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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
SPECIFIC AIMS
Apoptosis induced by oxidative stress has been shown to be involved in the development of neurodegenerative diseases. We hypothesized that since Bax is a key component in the apoptosis pathway, single-domain antibodies inhibiting Bax function should also be effective inhibitors of apoptosis in vivo. 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 to their mammalian host cells and rendered them highly resistant to oxidative-stress-induced apoptosis. These intrabodies would serve as drugs on their own in the context of gene therapy as well as a means for identifying small compound drugs for the degenerative diseases involving oxidative-stress-induced apoptosis.
PRINCIPAL FINDINGS
1. Bax-specific VHHs identified from a llama VHH phage display library inhibit Bax function in vitro
We initiated the search for single-domain antibodies with Bax-inhibiting properties by screening a naive llama VHH phage display library 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 (Fig. 1
A). All six VHHs bound strongly to Bax but not to control BSA antigen in phage enzyme-linked immunosorbent assays (ELISAs; Fig. 1B
). VHHs were expressed with C-terminal c-Myc-His5 tag in E. coli and purified to homogeneity by immobilized metal affinity chromatography for subsequent functional studies. The ability of the six VHHs to inhibit Bax in vitro was tested by monitoring reactive oxygen species (ROS) generation caused by recombinant Bax protein in isolated mitochondria. If the anti-Bax VHHs are inhibitory toward Bax preincubation in solution of isolated mitochondria with anti-Bax VHHs, followed by the addition of Bax should prevent this proapoptotic protein from permealizing the mitochondria, resulting in a reduction of ROS release from mitochondria.
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Indeed, for all the VHHs tested, we observed a significant decrease in ROS release from mitochondria incubated with VHHs and Bax compared with the ones incubated with Bax alone. Specifically, Bax3 and Bax5–2 showed greatest potential as specific Bax inhibitors, decreasing ROS production from mitochondria by 
55 and 90%, respectively. Recent observations have 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 variability of inhibition by different VHHs suggests that the VHHs should be blocking different sites on Bax. Specifically, Bax5–2 must be binding to a site critically involved in Bax function as it has the maximum inhibitory effect. Alternatively, the high inhibitory effect seen with Bax5–2 might be due to its higher affinity for Bax. Permeabilization of the mitochondria was also monitored through detection of cytochrome c release by Western blot. As in the previous ROS assay, isolated mitochondria in solution were preincubated with VHHs followed by the addition of Bax. We observed that mitochondria preincubated with VHHs had a significantly higher cytochrome c in its pellet fraction than the one incubated with Bax alone, demonstrating that the VHHs decreased the permeabilization of mitochondria initiated by Bax (data shown in the full-length article).
2. Bax-inhibiting single-domain antibodies are potent inhibitors of apoptosis in vivo as intrabodies
The assays performed with isolated mitochondria clearly indicated that the VHHs bind and inhibit Bax activity in vitro. Next, we studied the effects of these VHHs as intrabodies inside intact cells. SHSY-5Y cells were transfected with VHH genes in fusion with fluorescent tags [red fluorescent protein (RFP) or green fluorescent protein (GFP)] under the control of cytomegalovirus (CMV) promoter, and six stable cell lines were obtained each expressing a unique VHH. In addition, stable control cell lines containing genes for an irrelevant VHH, PTH50, or GFP were also established. Expression of the VHHs in each cell line was confirmed by Western blot in which cells transfected with only GFP produced a band at 27 kDa (lane 1), as expected. Cells transfected with GFP fused to a VHH produced a band with a mobility expected for its size, 41 kDa (lane 2), demonstrating the expression of the fusion protein in these cells (data shown in the full-length article).
To assess the protective capabilities of the six specific VHHs as intrabodies, we once again monitored the oxidative stress resistance. 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. Thus, we hypothesized that if the VHH intrabodies block Bax activity during oxidative stress, apoptosis should be prevented. In previous studies, it has been shown that exposure of SHSY-5Y cells to 100 µM H2O2 for 1 h results in a significant increase in the rate of apoptosis. By implementing this condition to the stable cell lines containing either VHHs or control genes, we monitored these cells 24 h after treatment for apoptotic features. Specifically, cells were stained with Hoechst reagent to monitor nuclear morphology; brightly stained and condensing nuclei were indicative of apoptotic cells. Cell viability counts for stable cell lines expressing anti-Bax intrabodies and treated with 100 µM H2O2 were virtually the same as those of nontreated/nontransfected cells and significantly higher than those for the control-treated cells: nontransfected GFP-transfected, RFP-transfected, and PTH50-RFP-transfected (Fig. 2
). Similar results were obtained for experiments with 200 µM H2O2/1 h treatment conditions.
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In addition, the ability of the anti-Bax VHHs to prevent mitochondrial destabilization and apoptosis was further confirmed using fluorescence staining JC-1 and Hoechst to monitor mitochondrial membrane potential and nuclear condensation, respectively. These findings were also supported by annexin V staining, lipid peroxidation, intrinsic ROS production, and caspase activation (data shown in the full-length manuscript).
CONCLUSIONS AND SIGNIFICANCE
In this study, we have reported 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 through blocking the proapoptotic effect of Bax on the mitochondria (Fig. 3
). This opens new opportunities for treating neurodegenerative diseases that involve cell death induced by oxidative stress and Bax activation.
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Currently, several research groups are working toward using intrabodies as therapeutic agents in various diseases. 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.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6306fje
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