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Leakage and aggregation of phospholipid vesicles induced by the Soybean Phospholipid


Leakage and aggregation of phospholipid vesicles induced by the Soybean Phospholipid

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									Eur. J. Biochem. 268, 48±55 (2001) q FEBS 2001

Leakage and aggregation of phospholipid vesicles induced by the BH3-only
Bcl-2 family member, BID
Dayong Zhai, Qi Miao, Xiaofeng Xin and Fuyu Yang
National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing, China

          BID is a BH3 domain-only member of the Bcl-2 family that acts as an apoptotic agonist in programmed cell
          death. After cleavage by caspase-8, the N-terminal of BID (N-BID) stays in the cytosol while the C-terminal of
          BID (C-BID) translocates to mitochondria, leading to cytochrome c release in vivo and in vitro. We have
          previously reported that BID or truncated BID (tBID) can induce the release of entrapped trypsin and
          cytochrome c from large unilamellar vesicles (LUVs). Further studies have been performed and are presented
          here; the results demonstrate that C-BID, like BID and tBID, induces vesicle leakage, whereas N-BID or the BID
          mutants BID (D59A) and BID (G94E) fail to have any significant effects. The affinity of the above-mentioned
          proteins for soybean phospholipid LUVs (SLUVs) decreased in an order similar to their leakage-inducing
          capability: tBID . BID . BID (D59A), while N-BID and BID (G94E) were unable to bind to the vesicles at all.
          BID-induced leakage was dependent on the lipid composition of vesicles. Acidic phospholipid (e.g. phosphatidic
          acid or phosphatidylglycerol) was necessary for BID-induced leakage while the presence of phosphatidylethano-
          lamine or cholesterol reduced the leakage. It was also found C-BID is better able to penetrate the soybean
          phospholipid monolayer than BID or tBID. A further finding was that tBID, but not full-length BID, could
          stimulate the aggregation of SLUVs. Finally, Bcl-xL, an apoptotic antagonist in programmed cell death, can
          prevent the aggregation of LUVs induced by tBID, but not the release of entrapped trypsin. It is postulated that
          two separate domains of tBID are responsible for inducing leakage and aggregation of phospholipid vesicles.
          Keywords: aggregation; apoptosis; BID; large unilamellar vesicles; leakage.

Apoptosis is an evolutionarily conserved process critical in                and BAD, can lead to mitochondrial damage. The ratio of these
various biological events, such as embryonic development,                   two groups determines the fate of mitochondria and the whole
maintenance of tissue homeostasis, removal of noninstructed,                cell [4,5]. The sequences of Bcl-2 family proteins share several
misinstructed and damaged cells, and immunological defense                  conserved motifs designated BH1±4 (Bcl-2 homology)
[1]. Various stimuli, including developmental and environ-                  domains [6]. Among them, the BH3 domain from pro-apoptotic
mental ones, deliver complex signals to promote apoptosis or                members of the Bcl-2 family is essential for the `killer' function
survival. A large number of pro-apoptotic and anti-apoptotic                [2]. NMR analysis of Bcl-xL ±BAK BH3 peptide complex has
molecules have been identified as playing principal roles in                revealed both hydrophobic and electrostatic interactions
apoptosis, in which members of the Bcl-2 family act as                      between the Bcl-xL pocket and a BH3 amphipathic a-helical
regulators [2].                                                             peptide from BAK [7]. Deletions in BAK and an extensive
  Mitochondria, which were once thought simply to generate                  mutation analysis of BAK suggest that the BH3 domain serves
energy for a cell, have been implicated as important sensors and            as a minimal `death domain' critical for both dimerization and
amplifiers in intracellular death signaling pathways by releas-             killing [8].
ing proteins such as cytochrome c and apoptosis-inducing                       The three-dimensional structure of Bcl-xL contains a bundle
factor [3]. Bcl-2 family proteins mainly located in the                     of seven a helices with two central, predominantly hydro-
mitochondrial outer membrane constitute a pivotal checkpoint                phobic, helices forming the core of the molecule while BID also
within the common part of the apoptotic pathway. The anti-                  has two central hydrophobic helices surrounded by six
apoptotic members of Bcl-2 family, such as Bcl-2 and Bcl-xL,                amphipathic helices [9±11]. Both of these resemble pore-
play important roles as protectors of mitochondrial integrity               forming bacterial toxins although their homologous sequence is
while various pro-apoptotic members, such as BAX, BAK, BID                  limited, suggesting they may have channel-forming potential.
                                                                            In fact, BAX, Bcl-xL and Bcl-2 have already been shown to
Correspondence to F. Yang, National Laboratory of Biomacromolecules,        have channel activity in artificial lipid membranes [12±14], and
Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing, 100101,   this has also been recently reported for BID [15]. The ability of
China. Fax: 1 86 10 64872026, Tel.: 1 86 10 64888574,                       these proteins to form ion channels has fostered the idea that
E-mail:                                               they may open pores or produce breaks in the mitochondrial
Abbreviations: DOPA, dioleoylglycerophosphatidic acid; DOPC,                outer membranes, allowing exit of cytochrome c [16]. More-
dioleoylglycerophosphocholine; DOPE,                                        over, BAX and BAK have been shown to stimulate the opening
dioleoylglycerophosphoethanolamine; DOPG,                                   of the voltage-dependent anion channel, a mitochondrial
dioleoylglycerophosphoglycerol; LUVs, large unilamellar vesicles; SLUVs,    channel through which cytochrome c permeates [17].
soybean phospholipid LUVs; tBID, truncated BID.                                BID is a BH3 domain-only protein that lacks transmembrane
(Received 24 July 2000, revised 16 October 2000, accepted                   domains and is predominantly localized in the cytosol [18]. It
20 October 2000)                                                            demonstrates unique and important properties after cleavage by
q FEBS 2001                                              BID-inducing leakage and aggregation of vesicles (Eur. J. Biochem. 268) 49

caspase-8; the 15-kDa BH3-containing C-terminal translocates       containing 10 mm KCl, 2 mm MgCl2, 1 mm EDTA, 1 mm
from cytosol to the mitochondrial membrane and leads to            EGTA, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl
cytochrome c release, while N-BID remains localized in the         fluoride (buffer A). After re-equilibration with buffer A, the
cytosol in vivo [19,20]. The data from the structure of BID and    pH was raised to 12 to obtain C-BID.
the yeast two hybrid experiments suggest that N-BID has an
inhibitory action associated with the C-terminal BH3 domain        Liposome preparation and detection of liposome leakage
[10,11,21], which may represent an important means of
regulating the activity of the whole BID molecule.                 LUVs were prepared as in Rietveld et al. [23]. Briefly, 2.5 mL
   It is still unclear how BID leads to cytochrome c release       diethyl ether and 0.5 mL Pipes buffer (10 mm Pipes, pH 7.0,
from mitochondria. Some authors speculate that BID may cause       50 mm NaCl, 0.2 mm EDTA) containing trypsin (2 mg´mL21)
cytochrome c release via formation of ion channels or pores        were added to a dry lipid film of 5 mm phospholipid. After
[15]. Others, however, argue that BID may directly perturb the     sonication for 20 min at 4 8C with a bath sonicator, and
membrane integrity by interacting with membrane lipids [20].       evaporation of ether under reduced pressure, the vesicles were
Another putative function of BID is that tBID can trigger the      dialyzed overnight at 4 8C in Pipes buffer. The vesicles
clustering of mitochondria in cells during the process of          formed were then fractionated by centrifugation in a
apoptosis [20]. Li et al. suggest that two independent pathways    Beckman Optima TLX table-top ultracentrifuge (TLA-100.3
for the BID-mediated destruction of mitochondria in cell death     rotor) at low speed (15 min, 5564 g, 4 8C) to discard the
may operate, based on analysis of the structure of BID [10].       multilamellar vesicles, and high speed (30 min, 42 000 g, 4 8C)
   The relative simplicity of the model system based on pure       to collect the LUVs. The LUVs were washed at least three
lipid bilayers invites its use in the study of the molecular       times to remove the nonenclosed trypsin. The phospholipid
mechanisms of membrane±protein interactions. We have               concentration was determined by perchloric acid destruction.
previously reported that BID can induce trypsin as well as            Various concentrations of the test proteins were added to
cytochrome c release from the internal medium of LUVs [22].        100 mL volumes of the LUV suspension, and pre-incubated at
Here, we used this model system to study the molecular             30 8C for 15 min. Ph-CO-Arg-OEt was introduced as substrate
mechanism underlying the actions of BID and other Bcl-2            to measure the degradation rate by the released trypsin at
proteins. Data show that in contrast to C-BID, the mutants of      253 nm by using a Shimadzu UV-2101PC spectrophotometer.
BID, BID (D59A), BID (G94E), and N-BID have almost no
leakage-inducing ability. Moreover, tBID rather than BID can       BID binding to LUVs
lead to the aggregation of LUVs. The leakage of vesicles by
                                                                   LUVs were prepared as above without entrapped trypsin. BID,
BID and the aggregation-inducing effect of tBID may be due
                                                                   BID (D59A), BID (G94E), or N-BID (0.1 mg´mL21) was
to the different nature of the two hydrophobic domains,
                                                                   incubated at different lipid:protein molar ratios in 100 mL Pipes
mimicking the functions of BID in vivo.
                                                                   buffer for 1 h at 30 8C. The proteins binding to the LUVs were
                                                                   separated from the free parts by centrifugation at 96 000 g in a
M AT E R I A L S A N D M E T H O D S                               Beckman Optima TLX table-top ultracentrifuge (TLA-100.3
                                                                   rotor) for 30 min at 30 8C. The protein concentration in the
Materials                                                          supernatant was determined by the Bradford method [24].
Human BID, BID (D59A), BID (G94E) and caspase-8
expression plasmids were kindly provided by X. Wang                Penetrating of the phospholipid monolayer by BID
(Department of Biochemistry, University of Texas South-            A film balance, type Han-2000, designed and made in our
Western Medical Center, Dallas, TX, USA). The Bcl-xL-GST           laboratory was used to study the membrane penetrating ability
expression plasmid was provided by J. Yuan (Department of          of proteins. Briefly, 3 mL of Pipes buffer as a subphase was
Cell Biology, Harvard Medical School, Boston, MA, USA).            added into the mini-trough, which has been described
The BAK-GST expression plasmid was obtained from Y.                previously [25]. The monomolecular lipid layer was spread to
Tsujinoto (Biomedical Research Center, Osaka University,           give the desired initial surface pressure by dropping aliquots of
Japan). Trypsin, proteinase K, cholesterol, dioleoylglycero-       lipid dissolved in chloroform on the aqueous surface. The
phosphatidic acid (DOPA), dioleoylglycerophosphoglycerol           surface pressure of the monolayer was measured by the
(DOPG), dioleoylglycerophosphoethanolamine (DOPE) and              Wilhelmy plate method using plates cut from filter paper and
dioleoylglycerophosphocholine (DOPC) were obtained from            rinsed with methanol prior to use. After the initial surface
Sigma. Ph-CO-Arg-OEt was from the Shanghai Institute of            pressure had stabilized to a plateau value, the desired
Biochemistry (Chinese Academy of Sciences, Shanghai, China).       amount of protein was injected to the mixing chamber
                                                                   (which contained a magnetic stir bar) through a 0.7-cm2
Expression and purification of recombinant proteins                hole in the edge. The samples were then rapidly mixed with
                                                                   bulk solution, and radically diffused to the upper monolayer-
Human BID, BID (D59A), BID (G94E), BAK, Bcl-xL and                 spreading disk. All measurements were performed at room
caspase-8 were expressed recombinantly as has been described       temperature. Usually, an increase of the surface pressure Dp
previously [19,20]. The plasmids for expression of the proteins    is measured as a function of the initial surface pressure p.
were transformed into bacteria BL21 (DE3) cells; proteins were     A plot of Dp versus p yields a straight line with negative
purified from the cell lysate using nickel affinity (Qiagen) or    slope that intersects the abscissa at the value named as the
GST affinity chromatography. A Bio-scale Q5 column (Bio-           limiting surface pressure.
Rad) was used for further purification. Truncated BID was
obtained by adding 1 : 50 (vol/vol) caspase-8, and incubating
                                                                   CD measurements
overnight at 4 8C. After dilution, the sample was loaded on to a
Bio-scale S5 column (Bio-Rad). Elution was performed with a        CD measurements were carried out on a JASCO J-720
linear gradient of 0 to 100 mm NaCl in 20 mm Hepes, pH 7.4,        instrument. Measurements were taken using a 1-mm path
50 D. Zhai et al. (Eur. J. Biochem. 268)                                                                                        q FEBS 2001

Fig. 1. SDS/PAGE analysis of purified BID, and its mutants and
fragments. Purified BID, BID (G94E), BID (D59A), tBID, C-BID and
N-BID were analyzed by SDS/PAGE in 15% gel and stained with
Coomassie blue. Molecular mass markers are indicated.

length. All spectra were recorded in 1.0-nm wavelength
increments with a 1-s time constant and a full-scale sensitivity
of 20 millidegrees. Each spectrum was the average of six scans
corrected for background solvent effects by subtraction of the
appropriate buffer blank. BID, BID (D59A), BID (G94E), and
N-BID were diluted to a concentration of 20 mm in NaCl/Pi
buffer; C-BID was diluted in 10 mm Tris buffer, pH 12. The
lipid concentration was 1 mm. Spectra were scanned in the far
UV from 250 to 190 nm.                                             Fig. 2. Effect of BID and lipid concentration on the leakage efficiency of
                                                                   LUVs. (A) O Shows the percentage of leakage after 15 min as a function of
                                                                   lipid concentration. The amount of lipid and BID was varied, a constant
                                                                   mole:mole ratio of 2000:1 being kept. X Shows the percentage of leakage
Turbidity measurement                                              after 15 min as a function of lipid concentration when BID was held
The aggregation of SLUVs after the proteins had been               constant at 0.1 mm. The lipid concentration was varied to obtain the desired
introduced was monitored spectrophotometrically at 600 nm          lipid:BID ratio. (B) Percentage of leakage after 15 min as a function of BID
in a cuvette with 1-cm path length using a Shimadzu UV-2101        concentration. The lipid concentration was kept constant at 300 mm while
PC spectrophotometer. Aliquots of BID, tBID or tBID (G94E)         the BID concentration was varied to obtain the desired ratio. In all cases, the
were added successively to the suspension of SLUVs (1 mm           extent of trypsin leakage was calculated as the slope of a fitted line drawn to
lipid concentration) in Pipes buffer. Truncated BID or tBID        the individual leakage curves.
(G94E) pre-incubated with Bcl-xL at 4 8C for 10 min (1:1,
mol:mol) was introduced and aggregation detected as above.
                                                                   95% as determined by SDS/PAGE analysis with Coomassie
The molar ratios of lipid to protein ranged from 2000:1 to
                                                                   blue staining (Fig. 1.).

                                                                   Comparison of trypsin release from LUVs induced by BID,
R E S U LT S                                                       C-BID, N-BID and its mutants
                                                                   Trypsin-containing LUVs have previously been used to monitor
Purification of BID, and its fragments and mutants
                                                                   protein translocation across the model membranes [26]. Here
Recombinant BID, BID (D59A), BID (G94E), BAK, Bcl-xL,              this system was used to assay the BID-induced leakage of
and caspase-8 were purified in accordance with previously          contents from the internal medium of the liposomes. Leakage is
published methods [19,20]. Truncated BID was prepared as the       studied as a function of a constant lipid:BID ratio, BID
product of BID cleaved by caspase-8 following overnight            concentration (lipid concentration kept constant), and lipid
incubation at 4 8C; N-BID was still tightly associated with        concentration (BID concentration kept constant). At a constant
C-BID in the buffer. After dilution, the tBID sample was loaded    lipid:BID ratio of 2000:1, the extent of leakage after 15 min
to the Bio-scale S5 column. The N-BID was easily washed out        reduced as the lipid concentrations decreased (Fig. 2A). This is
with 0.1 m NaCl, while C-BID could only be eluted when the         expected on the basis of the partitioning of the proteins between
pH value was above 12. The purity of the proteins was above        the bilayer and the aqueous phase; as the lipid concentration is
q FEBS 2001                                                       BID-inducing leakage and aggregation of vesicles (Eur. J. Biochem. 268) 51

                                                                             lowered, the fraction of the peptide that becomes vesicle-
                                                                             associated decreases. When BID concentration was held
                                                                             constant, increasing the lipid concentration led to a decrease
                                                                             in the extent of leakage (Fig. 2B). As the number of vesicles in
                                                                             suspension grows with increasing lipid concentration, the
                                                                             results are in agreement with the pore-forming hypothesis. In
                                                                             other words, a defined number of peptides is required for the
                                                                             formation of a channel or pore in the bilayer. These data also
                                                                             indicate that once in bilayer, BID does not rapidly redistribute
                                                                             between different vesicles. Finally, at constant lipid, increasing
                                                                             BID concentration induced a greater extent of leakage until a
                                                                             maximum value was reached (Fig. 2C). This result is consistent
                                                                             with a monomer±multimer assembly pore-forming process
                                                                             being involved in the mechanism of leakage. In all cases, the
                                                                             lipid:BID ratios were determined from the amount of vesicles
                                                                             and BID initially added to the cuvette. The effective ratio
                                                                             depends on the lipid:water partition coefficient of BID.
                                                                                When tBID is added to trypsin-encapsulated LUVs, it
                                                                             triggered release more efficiently than did the full-length
                                                                             BID. To investigate how BID destabilizes the LUVs, BID
                                                                             mutants and its fragments were examined. Both BID (D59A), a
                                                                             mutant that cannot be cleaved by caspase-8, and BID (G94E),
                                                                             which contains a point mutation in the BH3 domain, were
                                                                             unable to induce cytochrome c release from mitochondria.
                                                                             Strikingly, BID (D59A) and BID (G94E) were shown to lose
                                                                             the ability to induce trypsin release from LUVs at the same
                                                                             concentrations as BID did. A partial leakage of trypsin could
                                                                             only be observed when excessive amounts of these mutants
                                                                             were added (Fig. 3A).
                                                                                After cleavage by caspase-8, C-BID translocates to mito-
                                                                             chondria to induce the release of cytochrome c, whereas N-BID
                                                                             remains in the cytosol [19,20]. It is difficult to separate C-BID
                                                                             from N-BID in the experiments carried out in vitro, but a
                                                                             portion of C-BID could be precipitated by adjusting the pH of
Fig. 3. BID-induced release of entrapped trypsin from LUVs compris-          the medium to 7.0. The partially aggregated C-BID could
ing 20% DOPG and 80% 1,2-dioleoylphosphatidylcholine. LUVs                   trigger the release of entrapped trypsin from the liposomes. In
(300 mm lipid) were incubated for 15 min at 30 8C in the presence of test    contrast, N-BID was incapable of inducing leakage even at
protein in a 100-mL reaction mixture. After incubation, the substrate        higher concentrations (Fig. 3B).
Ph-CO-Arg-OEt was introduced to the supernatant to measure degradation          BAK, caspase-8 alone or BID proteolyzed with proteinase K
by the released trypsin at 253 nm. The same amount of buffer was used as a   were used as controls. None of these showed leakage-inducing
control. (A) BID (BID:lipid, 1:3000), BID (G94E) (protein:lipid, 1:100),     activity. To investigate further whether BID is able to induce the
and BID (D59A) (protein:lipid, 1:100). (B) BID (protein:lipid, 1:4000),      release of other molecules, fluorescein sulfonate (FS) or
C-BID (protein:lipid, 1:4000), and N-BID (protein:lipid, 1:50).              cytochrome c were entrapped in LUVs. A similar releasing
                                                                             effect could also be observed following BID treatment (data not

                                                                             BID-induced leakage of vesicles with different lipid
                                                                             To test whether the charge of the membrane constitutes an
                                                                             important factor in destabilizing liposomes, the effect of BID or
                                                                             tBID on leakage of LUVs composed of DOPA and
                                                                             1,2-dioleoylphosphatidylcholine, or DOPG and 1,2-dioleoyl-
                                                                             phosphatidylcholine, at ratios of 2 : 8, 3 : 7, 4 : 6, and 5 : 5
                                                                             (mol/mol), were studied. The results indicated that none of the
                                                                             lipid matrices tested changed the ability of the proteins to
                                                                             induce leakage (data not shown). This confirms that membranes
                                                                             containing varying amounts of negatively charged phospho-
                                                                             lipids allow the proteins to act similarly. Regarding
Fig. 4. Effect of lipid composition on the efficiency of leakage induced     1,2-dioleoylphosphatidylcholine-only LUVs entrapped with
by BID or tBID. Curves are shown for LUV-entrapped trypsin with lipid        trypsin, the efficiency of induction decreased drastically
composition: 20% DOPG, 80% DOPC (X); 20% DOPG, 10% DOPE, 70%                 (Fig. 4). It may deduced that a negative surface potential of a
DOPC (W); 20% DOPG, 30% DOPE, 50% DOPC(K); 20% DOPG, 30%                     lipid bilayer is necessary for BID or tBID to bind to membranes
cholesterol, 50% DOPC(L); and 100% DOPC(A).                                  during the initial step of the interaction, albeit that the motif
52 D. Zhai et al. (Eur. J. Biochem. 268)                                                                                              q FEBS 2001

                                                                               Fig. 6. Comparison of the initial surface pressure and the surface
                                                                               pressure increase of phospholipid monolayer following injection of
                                                                               different BID proteins. Test proteins (0.5 mg´mL21) were injected
Fig. 5. Binding of different BID to LUVs. BID preparations were                underneath a monolayer of asolectin at different initial pressures. The
incubated with LUVs for 1 h at 30 8C. Vesicles were collected by               limiting surface pressure of BID, tBID and C-BID were 36.4, 42 and 44
centrifugation, and protein associated with the liposome fraction was          mN/m, respectively. (A) Kinetic curves showing the change in surface
detected by the Bradford method. The continuous line represents the best fit   pressure. (B) p-Dp plots of surface pressure change.
of the data. (A) BID, tBID, BID (D59A), BID (G94E) and N-BID were
incubated with SLUVs. (B) BID and tBID were incubated with LUVs
comprising either 20% DOPA and 80% DOPC, or 20% DOPA, 30% DOPE                 Comparison of the LUV binding of BID, and its fragments and
and 50% DOPC.                                                                  mutants
                                                                               To study whether the discrepancy in the competency of these
                                                                               proteins to induce leakage was attributed to the difference in
directing leakage may be insensitive to the charge density on                  their binding activity, the affinities of the BID proteins to the
the membrane surface.                                                          same LUVs were determined and compared. The SLUVs were
   DOPE, the second most abundant phospholipid of mitochon-                    incubated with BID, BID (D59A), BID (G94E), tBID, and
dria, or cholesterol was introduced into vesicles to investigate               N-BID. After centrifugation, free proteins in the supernatant
whether lipid composition would alter the interaction of BID                   were analyzed. BID proteins associated with SLUVs were
with the vesicles. When the percentage of DOPE was about                       plotted as a function of the lipid concentration (Fig. 5A); it can
10%, the efficiency of BID induction was not obvious. Adding                   be observed that tBID has the highest affinity for SLUVs, BID
30% DOPE to bilayers containing 20% DOPG and 50% DOPC                          (D59A) has a lower affinity for the membrane than does the
resulted in a steep decrease in the leakage efficiency (Fig. 4),               wild type BID, and BID (G94E) and N-BID hardly bind to
indicated by an increase in the protein:lipid ratio needed to get              SLUVs.
the same final leakage (about ten-fold more protein was                           Binding of BID to vesicles comprising: 20% DOPG and 80%
required). Similar results were obtained in the case of SLUVS                  1,2-dioleoylphosphatidylcholine; 20% DOPG, 30% DOPE and
(data not shown). Vesicles comprising 30% cholesterol, 20%                     50% 1,2-dioleoylphosphatidylcholine; and DOPC only, was
DOPG and 50% DOPC also caused a clear drop in the                              also compared. As shown in Fig. 5B, BID had a higher affinity
induction efficiency of BID (Fig. 4). These vesicle leakage                    for DOPG/DOPC vesicles than for DOPG/DOPE/DOPC
results resemble those obtained using the pore-forming peptide                 vesicles, which was consistent with the disparity in the
synthetic GALA, which is similarly affected by DOPE and                        leakage-inducing activities with the two vesicle types. How-
cholesterol [27,28].                                                           ever, the discrepancy in binding affinity of tBID to these
q FEBS 2001                                                     BID-inducing leakage and aggregation of vesicles (Eur. J. Biochem. 268) 53

                                                                          increase when BID was pretreated with proteinase K, indicating
                                                                          that the rise in surface pressure reflected the association of BID
                                                                          with the monolayer.
                                                                             When using the BID protein-induced increase in surface
                                                                          pressure as a function of the initial surface pressure, an inverse
                                                                          linear relationship appears (Fig. 6B). The lipid-packing density
                                                                          of the liposomes used was equivalent to a monolayer surface
                                                                          pressure of 32±35 mN´m21 [25]. Our results indicate that
                                                                          BID could, conceivably, penetrate monolayers of soybean
                                                                          phospholipid. That the limiting surface pressure for C-BID,
                                                                          44 mN´m21, was much higher than that for BID, 36 mN´m21,
                                                                          or tBID, 41 mN´m21, (Fig. 6B) implies that BID exposes more
                                                                          hydrophobic residues after cleavage with caspase-8.

                                                                          Secondary structural changes in BID on interaction with
                                                                          The secondary structures of BID, BID (D59A), BID (G94E),
                                                                          tBID, N-BID and C-BID were determined and compared using
                                                                          CD spectra. The only minor difference was found in the shape
                                                                          or amplitude of the far-UV spectrum among BID, BID (D59A),
                                                                          BID (G94E), tBID and C-BID, while N-BID showed little a
                                                                          helix but an abundance of b structure (data not shown). No
                                                                          obvious differences occurred in any of the above proteins after
                                                                          lipid was introduced (data not shown). The high a-helix content
                                                                          of BID is reminiscent of the channel-forming Bcl-xL, Bcl-2 and
                                                                          BAX proteins, and the structurally related bacterial toxins such
                                                                          as the pore-forming colicins and diphtheria toxin [15].

                                                                          Aggregation of SLUVs induced by tBID
                                                                          In addition to inducing leakage from liposomes, it was
                                                                          interesting to note that tBID, but not full-length BID, could
                                                                          stimulate the aggregation of SLUVs, and that such aggregation
                                                                          was dependent on the protein concentration. Significant
                                                                          aggregation was observed only at tBID concentrations greater
                                                                          than 1 mm (tBID : lipid of approximately 1 : 1000, mol/mol).
                                                                          The line in Fig. 7A shows the time course of the tBID-induced
                                                                          aggregation. Neither proteinase K-treated tBID, caspase-8 nor
                                                                          BAK caused aggregation. The turbidity was unaffected even
Fig. 7. Aggregation of SLUVs induced by BID proteins. Aggregation was
                                                                          when these proteins were tested at ratios up to 1 : 25
monitored spectrophotometrically at 600 nm at 30 8C (A) Aggregation of
                                                                          (protein : lipid, mol/mol).
SLUVs (1 mm) on the addition of different amounts of tBID. The control       It has been reported that Bcl-xL can counteract the function
was tBID (10 nmol) pre-incubated with proteinase K for 10 min at 30 8C.   of BID, particularly tBID, due to the interaction of the
(B) Proteins were mixed with SLUVs (protein:lipid, 1:300, mol:mol)        hydrophobic cleft of the former with the BH3 domain of the
following pre-incubation of Bcl-xL with tBID or tBID (G94E) (1:1,         latter [19]. This is supported by the results described in Fig. 7B;
mol:mol) for 10 min at 4 8C.                                              Bcl-xL could prevent the aggregation of LUVs induced by
                                                                          tBID, but had no obvious influence on tBID (G94E), in which
                                                                          the BH3 domain is changed by point mutation. Li et al. have
vesicles was negligible. Compared with BID, tBID always                   described similar phenomena where overexpression of tBID has
showed higher affinity to the vesicles. Almost all of the BID             led to the clustering together of mitochondria in vivo [20].
proteins tested showed very weak binding affinity for the
DOPC vesicles (data not shown). These results showed that the             DISCUSSION
affinity of individual BID proteins to a particular membrane
parallels their capacity to induce leakage.                               BH3 domain-only proteins have been previously viewed as
                                                                          transdominant inhibitors that rely exclusively on dimerization
                                                                          with other Bcl-2 family proteins to exert effects on cell life and
Penetrance of the phospholipid monolayer
                                                                          death [15]. In this work, we have further demonstrated that BID
The experiments on vesicles were complemented by studying                 can directly induce the release of entrapped trypsin and
BID protein binding to a phospholipid monolayer. An increase              cytochrome c release from LUVs, as has been previously
in the surface pressure will be observed where the proteins have          reported [22]. The experiments described here were designed to
penetrated the phospholipid monolayer. As shown in Fig. 6A, at            understand the intrinsic activity of BID as a membrane-
an initial surface pressure of about 20 mN´m21, C-BID had the             integrating or pore-forming entity in the model membrane
highest penetrating ability among these proteins. The order was           system.
then tBID, BID and BID (D59A); BID (G94E) and N-BID had                      Firstly, we tested the hypothesis that BID causes leakage
almost no penetrating ability. The surface pressure did not               by a detergent-like mechanism. In our experiments with BID
54 D. Zhai et al. (Eur. J. Biochem. 268)                                                                                   q FEBS 2001

concentration kept constant, increasing the lipid concentration       experiments also show that C-BID has the highest potential
resulted in a decrease in the extent of leakage, while decreasing     ability to penetrate into membranes compared with BID or
the concentration of the lipid led to a reduction in leakage. This    tBID.
finding is in disagreement with the detergent-like hypothesis,           The N-terminal segment itself has no leakage-inducing
but is consistent with a monomer±multimer assembly pore-              ability, but may serve as an inhibitor of pro-apoptotic activity.
forming process [29]. Decreasing the lipid concentration lowers       The N-terminal domain remains intact at approximately the
the extent of leakage at a constant lipid:BID ratio, which would      same position and orientation after caspase-8 cleavage [10,11],
confirm this.                                                         but even in the uncleaved BID, the N-terminal helices may
   It has been reported that lipid composition may affect the         temporarily detach from the protein core, leading to some
efficiency of leakage induction by a pore-forming peptide. For        activity of the uncleaved protein [10]. Our results showed that
example, cholesterol significantly reduces the efficiency of          full-length BID contains much of its intrinsic function leading
leakage by the synthetic peptide GALA [27]. Our results also          to leakage of liposomes, which may be due to the temporary
showed that cholesterol can lead to a clear drop in the               detachment of N-terminal segment.
efficiency of BID-induced leakage. It can be envisaged that              In addition to inducing the passage of cytochrome c, tBID
penetration of BID into more rigid membranes is less favorable        can trigger the clustering of mitochondria around the nucleus to
for pore forming. 1,2-Dioleoylphosphatidylethanolamine also           form a ring in the early stage of apoptosis after transient
has an obvious effect on BID-induced leakage. Such a                  transfection of tBID-GFP into an ecdysone-inducible system
reduction is partially due to the decrease in the binding affinity    [20]. In the present paper, it is interesting to note that tBID, but
of BID protein for the liposomes, though it seems that for the        not full-length BID, shows the ability to aggregate SLUVs. The
same number of membrane-bound BIDs per vesicle, the amount            extent of aggregation depends on the amount of tBID added.
of leakage from DOPE-containing liposomes is still several-           These results mimic the function of tBID in vivo [20]. Full-
fold less than that from the DOPA/DOPC or DOPG/DOPC                   length BID can induce the leakage but is unable to aggregate
vesicles. During the preparation of the present paper, Szoka          LUVs.
et al. [28] reported that GALA-induced 1-aminonaphthalene-               Recently, the three-dimensional structures of both BID and
3,6,8-trisulfonic acid/p-xylene-bis-pyridinium bromide (ANTS/         tBID have been solved [10,11]. The surface electrostatic
DPX) leakage is also decreased when the vesicles contain              potential of BID does not reveal any unusually charged regions.
DOPE. They attribute the reduction to the lowering of bilayer         However, two hydrophobic patches appear on the surface. One
deformation energy caused by GALA aggregations that are               is the BH3 domain, where four partially conserved hydrophobic
adsorbed on the membrane surface in the presence of DOPE.             resides are exposed (I82, I86, L90, M97), the other is a large
Our results also show the influence of DOPE on the efficiency         hydrophobic cleft formed by L105, Y140, V150, L151 and
of leakage induced by BID. We postulate that the presence of          L154. The biological implication of the BID hydrophobic cleft
DOPE, which is well known as a nonbilayer-forming phos-               remains to be investigated. Inasmuch as cleavage by caspase-8
pholipid, may be unfavorable for the formation of pore                can greatly enhance the pro-apoptotic activity of BID, it has
structures, as in the case of GALA. It was reported previously        been hypothesized that such a dramatic activation is accom-
that proteins such as Bcl-2 and Bcl-xL are almost unable to           panied by a conformational change after cleavage. However,
form ion channels in neutral vesicles [12±15]. In our case, a         the overall structural integrity of BID is preserved on caspase-8
small amount of BID was found to bind with DOPC vesicles;             cleavage. Minor conformational change predominantly
hence only a small amount of entrapped molecules could be             occurred in the loop region near the cleavage site [10], but
released. The results obtained with DOPE, cholesterol and             the results from McDonnell et al. [11] suggest that a higher
1,2-dioleoylphosphatidylcholine-containing vesicles further           extent of the BH3 domain may be exposed following cleavage.
indicate that the pore-forming rather than the micellization          Considering the difference between BID and tBID in inducing
mechanism may account for the BID-induced leakage.                    the aggregation of LUVs, we postulate that different domains of
   The results of binding experiments with the monolayer may          tBID molecule may be responsible for the aggregation and
indicate that part of the differences between leakage-induction       leakage of LUVs. In other words, the BH3 domain of tBID
efficiencies of the different BID proteins is due to differences in   plays its main role in the clustering of liposomes, the BH3-
binding affinities for liposomes, particularly in the case of BID     independent hydrophobic cleft is more important for inducing
mutants and fragments. Compared with wild-type BID, an                leakage. Insertion of the hydrophobic H6 and H7 helical hairpin
excessive amount of BID (G94E) is required for the mutant to          perpendicularly into the lipid bilayer may form putative pores,
induce partial leakage. This result is in line with the lower         structurally similar to the pore-forming domain of bacterial
affinity of BID (G94E) to vesicles. Hence, we can conclude that       toxins [30]. This is further supported by the observation that
the BH3 domain of BID is necessary for its binding to                 Bcl-xL can counteract tBID-induced aggregation as the BH3
membranes.                                                            amphipathic helix of tBID binds to the hydrophobic cleft of
   The difference in the leakage activity of BID (D59A) and           Bcl-xL [20]. Adding Bcl-xL to tBID (G94E), which has a point
wild-type BID is unexpected, because the mutation site of BID         mutation in the BH3 domain, fails to prevent LUV aggregation.
(D59A) is not in the BH3 domain or other helices. Although its        Furthermore, it is interesting to note that, in contrary to the
binding activity with liposomes partially remains, it is no longer    results obtained with mitochondria in vivo, Bcl-xL cannot
capable of inducing leakage of LUVs. As a consequence of the          inhibit the activity of BID or tBID-induced leakage of LUVs.
point mutation, a suitable conformation of BID for pore               Possibly, in the former case, a BH3-dependent pathway for
formation may not be attained.                                        inducing leakage of mitochondrial membrane may co-exist
   Fragments of BID were used to identify which part may be           with a BH3-independent pathway [20].
responsible for inducing LUV leakage. The results showed that            The in vitro assays using model membrane systems have
C-BID keeps the leakage-inducing activity, while N-BID loses          facilitated the study of the mechanism by which BID damages
both the ability to bind to liposomes and leakage-inducing            mitochondria during apoptosis. Results from different
activity. We can propose that C-BID is responsible for inducing       approaches showed that the pore-forming rather than the
leakage of vesicles. Moreover, the results from monolayer             micellization mechanism may account for the BID-induced
q FEBS 2001                                                           BID-inducing leakage and aggregation of vesicles (Eur. J. Biochem. 268) 55

leakage of liposomes. Based on the results obtained, we                          14. Antonsson, B., Conti, F., Ciavatta, A., Montessuit, S., Lewis, S.,
tentatively suggest that the BH3 domain and the hydrophobic                           Martinou, I., Bermasconi, L., Bernard, A., Mermond, J.J., Mazzei, G.,
cleft of tBID may be responsible for the aggregation and                              Maundrell, K., Gambale, F., Sadoul, R. & Martinou, J.C. (1997)
leakage of phospholipid vesicles, respectively.                                       Inhibition of Bax channel-forming activity by Bcl-2. Science 277,
                                                                                 15. Schendel, S.L., Azimov, R., Pawlowski, K., Godzik, A., Kagan, B.L. &
ACKNOWLEDGEMENTS                                                                      Reed, J.C. (1999) Ion channel activity of the BH3 only Bcl-2 family
                                                                                      member, BID. J. Biol. Chem. 274, 21932±21936.
This work was financially supported by the National Natural Science
                                                                                 16. Green, D.R. (1998) Apoptotic pathways: the roads to ruin. Cell 94,
Foundation of China (39730130) and the Chinese Academy of Sciences. We
thank X. Wang, J. Yuan and Y. Tsujinoto for their generous gifts. We are
                                                                                 17. Shimizu, S., Narita, M. & Tsujlmoto, Y. (1999) Bcl-2 family proteins
also grateful to X. Han and X. Hang for their interesting discussions.
                                                                                      regulate the release of apoptogenic cytochrome c by the mitochon-
                                                                                      drial channel VDAC. Nature 399, 483±487.
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