1 Division of Nephrology and Dialysis, Department of Internal Medicine III, 2 Institute for Theoretical Chemistry and Molecular Structural Biology, and 3 Institute of Pharmacology, University of Vienna, A-1090 Vienna; and 4 Division of Nephrology and Dialysis, University of Innsbruck, A-6020 Innsbruck, Austria
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ABSTRACT |
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Bcl-2 protein family members are among the key regulators of the apoptosis effector phase. Therefore, we investigated the ability of synthetic peptides derived from proteins of the Bcl-2 family, namely, the NH2-terminal region of Bcl-2 (Bcl2_syn), a central domain of Bax (Bax_syn), and a central domain of Bak (Bak_syn) to interfere with the apoptotic process in LLC-PK1 cells. Apoptosis was induced by tacrolimus or lipopolysaccharide treatment, and microinjection of Bcl2_syn into stimulated LLC-PK1 cells significantly reduced the percentage of apoptotic cells detected within 4 h after the treatment. Microinjection of Bax_syn or Bax_syn, in contrast, induced apoptosis in otherwise untreated LLC-PK1 cells during the same period of time. A random sequence control peptide (Control_syn), which served as a negative control, as well as FITC-labeled dextran, which was coinjected in all experiments for visualization, were ineffective in either preventing or inducing apoptosis. These results suggest that synthetic peptides mimicking the functional domains of proteins of the Bcl-2 family are capable of regulating apoptosis when microinjected into LLC-PK1 cells in vivo. Analogs to these regulatory peptides could therefore provide valuable lead compounds in the therapeutical context.
apoptosis regulation; B cell lymphoma/leukemia-2; Bax; Bak; synthetic peptides
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INTRODUCTION |
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APOPTOSIS IS REGULATED BY external stimuli but also within the cell itself. Bcl-2 was first discovered because of its involvement in B cell malignancies, whereby chromosomal translocations activate the gene in the majority of follicular non-Hodgkin's B cell lymphomas (35). In 1988, Vaux et al. (36) were the first to report that Bcl-2 can prolong cell survival, and Hockenbery et al. (12) later demonstrated that this effect is mediated by its capacity to block apoptosis. As the Bcl-2 protein is expressed especially in long-lived cells and/or proliferating cell zones (13), it is not surprising that it is also found in human renal proximal tubule cells (29). Hence, Bcl-2 is an excellent candidate that may be involved in mechanisms that regulate apoptosis in kidney cells, such as LLC-PK1 (5). The Bcl-2 protein as well as other members of this family (such as Bag-1 and Bcl-xL) protects cells against apoptosis. In contrast, other Bcl-2-like proteins (such as Bak, Bad, and Bcl-xS) promote cell death. Thus, for any given apoptotic stimulus, the balance between death and survival appears to be determined by the ratio of apoptosis stimulating and suppressing Bcl-2 family members (30).
Cytoplasmic events, such as protease activation, rather than nuclear events, such as endonuclease activation, are assumed to play the initiating and primary role in mediating apoptotic cell death (24). Hence, a number of studies were carried out to elucidate the intracellular distribution of Bcl-2 homologues by antibody labeling and laser scanning microscopy techniques (17, 19, 22). The most prominent location of Bcl-2 proteins is the outer mitochondrial membrane, in which they show a patchy, nonuniform distribution. In addition, Bcl-2 is located at the endoplasmic reticulum but not in the plasma membrane, Golgi vesicles, or other organelles. A fraction of Bcl-2 is associated with nuclei as well (1), and the protein appears to span the inner and outer nuclear membrane, possibly forming a nuclear pore complex. The distribution of Bcl-2 over the nuclear membrane is not uniform, which suggests that it is targeted to specialized regions, although Bcl-2 lacks any of the common organelle-specific targeting sequences. Hence, Bcl-2 might specifically interact with certain protein complexes in intracellular membranes, such as the nuclear pore complex or the junctional complex in mitochondria. These complexes are both involved in protein transport across membranes (32).
Bcl-2 proteins display a complex domain structure, whereby the individual domains may subserve specific functions, such as homo- or heterodimerization, that are important in the apoptosis effector phase. This paper focuses on the distinct characteristics and functions of the three isolated domains Bcl2_syn, Bax_syn, and Bak_syn of the Bcl-2 family members Bcl-2, itself, Bax, and Bak, respectively. First, a structural and electrostatic analysis of the synthetically derived peptides in vitro is given, because both parameters are crucial to define specific molecular recognition sites. Thereafter, microinjection of these three peptides into renal proximal tubule epithelial cells was performed to investigate whether the three designed peptides might regulate apoptosis in vivo.
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EXPERIMENTAL PROCEDURES |
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Computational techniques. Secondary structure prediction of the peptide sequences Bcl2_syn, Bax_syn, and Bak_syn was performed by applying the neural network-based prediction routine PHDsec (33). The EMBL PredictProtein server was used to align the sequences of interest to homologous sequences with known secondary structures available in the SwissProt database.
Refinement of the secondary structure prediction as well as further analysis of the conformational space of the peptides was on the basis of a Monte Carlo simulated annealing (MCSA) algorithm using the program package MultiMize (Mayer B., personal communication). Peptide structure optimization was realized by using a combined optimization of potential energies (computed by means of the ECEPP-3 force field, see Ref. 28a) and free energies of solvation (calculated by means of a continuum approximation applying the Wesson-Eisenberg free solvation energy parameter set with the adjustment of Sharp; see Ref. 38) with an extended Metropolis criterion (see Ref. 25 for details of the method). In each MCSA step, one freely rotable bond was altered in the interval (Synthetic peptides. Bcl2_syn, Bax_syn, and Bak_syn were synthesized by Genemed Synthesis (South San Francisco, CA). Synthesis was checked by means of mass spectroscopy, and a purity of >95% was ensured by means of HPLC.
Bcl2_syn is defined by the sequence NH2-DNREIVMKYIHYKLSQRGYEW-COOH. This peptide resembles the BH4 domain of human Bcl-2 (9), in which 5 residues were shown to be critical for Bcl-2 activity, namely, Ile-14, Val-15, Tyr-18, Ile-19, and Leu-23, whereas the remaining 19 residues are not specifically required (20). A recent study analyzed the structure of this BH4 domain, and secondary structure prediction as well as circular dichroism spectroscopy suggested that this domain might be highlyCell cultures. LLC-PK1 are immortalized cells derived from pig renal proximal tubular epithelium (a kind gift from Drs. Walter Pfaller and Gerhard Gstraunthaler, University of Innsbruck). Nineteen 6-cm2 dishes with grid patterns forming 4-mm2 squares were used to seed the LLC-PK1 cells, which were cultured in 5 ml DMEM (GIBCO) containing 100 IU/ml penicillin-streptomycin (GIBCO), 4 mmol glutamine, and 5% FCS (GIBCO). Cells were incubated at 37°C in a humidified atmosphere and 5% CO2 for 48 h. For all experiments, confluent cell monolayers were used. The phenotype of the cells, the phenotypical change during adaptation to osmostress, and the typical appearance of apoptotic cells were determined previously (10, 39). Viability of cells was determined by light microscopy with and without trypan blue staining.
Before microinjection, DMEM was removed from LLC-PK1 cells and replaced by a buffer containing (in mM) 140 NaCl, 6.0 KCl, 2.0 CaCl2, 2.0 MgCl2, 20 glucose, and 10 HEPES, adjusted to pH 7.4 with NaOH.Apoptosis induction and microinjection.
Injection pipettes were made shortly before the experiments with a
Sutter P-97 programmable puller (Sutter Instruments). Microcapillaries (Type GB 100-TF8P, Science Products) were pulled to yield tip resistances of 50-80 M. Pipettes were loaded by retrograde
filling with 3 µl of a sterile filtered buffer [(in mM) 140 KCl,
1.59 CaCl2, 10 EGTA, and 10 HEPES, adjusted to pH 7.3 with
NaOH] containing 0.5% FITC-labeled dextran (70 kDa, 10 µM), to
which were added the peptides (in mM) 0.8 cytochrome c, 2.5 Bcl2_syn, 5.6 Bax_syn, 5.6 Bak_syn, and 5.6 Control_syn. An average of
100 cells were injected at a pressure of 100-200 hPa for 0.4 s, yielding an injection volume of ~1 fl of each of these solutions.
Control injections were done with FITC dextran in buffer alone as well
as with Control_syn (5.6 mM) and Bcl2_syn (2.5 mM) without externally
triggering apoptosis. In the experiments in which Bcl2_syn
injection was used to demonstrate its antiapoptotic effect in vivo,
cells were made apoptotic by preincubation with either 50 µM
tacrolimus (Fujisawa, Munich, Germany) for 2 h or 10 ng/ml
lipopolysaccharide (LPS) stimulation (LPS serotype O128:B12,
Sigma-Aldrich, Vienna, Austria) for 4 h (14, 26). The
time and concentration of tacrolimus and LPS were determined by kinetic
experiments using 1-100 µM tacrolimus between 1 and 4 h
(data not shown). Control injections with FITC-labeled cytochrome
c were used for comparison with the apoptotic effects of
Bax_syn and Bak_syn (3, 40).
Evaluation of apoptotic cells and viability. After 1, 2, 3, and 4 h, microinjected cells were counted by using fluorescence microscopy. Apoptotic cells were identified by morphological analysis using high-power light microscopy as suggested by Bortner and Cidlowski (2) and performed elsewhere (6), as well as by annexin V staining (Immunotech, Marseille, France). An anti-FITC antibody (Boehringer Mannheim, Germany) and the alkaline phosphatase anti-alkaline phosphatase technique with fast red as substrate was used to visualize the staining (DAKO, Vienna, Austria). Cells with morphological features suggestive of apoptosis were easily discriminated from nonapoptotic cells by the appearance of membrane blebbing and budding and the formation of apoptotic bodies. The number of annexin V-positive cells correlated well with the number of apoptotic cells obtained by morphological criteria (r2 = 0.93), although they were consistently higher. A total of 100 injected cells were counted in each individual experiment, and the fraction of apoptotic cells was recorded. Unless otherwise specified, the percentage of apoptotic cells was obtained by morphological criteria.
Statistics. Data are presented as means ± SE. ANOVA and Fisher's exact test were used to assess the significance of differences between the treatment and control groups. P < 0.05 was considered statistically significant.
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RESULTS |
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Structural properties of the isolated peptides.
Figure 1 gives the -helix formation
propensities [scaled in an interval (0, 9)] of the three
isolated peptides Bcl2_syn, Bax_syn, and Bak_syn applying PHDsec.
Clearly evident is the high
-helix propensity predicted for Bcl2_syn
and Bax_syn along the core of the peptides, i.e., the sequence
3REIVMKYIH11 for Bcl2_syn and
4KLSECLKRIGDEL16 for Bax_syn. Less well defined
is the
-helix in Bak_syn, showing overall decreased propensity and,
in addition, an
-helix break along amino acids 9-13 (IIGDD).
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Microinjection of Bcl2_syn peptide into tacrolimus- or LPS-treated
cells.
Tacrolimus treatment induced a time-dependent increase in the number of
apoptotic cells. The number of apoptotic cells was not
different between tacrolimus-treated uninjected cells and tacrolimus-treated cells that were injected with Control_syn (not shown); the percentages of apoptotic cells were roughly 3% at 1 h, 6% at 2 h, 9% at 3 h, and 10% at 4 h after
injection. Thus injection of the Control_syn peptide as well as
microinjection of FITC-dextran alone was not effective in preventing
tacrolimus-induced apoptosis. LPS stimulation led to a stronger
increase in apoptotic cell death compared with tacrolimus induction
(Fig. 2, A and B).
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Microinjection of Bax_syn, Bak_syn peptide, and cytochrome c.
The time course of the induction of apoptosis after
microinjection of various peptides is depicted in Fig. 2C. A
view of Bax_syn-injected apoptotic LLC-PK1 cells with
FITC and annexin V staining, as well as of nonapoptotic
LLC-PK1 cells injected with Control_syn cell, is given in
Fig. 3. For comparison, regularly
cultured nonmicroinjected native LLC-PK1 cells are also
shown. Cytochrome c led to a dramatic increase in the number
of apoptotic cells as early as 2 h after microinjection. The
percentages of apoptoctic cells 2, 3, and 4 h after microinjection
of cytochrome c were 8, 16, and 25% compared with 1, 2, and
4% for cells injected with Control_syn. As mentioned above, the time
course of spontaneous apoptosis in untreated, uninjected cells
was not statistically different from that in cells injected with
Control_syn and/or FITC-dextran (data not shown).
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DISCUSSION |
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We used synthetic peptides corresponding to the BH4 domain of Bcl-2 (Bcl2_syn) as well as peptides corresponding to functional segments of Bax and Bak (Bax_syn and Bak_syn, respectively) to interfere with the apoptosis effector phase. First, the secondary structures of these peptides were resolved to define sites potentially capable of molecular recognition, and then the peptides were microinjected into LLC-PK1 cells to investigate their functions in the regulation of apoptosis. The combination of these techniques allowed us to define basic requirements for the design of peptide mimetics as key regulatory elements of apoptosis.
A central issue of this study was the question of whether the peptides
may adopt stable secondary structures in solution to provide functional
molecular recognition sites. Our computational analysis demonstrated
the formation of extended -helices in Bcl2_syn. Previously, an X-ray
analysis of Bcl-xL (27), a protein highly homologous to Bcl-2, suggested a high degree of helicality for the BH4
region, which corresponds to the sequence of Bcl2_syn. This was
corroborated by circular dichroism spectroscopy, which indicated a
pronounced helicality for an isolated peptide corresponding to the BH4
region of Bcl-2 (20). Another important structural aspect
of Bcl2_syn is the presence of two opposing electrostatic elements. A
similar charge distribution has been reported for the BH4 domain of
Bcl-2 (20). Maintenance of the helical character as well
as of this characteristic charge must be assumed to be crucial for the
functions of the BH4 domain of Bcl-2 and, of course, for the isolated
peptide Bcl2_syn. The results obtained in our microinjection
experiments support this hypothesis by showing that Bcl2_syn is capable
of reducing the rate of apoptosis in tacrolimus-treated
LLC-PK1 cells.
Our computations also revealed a strong tendency to form -helices in
solution for Bax_syn (Figs. 1 and 2B). The sequence of this
peptide shows close homology to corresponding regions of Bak and Bik
(7) and is supposedly participating in Bcl-2 dimerization,
which may promote apoptosis. Bax_syn is also homologous to the
BH3 region of Bcl-2, and the BH3 domain also displays an
-helix in
Bcl-xL. Still, the BH3 region exhibits a function opposite to that of Bax_syn, which induced apoptosis in
LLC-PK1 cells. Charged amino acids within Bax_syn are
distributed in a way that the peptide exhibits an overall positively
charged NH2 terminus and a negatively charged COOH
terminus. This appears to represent a functional structural unit that
is sufficient to trigger apoptosis in LLC-PK1 cells.
The secondary structure of Bak_syn does not give a clear picture. Our
computational results indicate the formation of a random coil structure
in solution (Figs. 1 and 2C), and the electrostatics profile
also fails to provide any insight into predefined molecular recognition
sites. Nevertheless, an NMR study by Sattler et al. (34)
showed that this peptide is able to bind to the hydrophobic cleft
formed by the BH1, BH2, and BH3 regions of Bcl-xL, and, when complexed to Bcl-xL, this peptide may also form an
-helix. Our functional data showing the promotion of
apoptosis by Bak_syn support the idea that this peptide may
change from random coil to
-helix when recognizing and binding to a
respective complexation site on an apoptosis inhibitor (such as
Bcl-xL) of the Bcl-2 family.
During apoptosis, cytochrome c is released from mitochondria, and this release is supposed to be a critical trigger event for the activation of proapoptotic caspases. Accordingly, the microinjection of cytochrome c into the cytosol of human embryonic kidney 293 cells resulted in a dose-dependent induction of apoptosis (21). Similarly, microinjection of cytochrome c into normal kidney cells (NRK-52E) produced rapid apoptosis, which usually began within 30 min and reached a maximum of 60-70% after 3 h. Cells first showed shrinkage, then displayed multiple pseudopods, which rapidly extended and retracted, giving the cells a bosselated appearance (6). The same morphological features were detected in our studies on LLC-PK1 cells when injected with cytochrome c. In NRK-52E cells, apoptosis induced by cytochrome c was prevented by a caspase 3 inhibitor but not by the overexpression of Bcl-2, which indicates that cytochrome c acts further downstream within the apoptosis cascade than Bcl-2. Therefore, we used tacrolimus to provide an external trigger to stimulated apoptosis when investigating potentially antiapoptotic effects of Bcl2_syn.
Tacrolimus as well as LPS treatment and injection of Bax_syn and Bak_syn led to a significant increase in the number of apoptotic cells for as long as 4 h after injection. During this period of time, Bcl2_syn prevented apoptosis. Because the different peptides affected apoptosis in opposite directions at almost equimolar concentrations and the negative control peptide was ineffective in either direction, it is unlikely that intracellular osmotic or volume changes were responsible for the effects observed in Bax_syn- or Bak_syn-injected cells. The percentage of apoptotic cells after tacrolimus or Bax_syn and Bak_syn injection appear low on the first view. However, assuming a process time of 60-90 min from apoptosis induction to complete clearance and disappearance, a considerable number of cells would have undergone apoptosis after 4 h in culture (4, 8). This is especially important in the clinical setting of renal tubular cell injury, whereby a considerable number of cells are cleared by apoptosis.
Many studies have indicated the central role of members of the Bcl-2 family in the regulation of apoptosis in renal tubule cells in vivo (29). Bcl-2 is expressed in the normal and diseased kidney, particularly in distal and proximal tubule epithelial cells (5, 23, 28). Bax is expressed only faintly in healthy human renal tubule but is immediately upregulated after ischemic injury (data not shown). Bak, on the other hand, is expressed only in the distal convoluted tubule (19). Our present results indicate that synthetic peptides derived from members of the Bcl-2 family may be used to control the apoptotic process in renal tubule cells.
In conclusion, the artificial Bcl-2 peptide Bcl2_syn adopts a functional conformation in vitro and inhibits apoptosis in renal proximal tubular epithelial cells under quasi in vivo conditions. Bax_syn and Bak_syn are also functional peptides and induce apoptosis in these cells. Thus analogs to these regulatory peptides may provide valuable lead compounds in the therapeutic context.
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ACKNOWLEDGEMENTS |
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This study was supported by the Viennese Major Fonds (no. 1675), Austrian Science Fonds (FWF P-12037MED and P-13920MED), and Else Kröner Fresenius Fonds, Germany.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Oberbauer, Universitätsklinik für Innere Medizin III, Abteilung für Nephrologie und Dialyse, Währinger Gürtel 18-20, A-1090 Vienna, Austria (E-mail: rainer.oberbauer{at}akh-wien.ac.at).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 12, 2002;10.1152/ajprenal.00317.2001
Received 23 October 2001; accepted in final form 6 February 2002.
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