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* Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada; and
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada
1Correspondence: Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Ave., Windsor, Ontario, Canada, N9B 3P4. E-mail: spandey{at}uwindsor.ca
ABSTRACT
Bax is a proapoptotic protein implicated in cell death involved in several neurodegenerative diseases. Intracellularly expressed antibody (Ab) fragments (intrabodies) inhibiting Bax function would have potential for developing therapeutics for the aforementioned diseases and can serve as research tools. We report identification, cloning, and functional characterization of several Bax-specific single-domain antibodies (sdAbs). These minimal size Ab fragments, which were isolated from a llama VHH phage display library by panning, inhibited Bax function in in vitro assays. Importantly, as intrabodies, these sdAbs, which were stably expressed in mammalian cells, were nontoxic to their host cells and rendered them highly resistant to oxidative-stress-induced apoptosis. The intrabodies prevented mitochondrial membrane potential collapse and apoptosis after oxidative stress in the host cells. These anti-Bax VHHs could be used as tools for studying the role of Bax in oxidative-stress-induced apoptosis and for developing novel therapeutics for the degenerative diseases involving oxidative stress. —Gueorguieva, D., Li, S., Walsh, N., Mukerji, A., Tanha, J., Pandey, S. Identification of single-domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis.
Key Words: VHH • phage display library • mitochondria • neurodegenerative diseases
PROGRAMMED CELL DEATH or apoptosis is a physiological process essential for normal development and tissue homeostasis. Although the existence of cell death mechanisms is a protective measure for organisms that ensures the removal of unnecessary, damaged, or potentially dangerous cells, any deregulation or inappropriate induction of this process leads to the loss of healthy cells causing diseases. In particular, cell death in postmitotic tissues like brain and heart in adult organisms results in functional compromise, as is the case in Alzheimer’s disease, Parkinson’s disease (PD), and stroke. Cell death induced by oxidative stress has been shown to be involved in the development of these pathologies (1)
. Although the exact mechanism of cell death induced by oxidative stress is still unknown, mitochondria have been shown to play a central role in this process. Mitochondrial events such as opening of the permeability transition pores, mitochondrial membrane potential collapse, and release of proapoptotic factors such as cytochrome c and/or apoptosis inducing factors trigger the cascade of events leading to execution of apoptosis.
Bax is a 24 kDa protein of the Bcl-2 family with proapoptotic function. It normally resides in cytosol and translocates to mitochondria on induction of apoptosis, and it plays a key role in destabilizing mitochondria. Translocation of Bax to mitochondria followed by a conformational change in association with Bid leads to the release of cytochrome c, apoptosis-inducing factor, and caspase-9, which start the execution phase of apoptosis (2
, 3)
. Bax has been implicated in neuronal cell death during embryonic development and ischemia (4
, 5)
. Since Bax is involved in the early phase of apoptosis, developing specific blockers of Bax could protect neurons from oxidative-stress-induced apoptosis that is implicated in a number of neurodegenerative diseases.
Intrabodies to Bax with inhibitory action would have potential for the therapy of neurodegenerative disorders (6)
, in addition to being valuable tools for studying apoptosis. The efficacy of intrabodies critically depends on their stability (7
8
9
10)
. Since in the reducing environment of the cytoplasm intrabodies cannot form their stabilizing disulfide linkage(s), only those that are of sufficient stability can tolerate the absence of the disulfide linkage and be expressed in functional form (11)
. Traditionally, single chain Fvs (scFv, a molecule consisting of an Ab heavy chain variable domain, VH, and a light chain variable domain, VL, joined together by a linker) have been used as intrabodies. More recently, the feasibility of three types of sdAbs, VLs, VHs, and VHHs (VHs derived from camelid heavy chain antibodies), as intrabodies has also been demonstrated (10
11
12
13
14
15)
. Although they offer a comparable affinity (11
, 14
, 16)
, sdAbs have higher stability, solubility, and expression level than scFvs (16
17
18)
and thus are more efficacious as intrabodies (10
, 11
, 15)
. Intrabodies can be derived from monoclonal antibodies or Ab display libraries, e.g., Ab phage display libraries (6)
.
To combat neurodegenerative diseases resulting from death of postmitotic neuronal cells, it is important to understand how this process occurs. One proposed mechanism of apoptosis is the intrinsic reactive oxygen mediated pathway (19)
. Excess reactive oxygen species (ROS) released from the mitochondria has the ability to activate Bax leading to permealization of the outer mitochondrial membrane. As a result, various apoptotic-inducing factors and specific proapoptotic factors such as cytochrome c are released into the cytosol leading to a destructive apoptotic cycle and further ROS generation (20)
. We hypothesized that since Bax is a key component in this pathway, single-domain intrabodies inhibiting Bax function should also be effective inhibitors of apoptosis. We, thus, panned a llama VHH phage display library against Bax and identified several Bax-specific VHHs. Functional characterization of these VHHs showed that as intrabodies the VHHs were nontoxic in their mammalian host cells and effectively inhibited apoptosis induced by oxidative stress. These results also confirm the key role of Bax protein in apoptosis under oxidative stress.
MATERIALS AND METHODS
Panning and phage enzyme-linked immunosorbent assays
A llama VHH phage display library described previously was used in panning experiments (17)
. Panning against recombinant Bax protein was performed as described previously (17)
except that in the second and the third rounds, the phage elution additionally involved MgCl2/HCl treatment. First, the bound phages in the microtiter wells were eluted with 200 µl and neutralized with 100 µl 1 M Tris-HCl, pH 7.4 (17)
. Then, the emptied wells were subsequently incubated with 100 µl of 4 M MgCl2 at room temperature for 15 min. The eluted phage was removed and the wells were incubated with 100 µl of 100 mM HCl for 5 min at room temperature. The MgCl2/HCl-eluted phages were pooled, neutralized with 1.5 ml of 1 M Tris-HCl pH 7.4, and combined with the triethylamine-eluted phages. One milliliter of the combined phages was used to infect E. coli for overnight phage amplification, and the remaining 1 ml was stored at –80°C for future reference. VHH clones were identified from the titer plates by plaque-polymerase chain reaction and sequencing as described previously (21)
. After being panned, phage clones from titer plates were amplified in microtiter wells (21)
and screened for binding to Bax protein by standard enzyme-linked immunosorbent assays (ELISA) using a horse radish peroxidase/anti-M13 monoclonal antibody (mAb) conjugate (GE Healthcare, Baie d’Urfe, QC, Canada) as the detection reagent.
E. coli protein expression and purification
VHHs were cloned from the phage vector into the expression vectors by standard cloning techniques. E. coli expression of VHHs and subsequent purification by immobilized metal affinity chromatography were performed as described previously (21)
. Protein concentrations were determined by A280 measurements using molar absorption coefficients calculated for each protein (17)
.
Cell culture
Human neuroblastoma (SHSY-5Y) cells (American Type Culture Collection, Manassas, VA) were grown in complete medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) Ham’s F-12 media (Invitrogen Canada, Burlington, ON, Canada) with the addition of 2 mM L-glutamine (Invitrogen Canada) and 10% (v/v) FBS (Sigma, Oakville, ON, Canada) and 20 µg/ml gentamicin (Invitrogen Canada); 200 µg/ml Geneticin (G418; Invitrogen Canada) was added to all transfected cells. The cells were incubated at 37°C with 5% CO2 and 95% humidity.
Statistical analysis
P values for all graphs were calculated using Statistica Application for Windows 95, where P values <0.05 were assumed to be statistically different.
Mitochondria isolation and ROS measurement
SHSY-5Y cells were grown to 70% confluence in 10 ml Petri dishes. The intact mitochondria were isolated from these cells using a previously published method (22)
. Mitochondria were suspended in solution containing 0.25 M sucrose, 1 mM MgCl2, 10 mM HEPES, 4 mg/ml PHPA (p-hydroxyphenyl acetate), and 20 mM succinate (Sigma Canada). Mitochondrial ROS generation is measured by H2O2 generation rate, determined fluorimetrically by measurement of the oxidation of PHPA coupled to the reduction of H2O2 by horseradish peroxidase (Sigma Canada), based on a previously published protocol (22)
.
Detection of cytochrome c release by Western blot
Cytochrome c release was detected after incubating isolated mitochondria with VHH for 15 min followed by exposure to Bax for 5 min, in solution containing 0.25 M sucrose, 1 mM MgCl2, 10 mM HEPES, and 20 mM succinate. Samples were then spun down at 10,000 g for 5 min separating proteins of whole intact mitochondrial (pellet) and those released due to mitochondrial membrane permealization (supernatant). Pellet and supernatant fractions were solubilized in SDS-PAGE loading buffer, and proteins (50 mg protein/well) were then subjected to a 12% SDS-PAGE, followed by transfer on nitrocellulose membrane. The blots were probed with monoclonal anticytochrome c antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) followed by washing and a second incubation with horse radish peroxidase-conjugated anti-mouse antibodies. The blots were developed using a ChemiGlow West kit (Alpha Innotech, San Leonardo, CA) and recorded using an Alpha Innotech Imaging System. Integrated density values were calculated using Chemilmanager V5.5 program for Windows 95.
Mammalian cell transfection
Mammalian transfection of VHH fusion constructs was initiated by inserting the VHH genes in the HindIII/BamH I sites of pEGFP-N1 [VHH-green fluorescent protein (GFP) fusion], pDsRed1-N1 [VHH-red fluorescent protein (RFP) fusion] or HindIII/NotI site of pEGFP-N1 (VHH) (BD Biosciences, Mississauga, ON, Canada). The VHH recombinant vectors were propagated in E. coli and were purified using QIAprep Spin Miniprep kit according to the manufacturer’s instructions (QIAGEN, Mississauga, ON, Canada). The purified plasmids were subsequently used to transfect SHSY-5Y cells using Fugene 6 Transfection Reagent (Hoffmann-La Roche Ltd., Mississauga, ON, Canada) following the manufacturer’s protocol. Forty-eight hours after transfection, cells were transferred to complete DMEM media (as described above) containing 300 µg/ml geneticin for selection of positive transfected cells for 1–2 wk. Stable cell lines were subsequently maintained in complete DMEM media as described above with 200 µg/ml geneticin.
Detection of VHH expression in mammalian cells by Western blot
Equal amounts of protein extract (50 µg) from control cells containing GFP only and cells expressing specific GFP linked anti-Bax VHHs were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed with monoclonal anti-GFP antibodies (Sigma, St. Louis, MO) after which they were washed and incubated with horseradish peroxidase-conjugated anti-mouse antibodies. The blots were developed as described above.
Induction of oxidative stress
Working solutions of H2O2 were made by diluting a 10 M stock of H2O2 solution with distilled water to a concentration of 100 mM. SHSY-5Y cells were grown to 
70% confluence. Oxidative stress was induced by incubating the cells in complete media containing either 100 µM or 200 µM H2O2 for 1 h or 3 h at 37°C. The media were then replaced with fresh, complete media (without H2O2), and the cells were incubated for different time periods to monitor apoptotic features and oxidative stress parameters.
Monitoring nuclear morphology
Nuclear morphology was monitored as an indicator for apoptosis in cells by staining cells with Hoechst 33342 (Invitrogen Canada) to a final concentration of 10 µM. After incubating for 10 min at 37°C, the cells were then examined under a fluorescence microscope (Zeiss Axioskope 2 Mot plus, Gottingen, Germany) and fluorescence pictures were taken using a camera (QImaging, Gottingen, Germany). The images were processed using Improvision OpenLab v3.1.2, Jasc Paint Shop Pro v8.00, and Adobe Photoshop v8.0.
Mitochondrial membrane potential detection and measurement
Mitochondrial membrane potential was detected using JC-1 mitochondrial specific dye (Invitrogen Canada). The cells were treated with 10 µM JC-1 and incubated for 40 min at 37°C. The cells were observed under the fluorescent microscope, and fluorescence pictures were taken and processed as described above. Alternatively, mitochondrial membrane potential stability was also quantified using Dual Sensor: MitoCasp Assay (Cell Technology, Mountain View, CA, USA) as per manufacturer’s instructions.
Monitoring plasma membrane flipping
Annexin V (Invitrogen Canada) was used to monitor plasma membrane flipping in cells according to the manufacturer’s instructions. After being incubated for 15 min at 37°C, the cells were examined under the fluorescence microscope and fluorescence pictures were taken and processed as described above.
Lipid peroxidation determination
Lipid peroxidation in cells was determined using the thiobarbituric acid-reactive substances (TBARS) reaction with malondialdehyde and related compounds as described previously (23)
.
Caspase 3/7 activation measurement
The activation of caspase 3/7 was measured in cells using Apo 3/7 HTS High Throughput Screen Assay kit (Cell Technology) as per manufacturer’s instructions.
RESULTS
Identification of anti-Bax VHHs
We initiated the search for antiapoptotic single-domain intrabodies by panning a naive llama VHH phage display library (17)
against Bax. Screening of 38 colonies from the second and the third rounds of panning gave six different VHH sequences, namely, Bax1, Bax2, Bax3, Bax4, Bax5–1, and Bax5–2, occurring at frequencies of 9, 24, 2, 1, 1, and 1, respectively (Fig. 1
A). All six VHHs bound strongly to Bax but not to a control BSA in phage ELISAs (Fig. 1B
). VHHs were expressed in fusion with C-terminal c-Myc-His5 tag in E. coli and purified to homogeneity for subsequent functional studies.
|
Functional characterization of VHHs in vitro: inhibition of Bax activity in isolated mitochondria
The ability of the six VHHs to inhibit Bax was tested by monitoring Bax-induced ROS generation from isolated mitochondria, which as mentioned above correlates with mitochondrial destabilization (20)
. We hypothesized that if the anti-Bax VHHs are inhibitory toward Bax, preincubation of isolated mitochondria with anti-Bax VHHs, followed by the addition of Bax should prevent Bax from permealizing the mitochondria and lead to a reduced ROS release from mitochondria into the solution. Indeed, for all the VHHs (Bax2, Bax 3, Bax 4, Bax 5–1, and Bax 5–2) tested, we observed a significant decrease in ROS release from mitochondria incubated with VHHs and Bax compared to the fractions of mitochondria incubated with Bax alone or with Bax and an irrelevant VHH (P<0.05; Fig. 2
A). Specifically, Bax3 and Bax5–2 VHHs showed greatest potential as Bax inhibitors decreasing ROS production from mitochondria by 55 and 90%, respectively.
|
Permeability of the mitochondria was also monitored through detection of cytochrome c release by Western blot. Cytochrome c is released from the inner mitochondrial space into the solution when this organelle is destabilized (19)
. Thus, stable and healthy mitochondria are expected to show strong retention of cytochrome c. As in the previous ROS assay, isolated mitochondria in solution were preincubated with VHHs followed by the addition of Bax (mitochondria alone and in presence of recombinant Bax protein only were used as controls). When incubated with Bax, significantly higher cytochrome c release was seen in the supernatant fraction of the mitochondria incubated with recombinant Bax protein alone compared to those incubated with VHHs and Bax (Fig. 2B
); these findings were further confirmed by calculating the percent integrated density value of each band intensity (Fig. 2C
). Equal protein sample loading was confirmed in both instances by Ponseau S staining of the blots before incubation with blocking solution (data not shown). Conversely, mitochondria preincubated with VHHs showed significantly more cytochrome c in their pellet fraction (representing intact mitochondrial membrane) than the ones with no preincubation, demonstrating that the VHHs decreased the permeability of mitochondria initiated by Bax (data not shown).
Functional characterization of anti-Bax VHHs in situ inhibition of apoptosis by VHHs when expressed as intrabodies in mammalian cells
The assays performed on isolated mitochondria clearly indicated that the VHHs can bind and prevent Bax activity in solution and in isolated mitochondria. We further studied the effects of the VHHs as intrabodies inside intact cells. SHSY-5Y cells were transfected with all six VHH genes in fusion with RFP and GFP (Fig. 3
A) and 12 stable cell lines, each expressing a unique VHH fusion protein, were obtained. In addition, stable control cell lines containing genes for an irrelevant VHH (PTH50, a parathyroid-hormone binding VHH) or fluorescent proteins alone were established. Expression of the VHHs from both transient and stable transfections was confirmed by Western blot analysis of cell lysates using anti-GFP Ab (Fig. 3B
). In lane 1, cells containing GFP produce a band just under 30 kDa, consistent with the GFP molecular mass 27 kDa (24)
, although cells expressing each of the six anti-Bax VHH-GFP fusion proteins produce a band at 40 kDa, very close to the theoretical value, 41 kDa (Bax5–2, Bax3, and Bax2 are shown). Fluorescence microscopy was also used to "visualize" expression of VHH-RFP or VHH-GFP fusion proteins in cells (Fig. 3C
, Bax4-RFP is shown).
|
To assess the protective capabilities of the six Bax-specific VHHs in context of intrabodies, we again monitored the resistance of cells to apoptosis under oxidative stress. As previously discussed, oxidative stress due to mitochondrial ROS elevation has been linked to the activation of Bax and ultimate destabilization of the mitochondria leading to apoptosis (19, 25). Thus, we hypothesized that if the VHH intrabodies block Bax activity during oxidative stress, apoptosis would be prevented. In previous studies, it was shown that exposure of SHSY-5Y cells to 100 µM H2O2 for 1 h results in a significant increase in the rate of apoptosis (26)
. By implementing this condition to the stable cell lines containing either a VHH gene or a control gene, we monitored the cells 24 h after H2O2 treatment for apoptotic features. To this end, the cells were stained with Hoechst reagent where brightly stained and condensing nuclei would be indicative of apoptotic cells (Fig. 4
).
|
Untreated SHSY-5Y cells not expressing any VHH were used as a positive control with 96% cell viability (Fig. 5
). When the three negative control cell lines (nontransfected, transfected with GFP, or RFP only or transfected with PTH50) were exposed to 100 µM H2O2, a significant number of cells underwent apoptosis 24 h after the treatment as indicated by brightly stained condensed nuclei. Thus, the number of nonapoptotic healthy cells was reduced to 50% (Fig. 5)
. Interestingly, the cells containing any of the six anti-Bax VHH intrabodies in fusion with GFP or RFP showed very good resistance to the similar treatment with 87–93% viabilities and varied significantly from all the control treated cell lines (P<0.05). These values are very close to the viability values for the untreated, nontransfected SHSY-5Y cells (96%), demonstrating the effectiveness of the VHH intrabodies in preventing apoptosis.
|
Annexin V in parallel with Hoechst staining (Fig. 6
) was used to monitor plasma membrane flipping (another indicator of the early phase of apoptosis) and nuclear condensation, respectively. These assays further confirmed the above finding that apoptosis was inhibited in the cells expressing anti-Bax VHHs. Moreover, the expression of VHHs was nontoxic to their host cells as growth and proliferation was not hindered. The VHH-containing cells that survived the oxidative stress were viable and fully functional after several days after H2O2 treatment (Fig. 7
), as their growth rates were similar to those without any oxidative stress.
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Intrabodies prevent mitochondrial membrane potential collapse after oxidative stress and render the host cells resistance to apoptosis at higher level of oxidative stress
To further assess the degree of potency of the VHH intrabodies in preventing apoptosis, we increased the stress conditions. When the H2O2 (100 µM) exposure time was increased to 3 h, cells expressing VHHs showed almost identical survival rates as when exposed for 1 h (data not shown). When treated with 200 µM H2O2 for 1 h, control cell lines (containing no or the irrelevant VHH) showed a very high degree of apoptosis and poor survival as measured by Hoechst staining and trypan blue exclusion assay. Conversely, almost all cell lines contain each of the VHHs showed significant survival, with the most promising being the cells containing Bax1, Bax2, Bax3, and Bax5–2 VHHs (Fig. 8
). As before, the cell viabilities of the anti-Bax VHH expressing cells were shown to be statistically higher than all the control treated cell lines (P<0.05). Importantly, these results are in agreement to the ROS data (Fig. 2A
) obtained using VHHs in presence of isolated mitochondria. In addition, JC-1 staining of mitochondrial membrane potential after the 200 µM H2O2/1 h treatment (Fig. 9
) showed stable mitochondrial potential in cells expressing the anti-Bax VHHs comparable to the nontransfected, nontreated control SHSY-5Y. These results were further quantified using a newly developed mitochondrial membrane potential assay called MitoCasp assay. In this assay, a cell permeable cationic dye when exposed to cell fractions is accumulated in healthy mitochondria and exhibits a strong fluorescence signal (in the red), which can be measured using a fluorescence plate reader. Collapse of mitochondrial membrane potential leads to a decrease in the fluorescence. Results shown in Fig. 10
indicated that there was a considerable decrease in the fluorescence in the nontransfected SHSY-5Y cells after 100 µM H2O2/1 h treatment, compared to the nontreated control cells and the treated Bax5–2 expressing cells (P<0.05). Conversely, cells expressing Bax5–2 VHH produced strong fluorescence comparable to control nontransfected/nontreated SHSY-5Y cell (P>0.05). These data further confirm the ability of these intrabodies to protect the mitochondria from Bax-mediated permealization in the presence of oxidative stress (Fig. 10)
.
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In addition, we also monitored lipid peroxidation, another indicator of oxidative stress. When cells are exposed to higher ROS levels, most commonly due to mitochondrial damage, lipid deterioration is observed (23)
. Lipid peroxidation was assessed for cells expressing anti-Bax VHHs as well as nontransfected cells (with and without treatment), 24 h after exposure to 100 µM H2O2 for 1 h. We observed a significant decrease in lipid peroxidation in cells expressing anti-Bax VHHs, compared to nontransfected/treated SHSY-5Y cells (Fig. 11
; Bax3 and Bax5–2 are shown; P<0.05). This indicates that the VHHs are able to block mitochondrial permeabilization by Bax, thus, limiting the leakage of various apoptotic inducing factors and ultimately preventing cell death.
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Activation of executioner caspases 3/7 was also monitored in VHH-transfected cells using a high throughput screen for caspases 3/7 assay kit measuring the activity of these proteases as an increase in fluorescence. Specifically, this kit utilized a quenched (z-DEVD)2-R110 peptide, which is cleaved by active caspases 3/7, to release R110 free dye from the quenching caspase substrate DEVD. In this way the increase in fluorescence is indicative of capsase 3/7 activation in vivo. For this assay, oxidative stress was induced in control nontransfected (nontreated or treated) cells and Bax5–2 VHH-expressing cells (100 µM H2O2/1 h) and caspase activation was measured after 6 h. We observed a significant increase in fluorescence indicative of strong caspase activation in control nontransfected/treated SHSY-5Y cells, which was significantly lower in nontransfected/ nontreated and Bax5–2-expressing cells (P<0.05; Fig. 12
).
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Presence of GFP or RFP as fusion proteins does not alter the antiapoptotic activities of anti-Bax intrabodies
To further show that antiapoptotic activities of the anti-Bax VHHs are independent of their fusion context, cell lines of VHH intrabodies without fusion to GFP or RFP were also established. These cells were also monitored for their ability to resist oxidative stress induced apoptosis by treatment with 200 µM H2O2 for 1 h. Apoptosis was monitored after 24 h using Hoechst staining to detect apoptotic nuclei. As shown in Fig. 13
, cells expressing the "unfused" anti-Bax VHHs have cell viability rates that were significantly higher than all the treated control cell lines (P<0.05), indicating that the cells transfected without the fluorescent marker protein have comparable cell survival rates to their respective cells with fused VHH. These results clearly indicate that inhibition of apoptosis was solely due the VHHs and not due to the marker fusion protein.
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DISCUSSION
Here we report for the first time the identification of several nontoxic, Bax-inhibiting VHH intrabodies that phenotypically transform their host cells into cells that are resistant to oxidative-stress-induced apoptosis. This opens new opportunities for treating neurodegenerative diseases that involve cell death induced by oxidative stress and Bax activation. Recent observations by Kuwana et al. (27)
indicated that Bax initiates mitochondrial permeabilization by associating with Bid and lipids. Therefore, VHHs could inhibit the Bax function by binding to Bax at several sites: on the Bid-binding site, on the transmembrane domain, or at the site involved in Bax-Bax dimerization and activation. Since for all VHHs the same concentration of VHH was used, the differences in the degree of inhibition by different VHHs suggest that the VHHs should be blocking different sites on Bax. Specifically, Bax5–2 must be binding at the site critically involved in Bax function as it had the maximum inhibitory effect (in both in vitro and in vivo studies). Alternatively, the differences in the degree of inhibition by different VHHs may be due to their differences in their affinity for Bax.
The efficacy of intrabodies is determined by their affinity, stability, and expression levels inside cells. VHHs are preferable over scFvs for intrabody applications because of higher stability and expression levels. Binders obtained previously from the naive VHH phage display library (17)
used in this study have had KDs in the low micromolar range with KDs of a few micromolars at best (28
, 29)
. In accordance with this, one of the anti-Bax VHHs analyzed by surface plasmon resonance had a KD of 40 µM (data not shown). In the light of their low affinity, the VHHs in this study must have owed their potency to their high stability and expression levels. This underlines once again the importance of Ab stability, expression levels, and the suitability of VHHs as intrabodies. Moreover, the inherent stability of VHHs eliminates the need for further screening of lead intrabodies for stability (9
, 30)
. The smaller size of the VHHs is an advantage in cases where the intrabody needs to travel through intracellular membrane pores to exert its effect (15)
. If required, the intrabody potency of the current VHHs can be further enhanced by increasing their affinity by in vitro affinity maturation experiments.
There are several well-known and accepted apoptotic pathways, including extrinsic pathways involving cell surface receptors, as well as intrinsic pathways stemming from caspase-dependent and -independent mechanisms. One important intrinsic apoptotic pathway is ROS-induced apoptosis which results in destabilization of the mitochondria and release of proapoptotic proteins resulting in cell death. Due to the extreme vulnerability of the brain to oxidative stress, targeting this particular ROS-mediated pathway is of primary importance for the treatment of neurodegenerative diseases. The sensitivity of neuronal cells can be attributed to the large O2 exposure as well as limited antioxidants in the brain. In addition, a significant fraction of all brain lipids is polyunsaturated and, thus, easily oxidizable, and finally, the presence of iron in various regions of the brain has also been implicated in oxidative damage because of its catalytic role in production of radicals (31)
. Observations that the VHHs showed specific binding to Bax protein and were capable of inhibiting Bax-induced mitochondrial dysfunction in in vitro assays suggest that inhibition of apoptosis by these intrabodies must be due to their Bax-modulating activity in the cells.
To rule out the possibility that intrabodies might be acting simply as general antioxidants, we repeated our experiments using cells expressing an irrelevant VHH intrabody PTH50. The results clearly indicated that there was no protection against apoptosis by PTH50. Therefore, we can conclude that the specific binding to Bax and subsequent modulation of this proapoptotic protein by the anti-Bax VHHs is responsible for protection against oxidative-stress-induced apoptosis. Furthermore, we have shown that the presence of the anti-Bax intrabodies neither inhibit cell growth nor affect cellular morphology, suggesting that the VHHs are well tolerated in the cells and might not interfere with any other processes.
The interaction of Bax with VDAC on the mitochondrial outer membrane has been reported as a possible mechanism for Bax-induced permealization of the mitochondria and induction of apoptosis (33)
. Our in vitro and in vivo results have both shown that the presence of the anti-Bax VHHs can protect the mitochondria from Bax associated damage (by decreasing ROS generation and preventing MMP collapse, respectively). Our preliminary immunoprecipitation studies using anti-VDAC antibodies to coprecipitate Bax with VDAC from cell lysates of nontransfected and Bax5–2 expressing cells have shown that there is a disruption of Bax-VDAC association in the Bax5–2-expressing cells. This suggests that at least in the case of Bax5–2, the anti-Bax VHH may work by preventing the interaction between Bax and VDAC, thus inhibiting the opening of the PTP and overall apoptosis induced by oxidative stress.
Currently, several research groups are working toward utilizing intrabodies as therapeutic agents in various diseases (6)
. Beside their direct use as intrabodies in the context of gene therapy, the present VHHs could also be used as means to fish out specific and nontoxic small molecular mass inhibitors of Bax from pharmacophore libraries. Furthermore, the anti-Bax VHHs and the oxidative-stress-resistant stable cell lines in this study would be valuable research tools to elucidate the mechanism of mitochondrial permeabilization and apoptosis in general. Finally, the work presented here can be extended to other apoptotic proteins for obtaining a versatile pool of VHHs as a source of therapeutics and probes for delineating the mechanism of apoptosis.
ACKNOWLEDGMENTS
We thank Natasha Sharda and Ariel Burns for technical assistance. We thank Dr. M. Bani for technical assistance and scientific input and Dr. Roger MacKenzie and Tomoko Hirama for performing SPR experiments. We thank Tom Devecseri for helping with photography and imaging. This research project was supported by a Research in Aid Grant from Heart and Stroke Foundation of Ontario to S. Pandey and J. Tanha, a NSERC Discovery Grant to S. Pandey and HSFO graduate scholarship to Deyzi Gueorguieva.
Received for publication May 5, 2006. Accepted for publication July 5, 2006.
REFERENCES
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