , Espectroscopía de Resonancia por Spin del Electrón/métodos , Electrones , Humanos , Ratones , Marcadores de SpinRESUMEN
In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the 'α3/α5' interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known 'α6:α6' interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH.
Asunto(s)
Mitocondrias/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Animales , Apoptosis , Sitios de Unión , Células Cultivadas , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Fibroblastos/citología , Fibroblastos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Ratones , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane alpha-helices in Bax and Bak, mature MAC pores would typically contain approximately 9 monomers and have diameters of 5.5-6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Modelos Biológicos , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/química , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular , Citocromos c/genética , Citocromos c/metabolismo , Liposomas/química , Ratones , Ratones Noqueados , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Permeabilidad , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Although rarely elicited during natural human infection, the most broadly neutralizing antibodies (BNAbs) against diverse human immunodeficiency virus (HIV)-1 strains target the membrane-proximal ectodomain region (MPER) of viral gp41. To gain insight into MPER antigenicity, immunogenicity, and viral function, we studied its structure in the lipid environment by a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and surface plasmon resonance (SPR) techniques. The analyses revealed a tilted N-terminal alpha helix (aa 664-672) connected via a short hinge to a flat C-terminal helical segment (675-683). This metastable L-shaped structure is immersed in viral membrane and, therefore, less accessible to immune attack. Nonetheless, the 4E10 BNAb extracts buried W672 and F673 after initial encounter with the surface-embedded MPER. The data suggest how BNAbs may perturb tryptophan residue-associated viral fusion involving the mobile N-terminal MPER segment and, given conservation of MPER sequences in HIV-1, HIV-2, and SIV, have important implications for structure-guided vaccine design.
Asunto(s)
Antígenos Virales/química , Anticuerpos Anti-VIH/química , Proteína gp41 de Envoltorio del VIH/química , Antígenos Virales/inmunología , Espectroscopía de Resonancia por Spin del Electrón , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Espectroscopía de Resonancia Magnética , Estructura Secundaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed cell death. Among the "BCL-2 homology (BH) 3-only" members of pro-apoptotic proteins, truncated BID (tBID) has been implicated in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 peptide alone at submicromolar concentrations cannot activate BAX or complement BID BH3 mutant-tBID in mitochondrial and liposomal release assays. Because tBID contains structurally defined membrane association domains, we investigated whether membrane targeting of BID BH3 peptide would be sufficient to restore its pro-apoptotic activity. We developed a Ni(2+)-nitrilotriacetic acid liposomal assay system that efficiently conjugates histidine-tagged peptides to a simulated outer mitochondrial membrane surface. Strikingly, nanomolar concentrations of a synthetic BID BH3 peptide that is chemically tethered to the liposomal membrane activated BAX almost as efficiently as tBID itself. These results highlight the importance of membrane targeting of the BID BH3 domain in tBID-mediated BAX activation and support a model in which tBID engages BAX to trigger its pro-apoptotic activity.
Asunto(s)
Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/química , Histidina/química , Humanos , Técnicas In Vitro , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Péptidos/química , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Recombinantes/química , Proteína bcl-X/metabolismoRESUMEN
BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/fisiología , Proteína X Asociada a bcl-2/fisiología , Secuencia de Aminoácidos , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/química , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Células Jurkat , Liposomas/química , Liposomas/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Proteína X Asociada a bcl-2/química , Proteína bcl-X/químicaRESUMEN
The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.