Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Proapoptotic Bid binds to monolysocardiolipin,a new molecular connection between mitochondrial membranes and cell death

Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL). MCL, normally present in the remodelling of mitochondrial lipids, progressively increases in mitochondria during Fas-mediated apoptosis as a by-product of cardiolipin degradation, and also enhances Bid binding to membranes. MCL may thus play a crucial role in connecting lipid metabolism, relocation of Bid to mitochondria and integrated action of Bcl-2 proteins on mitochondrial membranes. We propose that Bid interaction with MCL 'primes' the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors.     

The interaction between tBid and cardiolipin or monolysocardiolipin

Bid, a BH3-only pro-apoptotic member of the Bcl-2 family, is cleaved by caspase 8 in apoptosis induced by death domain receptors. The carboxyl terminus of the cleavage product, tBid, remains associated with the amino terminal fragment (nBid) after cleavage. Dissociation of tBid from nBid occurs during targeting of tBid to mitochondria. We use an in vitro system and demonstrate that cardiolipin is sufficient for the dissociation. Monolysocardiolipin, a metabolite of cardiolipin that increases in mitochondria during apoptosis, has the same affinity to tBid as cardiolipin and is also capable of inducing dissociation of tBid from nBid. In contrast, phosphatidylethanolamine could not induce dissociation of tBid from nBid. To determine the site of tBid that interacts with cardiolipin, we performed mutational analysis by eliminating the positive-charged residues in helices 4-6. None of the single mutations can abolish the ability of tBid to target to mitochondria and to induce cytochrome c release, suggesting that positive-charged residues in helices 4-6 may not be required for mitochondrial targeting of tBid.     

The roles of Bid

Bid is an abundant pro-apoptotic protein of the Bcl-2 family that is crucial for death receptor-mediated apoptosis in many cell systems. Bid action has been proposed to involve the mitochondrial re-location of its truncated form, tBid, to facilitate the release of apoptogenic proteins like cytochrome c. However, the precise mechanism of (t)Bid action is unknown. To advance our knowledge, this review evaluates the basic steps of Bid activation—caspase cleavage, dissociation of tBid, and lipid-mediated mitochondrial relocation—and their structure-function aspects. Relevant current information is thoroughly examined to outline the problems that hamper our understanding of the possible roles of Bid in cell life and death, and suggest valuable directions for obtaining a clarification of its pro-apoptotic mechanism.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Cardiolipin provides specificity for targeting of tBid to mitochondria

Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unknown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Insight into the apoptosis-inducing action of alpha-bisabolol towards malignant tumor cells: involvement of lipid rafts and Bid

In a precedent report we showed that α-bisabolol, a sesquiterpene present widely in the plant kingdom, exerts a rapid and efficient apoptosis-inducing action selectively towards human and murine malignant glioblastoma cell lines through mitochondrial damage. The present study extends these data demonstrating the apoptosis-inducing action of α-bisabolol towards highly malignant human pancreatic carcinoma cell lines without affecting human fibroblast viability. The present study further shows the preferential incorporation of α-bisabolol to transformed cells through lipid rafts on plasma membranes and, thereafter, direct interaction between α-bisabolol and Bid protein, one of pro-apoptotic Bcl-2 family proteins, analyzed either by Surface Plasmon Resonance method or by intrinsic fluorescence measurement. Notions that lipid rafts are rich in plasma membranes of transformed cells and that Bid, richly present in lipid rafts, is deeply involved in lipid transport make highly credible the hypothesis that the molecular mechanism of α-bisabolol action may include its capacity to interact with Bid protein.     

Interaction of 14-3-3 with Bid during seizure-induced neuronal death

Seizure-induced neuronal death may involve coordinated intracellular trafficking and protein-protein interactions of members of the Bcl-2 family. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3-interacting domain death agonist (Bid) may contribute to seizure-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid during seizure-induced neuronal death. Brief seizures were evoked in rats by intraamygdala microinjection of kainic acid to elicit unilateral hippocampal CA3 neuronal death. Coimmunoprecipitation analysis demonstrated that although Bcl-2-associated death promoter (Bad) constitutively bound 14-3-3, there was no interaction between Bid and 14-3-3 in control brain. Seizures triggered Bid cleavage and a commensurate increase in binding of Bid to 14-3-3 within injured hippocampus. Casein kinases I and II, which can inactivate Bid by phosphoserine/threonine modification, did not coimmunoprecipitate with Bid. The largely uninjured contralateral hippocampus did not exhibit Bid cleavage or binding of 14-3-3 to Bid. In vitro experiments confirmed that 14-3-3beta is capable of binding truncated Bid, likely in the absence of phosphoserine/threonine modification. These data suggest 14-3-3 proteins may target active as well as inactive conformations of pro-apoptotic Bcl-2 death agonists, highlighting novel targets for intervention in seizure-induced neuronal death.     

The cardiolipin-binding domain of Bid affects mitochondrial respiration and enhances cytochrome c release

Bid is cleaved by caspase 8 during apoptosis and the truncated Bid (tBid) translocates to mitochondria by targeting cardiolipin. Amino acids 103-162 of Bid were reported as the cardiolipin-binding domain (CBD). The EGFP-CBD fusion protein targets to mitochondria and induces apoptosis. Using [(3)H]cardiolipin, we proved that recombinant CBD binds cardiolipin similar to tBid and tBid(G94E), a mutant with a defective BH3 domain. CBD could induce cytochrome c release from isolated mitochondria, but much less potent than tBid. Free cardiolipin inhibited the CBD-induced cytochrome c release, suggesting that it may be mediated by interfering with mitochondrial cardiolipin, especially with the interaction between cytochrome c and cardiolipin. This is consistent with the findings that CBD induced cytochrome c release in Bax-deficient cells, and that CBD suppressed mitochondrial respiration through directly interfering with cardiolipin, a critical lipid involved in oxidative phosphorylation. These results indicate the functional importance of CBD in tBid-induced apoptosis.     

Apoptosis: mitochondrial membrane permeabilization--the (w)hole story?

One critical step of apoptosis is the release of mitochondrial proteins through the outer mitochondrial membrane. Recent work shows that two pro-apoptotic Bcl-2 family proteins, Bax and Bid, as well as the mitochondrion-specific lipid cardiolipin may cooperate in chemically defined liposomes to generate a protein-permeable conduit, relaunching the debate on the identity of the pore responsible for mitochondrial membrane permeabilization during apoptosis.     

The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites

Background

Following cleavage by caspase 8, the C-terminus of Bid translocates from the cytosol to the mitochondria that is dependent upon structures formed by the mitochondrial-specific lipid cardiolipin. Once associated with mitochondria, truncated Bid (tBid) causes the potent release of cytochrome c, endonuclease G, and smac.

Results

We investigated whether tBid localizes specifically to the contact sites of mitochondria purported to be rich in cardiolipin. A point mutation changing the glycine at position 94 to glutamic acid in the BH3 domain of tBid (tBid_G94E) was principally used because mitochondria treated with this mutant tBid displayed better preservation of the outer membrane than those treated with wild type tBid. Additionally, tBid_G94E lowers the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria. Electron microscope tomography coupled with immunogold labeling was used as a new hybrid technique to investigate the three-dimensional distributions of tBid and tBid_G94E around the mitochondrial periphery. The statistics of spatial point patterns was used to analyze the association of these proteins with contact sites.

Conclusions

Immunoelectron tomography with statistical analysis confirmed the preferential association of tBid with mitochondrial contact sites. These findings link these sites with cardiolipin in tBid targeting and suggest a role for Bcl-2 family members in regulating the activity of contact sites in relation to apoptosis. We propose a mechanism whereby Bcl-2 proteins alter mitochondrial function by disrupting cardiolipin containing contact site membranes.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

Regulation of ceramide channels by Bcl-2 family proteins

Mitochondrial outer membrane permeabilization to proteins, an irreversible step in apoptosis by which critical proteins are released, is tightly regulated by Bcl-2 family proteins. The exact nature of the release pathway is still undefined. Ceramide is an important sphingolipid, involved in various cellular processes including apoptosis. Here we describe the structural properties of ceramide channels and their regulation by the anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. The evolutionarily conserved regulation of ceramide channels by Bcl-2 family proteins, consistent with their role in apoptosis, lends credibility to the notion that ceramide channels constitute the protein release pathway.     

Structural biology of the Bcl-2 family of proteins

The proteins of the Bcl-2 family are important regulators of programmed cell death. Structural studies of Bcl-2 family members have provided many important insights into their molecular mechanism of action and how members of this family interact with one another. To date, structural studies have been performed on six Bcl-2 family members encompassing both anti- (Bcl-x(L), Bcl-2, KSHV-Bcl-2, Bcl-w) and pro-apoptotic (Bax, Bid) members. They all show a remarkably similar fold despite an overall divergence in amino acid sequence and function (pro-apoptotic versus anti-apoptotic). The three-dimensional structures of Bcl-2 family members consist of two central, predominantly hydrophobic alpha-helices surrounded by six or seven amphipathic alpha-helices of varying lengths. A long, unstructured loop is present between the first two alpha-helices. The structures of the Bcl-2 proteins show a striking similarity to the overall fold of the pore-forming domains of bacterial toxins. This finding led to experiments which demonstrated that Bcl-x(L), Bcl-2, and Bax all form pores in artificial membranes. A prominent hydrophobic groove is present on the surface of the anti-apoptotic proteins. This groove is the binding site for peptides that mimic the BH3 region of various pro-apoptotic proteins such as Bak and Bad. Structures of Bcl-x(L) in complex with these BH3 peptides showed that they bind as an amphipathic alpha-helix and make extensive hydrophobic contacts with the protein. These data have not only helped to elucidate the interactions important for hetero-dimerization of Bcl-2 family members but have also been used to guide the discovery of small molecules that block Bcl-x(L) and Bcl-2 function. In the recently determined structure of the anti-apoptotic Bcl-w protein, the protein was also found to have a hydrophobic groove on its surface capable of binding BH3-containing proteins and peptides. However, in the native protein an additional carboxy-terminal alpha-helix interacts with the hydrophobic groove. This is reminiscent of how the carboxy-terminal alpha-helix of the pro-apoptotic protein Bax binds into its hydrophobic groove. This interaction may play a regulatory role and for Bax may explain why it is found predominately in the cytoplasm prior to activation. The hydrophobic groove of the pro-apoptotic protein, Bid protein, is neither as long nor as deep as that found in Bcl-x(L), Bcl-2, or Bax. In addition, Bid contains an extra alpha-helix, which is located between alpha1 and alpha2 with respect to Bcl-x(L), Bcl-2, and Bax. Although there are still many unanswered questions regarding the exact mechanism by which the Bcl-2 family of proteins modulates apoptosis, structural studies of these proteins have deepened our understanding of apoptosis on the molecular level.     

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.     

Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax

Following caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins.     

Three novel Bid proteins generated by alternative splicing of the human Bid gene

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. Bid(S) contains the N-terminal regulatory domains of Bid without the BH3 domain; Bid(EL) corresponds to full-length Bid with additional N-terminal sequence; and Bid(ES) contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies Bid(EL) induces apoptosis, whereas Bid(S) abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. Bid(ES) induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.     

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis¹. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)¹. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues².In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)^3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation^7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members^7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization¹⁰. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)¹⁰. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)¹¹. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.     

促细胞凋亡因子Bid蛋白的研究进展

Bid蛋白是Bcl-家族中促凋亡类的蛋白。它具有可被caspase8酶切高控、高效地诱民细胞色素c从线粒体泄漏到胞浆中的功能,从而在细胞凋亡中起着重要的作用,因而倍受人们的重视。Bid蛋白的功能被发现以来,短短几年间,人们从分子生物学、细胞学、结构分析以及利用脂质体模型膜体系等各方面对Bid蛋白进行研究,取得了很大的进展。