Results and Discussion Proton ion-induced Ca2+release measurement using indo-1 fluorescence and45Ca2+ We have previously suggested that BI-1 is a pH-depen-dent regulator of Ca2+ channel
Trang 1inhibitor-1 reconstituted into liposomes
Taeho Ahn1, Chul-Ho Yun2, Ho Zoon Chae2, Hyung-Ryong Kim3and Han-Jung Chae4
1 Department of Biochemistry, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea
2 School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea
3 Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Korea
4 Department of Pharmacology and Institute of Cardiovascular Research, Medical School, Chonbuk National University, Jeonju, Korea
Bax inhibitor-1 (BI-1; also known as ‘testis-enhanced
gene transcript’) is an antiapoptotic protein capable of
inhibiting Bax activation and translocation to
mito-chondria [1] Cells isolated from BI-1) ⁄ )mice exhibited
hypersensitivity to apoptosis induced by endoplasmic
reticulum (ER) stress [2] In BI-1) ⁄ ) mice, the
ische-mia⁄ reperfusion-induced unfolded protein response
increased significantly, leading to increased cell death
[3] This ubiquitously expressed protein has 237 amino
acids and a molecular mass of 26 kDa Computer
predictions and experimental observations suggest that
BI-1 is a membrane-spanning protein with six to seven
transmembrane domains and a cytoplasmic C-terminus localizing to the ER and nuclear envelope [4] Sequence homology among several species indicates that the characteristic hydrophobicity and ER mem-brane localization have been evolutionarily conserved [5] Functionally, BI-1 affects the leakage of calcium ions from the ER, as measured with Ca2+-sensitive, ER-targeted fluorescent proteins and Ca2+-sensitive dyes [2] BI-1 also regulates the production of reactive oxygen species through functional inhibition of Bax [6,7] In the BI-1 overexpression system, the increase in heme oxygenase-1 expression was also suggested as a
Keywords
antiporter; Bax inhibitor-1; Ca 2+ -release;
proteoliposome; reconstitution
Correspondence
T Ahn, Department of Biochemistry,
College of Veterinary Medicine, Chonnam
National University, Gwangju 500-757, Korea
Fax: +82 62 530 2809
Tel: +82 62 530 2823
E-mail: thahn@chonnam.ac.kr
H.-J Chae, Department of Pharmacology
and Institute of Cardiovascular Research,
Medical School, Chonbuk University, Jeonju,
Chonbuk 561-181, Korea
Fax: +82 63 275 2855
Tel: +82 63 270 3092
E-mail: hjchae@chonbuk.ac.kr
(Received 11 November 2008, revised 30
December 2008, accepted 10 February
2009)
doi:10.1111/j.1742-4658.2009.06956.x
We investigated the functional activity of recombinant Bax inhibitor-1 reconstituted into liposomes When proteoliposomes were suspended in acidic solutions, encapsulated Ca2+ was released from the membranes, as previously suggested [Kim HR, Lee GH, Ha KC, Ahn T, Moon JY, Lee
BJ, Cho SG, Kim S, Seo YR, Shin YJ et al (2008) J Biol Chem 283, 15946–15955] Concomitantly, proton ions were internalized when assayed using the time-dependent change in the fluorescence of the pH-sensitive dye oxonol V entrapped in the proteoliposomes The influx of proton ions was confirmed by observing tritium accumulation in the membranes However, the external acidity of the membranes per se did not induce proton ion influx without internalized Ca2+ These results suggest that reconstituted Bax inhibitor-1 has a Ca2+⁄ H+antiporter-like activity
Abbreviations
BI-1, Bax inhibitor-1; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; PICR, proton ion-induced Ca 2+ release.
Trang 2regulatory mechanism for reactive oxygen species
through the activation of Nrf2 transcription factor [8]
Recently, we also suggested that BI-1 acts as a
pH-dependent Ca2+ channel in the ER, which
increases Ca2+ leakage via a mechanism dependent on
both the pH and the C-terminal cytosolic region of the
protein [9] However, the precise role of BI-1 remains
unknown Our results, following the reconstitution of
recombinant BI-1 into membranes, support a role for
BI-1 as a Ca2+⁄ H+antiporter
Results and Discussion
Proton ion-induced Ca2+release measurement
using indo-1 fluorescence and45Ca2+
We have previously suggested that BI-1 is a
pH-depen-dent regulator of Ca2+ channel activity [9] To
ascer-tain the validity of the proper refolding and
reconstitution of recombinant BI-1 into lipid bilayers,
a functional assay was performed using
proteolipo-somes to monitor proton ion-induced Ca2+ release
(PICR) from the liposomes Rapid dilution of
BI-1-reconstituted vesicles in acidic solution induced the
release of entrapped Ca2+ (Fig 1), confirming BI-1
activity as a Ca2+ channel In kinetic analyses
(Fig 1A), the relaxation time was 37 s Using other
pH values (e.g pH 6.5 and 5.0), relaxation times were
calculated to be in the region of 35)42 s (results not
shown), in which any tendency for acidity was not
observed The amounts of Ca2+ released were similar
when assayed using a fluorescent dye and 45Ca2+
(Fig 1B) These results also suggest that increasing
acidity is linked to increased Ca2+release by the
pro-teoliposomes, implying that concomitant refolding and
reconstitution of BI-1 can be used as a functional
assay of the recombinant protein As a control
experi-ment, Ca2+efflux from proteoliposomes was measured
under alkaline conditions, such as pH 8.5 and 9.0, but
any remarkable Ca2+ release was not observed PICR
was also performed with liposomes in the absence of
reconstituted BI-1 Acidic or alkaline conditions did
not induce the release of encapsulated Ca2+ in the
absence of BI-1, when assayed using indo-1 and
45Ca2+ This suggests that the liposome was stable in
acidic or alkaline solutions and that the PICR
described in Fig 1 was entirely mediated by
reconsti-tuted BI-1
External Ca2+effect on PICR of reconstituted BI-1
External (cytosolic) Ca2+ is a well-known modulator
of the channel activity of the inositol
1,4,5-trisphos-phate (InsP3) receptor [10,11] To compare the pattern for stimulus-induced Ca2+ release between BI-1 and the InsP3receptor, we investigated the effect of external
Ca2+ on the PICR External Ca2+ consistently inhib-ited the PICR, regardless of the pH value outside the liposomes, when the Ca2+concentration was increased
Fig 1 Acidic or alkaline pH induced Ca 2+ release from BI-1 recon-stituted into lipid vesicles (A) The change in the fluorescence inten-sity of indo-1 was recorded kinetically after the rapid dilution of proteoliposomes into an acidic solution (pH 5.5) (B) The amounts
of Ca2+released by acidic or alkaline pH were expressed as a per-centage of the maximum releasable Ca 2+ using the fluorescence and the radioactivity of 45 Ca 2+ , and were compared with those caused by the addition of 1% Triton X-100 (TX-100), which was set
to 100% (C)pH 5.0 and (C)pH 9.0 represent the Ca 2+ release at pH 5.0 and 9.0 in the absence of reconstituted BI-1, respectively Data shown are the mean ± SE of five independent experiments.
Trang 3to 5 lm (Fig 2) It has been suggested that a biphasic
mode of external Ca2+ on the channel activity of the
InsP3receptor is important in generating characteristic
Ca2+ signaling within cells [12–15] Moreover,
although the effect of external Ca2+ differs according
to the type of InsP3 receptor, Ca2+ efflux exhibits a
biphasic pattern, with a maximal peak near
0.2)0.3 lm of cytoplasmic Ca2+[11,12] Therefore, the
mode of regulation by external Ca2+ suggests that
BI-1 shows Ca2+ signaling different from that of the
conventional InsP3 receptor, even though the other
regulation modes were not experimentally compared
Proton influx into proteoliposomes by exchange
with Ca2+efflux
To obtain more insight into the functional influence of
proton ions on the PICR and to test the possibility
that BI-1 incorporated into membranes might exhibit a
Ca2+⁄ H+ antiporter-like function, we measured the
changes in emission fluorescence for encapsulated
oxo-nol V (a pH-sensitive and non-permeable fluorescent
probe), which was entrapped in membranes in the
presence of internalized Ca2+, as described previously
[16] Figure 3A shows the time course of the decrease in fluorescence of the probe upon mixing proteoliposomes with acidic solutions Relaxation times were calculated to be in the range of 32)45 s,
Fig 2 Effect of external Ca 2+ on PICR of reconstituted BI-1 The
effects of external Ca 2+ on the PICR were plotted as external Ca 2+
concentration versus the amount of45Ca2+ released from
proteo-liposomes The external Ca 2+ concentration was buffered by the
EGTA–Ca 2+ chelating system and the experiment was performed
using the method described in Fig 1 Inset: the radioactivity by
45 Ca 2+ efflux was normalized to 100% upon the absence of
exter-nal Ca 2+ and expressed as a relative percentage.
Fig 3 Proton influx into proteoliposomes (A) The time course for the decrease in fluorescence of oxonol V encapsulated into proteo-liposomes was measured After rapid mixing of proteolipsomes containing internalized Ca2+with acidic solutions, the emission fluo-rescence at 630 nm (excitation at 610 nm) was measured as a function of time Lines a, b, c and d indicate an exterior pH of 6.5, 6.0, 5.5 and 5.0, respectively Line e represents the emission fluo-rescence in the absence of entrapped Ca 2+ (B) The amount of pro-ton uptake into liposomes as a result of the decrease in pH was assayed by measuring the radioactivity of tritium, as described in Materials and methods (In the figure, the y-axis represents the cpm values of pellet) Black and hatched bars represent the radio-activities of liposomes consisting of pure phospholipids without reconstituted BI-1 protein and the cpm values of proteoliposomes without internalized Ca 2+ , respectively, after the same procedure described above was performed White and gray bars represent the cpm values of proteoliposomes after mixing the vesicles with
pH 7.4 and each indicated acidic solution, respectively.
Trang 4which were very similar to the rate of Ca2+ release.
However, no correlation between the relaxation time
and the acidity was apparent As a control experiment,
the change in fluorescence was also analyzed under the
conditions described above, without encapsulated
Ca2+ In this case, we did not observe any significant
decrease in the emission fluorescence of oxonol V
(Fig 3A, line e) This indicates the importance of
entrapped Ca2+for the uptake of protons into
proteo-liposomes However, the experiment was not
per-formed in the presence of various concentrations of
internalized Ca2+ Therefore, it is not, at present,
pos-sible to determine how many proton ions are moved
into liposomes in response to entrapped Ca2+ions
Proton influx was also ascertained by measuring the
radioactivity of proteoliposomes containing
encapsu-lated Ca2+ (Fig 3B) When the proteoliposomes were
suspended in the indicated acidic buffer solutions
containing [3H], the radioactivity inside the
proteolipo-somes increased with decreasing external pH,
indica-tive of a pH-dependent proton uptake into the
proteoliposomes However, at pH 5.0, the radioactivity
of the proteoliposomes decreased, compared with the
radioactivity measured at pH 5.5, which was not
con-sistent with the results obtained using oxonol V As
expected, without reconstituted BI-1, the remarkable
proton influx could not be observed, regardless of the
external acidity By contrast, significant tritium
accu-mulations were shown with proteoliposomes in the
absence of internal Ca2+, which is different from the
change in oxonol V-mediated fluorescence described in
Fig 3A Furthermore, the cpm values increased with
increasing acidity This may be related to the finding
that the C-terminal region of BI-1, which contains a
cluster of charged residues, EKDKKKEKK, is
impor-tant in the protein function as a pH sensor [9], similar
to observations made for the TREK-1 potassium
chan-nel [17] Therefore, this cytoplasmic motif may bind to
proton ions and result in the increased cpm values (as
shown in Fig 3B), without proton uptake into
proteo-liposomes in the absence of internal Ca2+ However,
at present, there is no direct evidence for the
associa-tion of BI-1 with proton ions Taken together, these
results suggest that encapsulated Ca2+ facilitates
proton movement into proteoliposomes
As a control experiment, liposomes without
reconsti-tuted BI-1 protein displayed background levels of
radioactivity, suggesting that proton uptake was not
induced by differences in the concentration of proton
ions between the interior and exterior of
proteolipo-somes per se (black bars in Fig 3B) Collectively, these
results support the possible role of reconstituted BI-1
as a Ca2+⁄ H+antiporter
Proteolytic cleavage of reconstituted BI-1
On the basis of the result described in Fig 3, it was considered that the proton influx associated with Ca2+ efflux resulted from the membrane topology of BI-1, because recombinant BI-1 proteins could be incorpo-rated into artificial lipid bilayers with different confor-mations during proteoliposome formulation To test this possibility and to obtain insight into the confor-mation of reconstituted BI-1, proteolytic cleavage was performed with the prepared proteoliposomes using carboxypeptidase B, which was added externally The protease preferentially catalyzes the hydrolysis of the basic amino acids lysine, arginine and ornithine from the C-terminal end of polypeptides After cleavage, SDS⁄ PAGE analysis revealed that nearly all the recon-stituted BI-1 was partially digested by the protease and intact BI-1 could not be observed (Fig 4) This suggests that most recombinant BI-1 proteins have a protease-accessible structure in membranes during the reconstitution procedure although the precise confor-mation of BI-1 in a lipid bilayer is not currently known In relation to the membrane topology of BI-1, previous observations also support that the C-terminus
of BI-1 is outside the ER [18,19] After proteolytic digestion, the PICR was examined with the resulting proteoliposomes However, we could not detect any remarkable Ca2+release and proton influx (results not shown) Considering the proteolytic pattern on SDS⁄ PAGE, these results imply that the C-terminal region of BI-1 recognizing external acidity may be exposed to the outer surface of proteoliposomes, where they can be cleaved by the protease
In terms of the BI-1-induced protective function, the interplay of Ca2+ and H+ needs to be carefully
Fig 4 Proteolytic cleavage of proteoliposomes Reconstituted BI-1 was cleaved by carboxypeptide B and the products were analyzed
by SDS ⁄ PAGE Lanes 1 and 2 represent the reconstituted BI-1 and the digested sample, respectively.
Trang 5interpreted At normal pH, BI-1 has been suggested to
leak Ca2+, leading to a decrease in the Ca2+
concen-tration in the ER [9,20] In this study, a unique
func-tion for the BI-1–Ca2+⁄ H+antiporter is suggested At
normal pH, Ca2+ homeostasis may be maintained
even in the presence of other stimuli such as ER stress,
because of Ca2+porter activity In view of pH
homeo-stasis, proton uptake may be accelerated by a
Ca2+⁄ H+antiporter-like function of BI-1, leading to a
cell-protective function Therefore, it may be
antici-pated that the antiporter activity of BI-1 contributes to
adaptation of the channel protein and⁄ or ER Ca2+
regulation However, in exceptional surroundings, such
as at severely acidic pH, BI-1-overexpressed cells may
be highly sensitized to cell death, consistent with an
in vivo cell-based study [9] More detailed studies are
needed to establish the functional relevance of BI-1
antiporter activity to cell death
Conclusion
Proton ions induce Ca2+ efflux from
BI-1-reconsti-tuted liposomes containing entrapped Ca2+, and
con-comitantly proton influx Taking into account that the
movement of these ions was not observed without
encapsulated Ca2+and acidity, our results suggest that
the reconstituted recombinant BI-1 has a Ca2+⁄ H+
antiporter-like function
Materials and methods
All phospholipids were purchased from Avanti Polar Lipids
(Alabaster, AL, USA) The fluorescent Ca2+ indicator,
indo-1, and the pH-sensitive dye, oxonol V, were purchased
from Invitrogen (Carlsbad, CA, USA) Radioactive
materi-als45Ca2+and3H were obtained from GE Healthcare
Bio-Sciences (Piscataway, NJ, USA)
Construction of Bax inhibitor-1 expression
plasmid
Human BI-1 cDNA was subcloned into the pET12a
expres-sion vector to generate pET12a⁄ BI-1 using PCR PCR
amplification was designed to include BamHI and HindIII
restriction enzyme sites for forward and reverse primer
oli-gonucleotides, respectively (forward: 5¢-GACGGATCCAT
GAACATATTTGATCGAAAG-3¢, reverse: 5¢-GACAAGC
per-formed with Pfu polymerase (Stratagene, La Jolla, CA,
USA) The mixture was preincubated for 5 min at 94C
before the addition of the polymerase, followed by
amplifi-cation for 30 cycles: 94C for 90 s, 60 C for 90 s and
72C for 3 min The resulting PCR product was purified,
digested with BamHI and HindIII, and then ligated into a pET-12a vector treated with the same restriction enzymes The nucleotide sequence of the entire region including the BI-1 gene was analyzed by dideoxy sequencing
Expression of recombinant BI-1 protein
Cultures of Escherichia coli BL21(DE3) containing pET12a⁄ BI-1 were grown at 37 C in 500 mL of Luria–Ber-tani⁄ ampicillin (50 lgÆmL)1) until an attenuance at 600 nm
of 0.5 was attained Induction of the recombinant protein was carried out by the addition of 0.5 mm isopropyl-b-d-thiogalactopyranoside and further incubation for 4 h To obtain inclusion bodies of expressed BI-1, harvested bacte-rial pellets were resuspended in lysis buffer consisting of
25 mm Tris⁄ HCl (pH 8.0), 100 mm NaCl, 1 mm phen-ylmethanesulfonyl fluoride and 5 lgÆmL)1 of benzamidine, leupeptin and pepstatin The cells were lysed by passage through an Amicon French pressure cell (Millipore,
Billeri-ca, MA, USA) and the lysates were centrifuged (12 000 g,
10 min, 4C) to recover the inclusion bodies The pellets were resuspended with 5 mL of the lysis buffer using probe sonication and were centrifuged (12 000 g, 10 min, 4C) The resuspension and centrifugation steps were repeated five times The final inclusion body-containing pellet was dissolved in buffer A, which consisted of 20 mm Hepes (pH 7.4), 8 m urea, 0.1 mm dithiothreitol, 1 mm CaCl2 and 1.5% Chaps by incubating the sample at 25C for 10 min with gentle shaking
Refolding and reconstitution of BI-1 inclusion bodies into proteoliposomes
Chloroform solutions of lipids were stored in sealed amp-ules under argon gas at)20 C Phosphatidylcholine, phos-phatidylethanolamine and phosphatidylserine (all from bovine brain) dissolved in chloroform were mixed to give a respective molar ratio of 5 : 3 : 2 The phospholipid con-centrations were determined using a phosphorus assay [21]
A phospholipid concentration of 5 mm was used to recon-stitute the BI-1 protein The solvent was evaporated under
a stream of argon gas and the residual chloroform was removed by centrifugal lyophilization The dry lipids were hydrated with 1 mL of buffer A containing 20 lg BI-1 The mixtures were dialyzed for 12 h against an excess vol-ume of buffer B (buffer A without Chaps) containing 2 m urea The dialysis step was repeated for 12 h against an excess volume of buffer C (buffer B without urea) The resulting proteoliposomes were pelleted by centrifugation at
100 000 g for 30 min at 4C and washed with buffer D (20 mm Hepes, pH 7.4, 100 mm NaCl, 0.1 mm dithiothrei-tol, 0.5 mm EGTA, 1 m KCl) and then were dialyzed against buffer E (buffer D without 1 m KCl and 0.5 mm EGTA) for 12 h at 4C The resulting proteoliposomes
Trang 6were passed through Chelex 100 (Bio-Rad, Hercules, CA,
USA) to remove free Ca2+ The formation of
proteolipo-somes was monitored spectrofluorometrically by
measure-ment of light scattering during dialysis at a wavelength of
450 nm The amounts of reconstituted BI-1 protein were
determined using the NanoOrange protein quantitation
kit (Invitrogen)
Hydrogen ion-mediated Ca2+release from
proteoliposomes using indo-1 fluorescence and
45Ca2+
Ca2+ release from the proteoliposomes was measured as
described previously [4] Briefly, Ca2+efflux was observed
by measuring the fluorescence changes of external indo-1
(5 lm) after rapid dilution of the proteoliposomes with
acidic solutions (50 mm sodium phosphate pH 6.5 or
50 mm sodium citrate pH 6.0 and 5.5) at a ratio of 1 : 20
(v⁄ v) The fluorescence intensity was measured at emission
and excitation wavelengths of 393 and 355 nm, respectively
To quantify the proton ion-mediated release of Ca2+from
proteoliposomes, the acidity-induced fluorescence intensity
of indo-1 was compared with the fluorescence intensity
after addition of Triton X-100 to a final concentration of
1% (v⁄ v) Proteoliposomes were also prepared in the
pres-ence of 45Ca2+to include 20 000 cpm of 45Ca2+ under
the same conditions After the dilution of proteoliposomes
with acidic solutions to induce Ca2+efflux, the sample was
centrifuged (100 000 g, 30 min, 30C) The amount of
pro-ton-mediated Ca2+ release was determined by the
radioac-tivity of the pellet and supernatant using scintillation
counting
Measurement of proton ion influx into
proteoliposome
To analyze proton influx into proteoliposomes coupling
Ca2+ efflux, the pH-sensitive fluorescent probe oxonol V
was encapsulated inside proteoliposomes (pH 7.4) during
membrane formulation in the presence of Ca2+, as
previ-ously described [16] After rapid mixing of the
proteolipo-somes with each of the indicated buffer solutions (pH 9.0,
8.5, 6.5, 6.0, 5.5, and 5.0 as described above), the decrease
in fluorescence was measured at an emission wavelength of
630 nm (excitation at 610 nm) Proton uptake was also
investigated by measuring the tritium radioactivities
Prote-oliposomes with an interior pH of 7.4, in the presence or
absence of internalized Ca2+, were incubated with each
indicated buffer solution containing [3H] ( 20 000 cpm)
for 20 min at 30C The radioactivities of the pellet and
supernatant fractions were measured after centrifugation of
reaction samples using a Beckman TLA 100.2 rotor
(Beck-man Coulter, Palo Alto, CA, USA) at 70 000 rpm for
30 min
Removal of Ca2+contamination
Removal of Ca2+ contamination was conducted as described previously [22] Ca2+ contamination during all experiments was checked using the fluorescence of the
Ca2+indicator indo-1 before measurements
Proteolytic cleavage of reconstituted BI-1 with carboxypeptidase B
After reconstitution of BI-1, proteoliposomes were ultracen-trifuged as described above The pellet was suspended in
20 mm Hepes (pH 7.4) containing carboxypeptidase B at a BI-1⁄ carboxypeptidase B ratio of 50 : 1 (w ⁄ w) The sample was incubated at 25C for 20 min and the reaction was ter-minated by the addition of 1 mm EDTA (final concentra-tion) The products were analyzed by 12.5% SDS⁄ PAGE and followed by Coomassie Brilliant Blue staining of the resolved proteins
Acknowledgements
This work was supported by Korea Research Foun-dation Grant funded by the Korean Government (KRF-2008-314-C00231) and by Korea Science and Engineering Foundation Grant (R01-2006-000-10422-0)
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