Abstract
Mutations in the ABCD1 gene that encodes peroxisomal ABCD1 protein cause X-linked adrenoleukodystrophy (X-ALD), a rare neurodegenerative disorder. More than 70% of the patient fibroblasts with this missense mutation display either a lack or reduction of the ABCD1 protein because of posttranslational degradation. In this study, we analyzed the stability of the missense mutant ABCD1 proteins (p.A616T, p.R617H, and p.R660W) in X-ALD fibroblasts and found that the mutant ABCD1 protein p.A616T has the capacity to recover its function by incubating at low temperature. In the case of such a mutation, chemical compounds that stabilize mutant ABCD1 proteins could be therapeutic candidates. Here, we prepared CHO cell lines stably expressing ABCD1 proteins with a missense mutation in fusion with green fluorescent protein (GFP) at the C-terminal. The stability of each mutant ABCD1-GFP in CHO cells was similar to the corresponding mutant ABCD1 protein in X-ALD fibroblasts. Furthermore, it is of interest that the GFP at the C-terminal was degraded together with the mutant ABCD1 protein. These findings prompted us to use CHO cells expressing mutant ABCD1-GFP for a screening of chemical compounds that can stabilize the mutant ABCD1 protein. We established a fluorescence-based assay method for the screening of chemical libraries in an effort to find compounds that stabilize mutant ABCD1 proteins. The work presented here provides a novel approach to finding therapeutic compounds for X-ALD patients with missense mutations.
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Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (16K09961), by the Platform Project for Supporting in Drug Discovery and Life Science Research from Japan Agency for Medical Research and Development (AMED), and by JSPS Core-to-Core Program, B. Asia-Africa Science Platforms. D.G.K. acknowledges the Matsumae International Foundation, Takeda Science Foundation, and Goho Life Sciences International Fund for fellowships. Pacific Edit reviewed the manuscript prior to submission.
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Communicated by: Nancy Braverman, M.D., M.Sc.
Take-Home Message
A novel approach to finding therapeutic compounds for X-ALD patients.
Conflict of Interest
Masashi Morita, Shun Matsumoto, Airi Sato, Kengo Inoue, Dzmitry G. Kostsin, Kozue Yamazaki, Kosuke Kawaguchi, Nobuyuki Shimozawa, Stephan Kemp, Ronald J. Wanders, Hirotatsu Kojima, Takayoshi Okabe, and Tsuneo Imanaka declare that they have no conflict of interest.
Informed Consent
All procedures were in accordance with the ethical standards of the responsible committee on human studies (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Written informed consent was obtained from all patients for being included in the study.
Animal Rights
The article does not contain animal subjects.
Author Contributions
TI conceived and supervised the study; TI, MM, AH, HK, TO, NS, SK, and RJW designed the experiments; MM, SM, AS, KI, DGK, and KY performed the experiments; and MM, SM, and DGK wrote the manuscript, which was discussed by all authors.
Electronic Supplementary Material
Suppl. Fig. 1
ABCD1-GFP and missense mutations in X-ALD patients. The mutated positions in the ABCD1 protein in fusion with GFP are used in this study (TIFF 1521 kb)
Suppl. Fig. 2
Recovery of mutant ABCD1-GFP by the treatment of MG132. CHO/mutABCD1-GFP (A616T, R617H and R660W) cells were cultured in the presence of MG132 (20 μM) for 20 h. After the incubation, cells were harvested and subjected to immunoblot analysis as in Fig. 2 (TIFF 1521 kb)
Suppl. Fig. 3
Recovery of mutant ABCD1-GFP (A616T) by incubating low temperature. CHO/mutABCD1-GFP (A616T) cells were cultured by incubating at 30°C for up to 5 days. After the incubation, cells were harvested and subjected to immunoblot analysis as in Fig. 2 (TIFF 1521 kb)
Suppl. Fig. 4
Stability of mutant ABCD1-GFP (A616T) in peroxisomes. CHO/mutABCD1-GFP (A616T) cells were incubated at 30°C for 5 days and incubated at 37°C for further 0, 12, 24, and 48 h. After fixation, cells were stained as in Fig. 2b (TIFF 1521 kb)
Suppl. Fig. 5
Measurement of fluorescence intensity with a spectrofluorometer. CHO/mutABCD1-GFP (A616T) cells were cultured on 96 well plate and incubated at 30°C for 5 days or with MG132 (20 μM) for 20 h. After the incubation, the cells were washed with 1 × HBSS and directly analyzed the fluorescence intensity. The fluorescence images of CHO/wildABCD1-GFP cells and CHO/mutABCD1-GFP (A616T) cells were also shown in the figure (TIFF 1521 kb)
Suppl. Fig. 6
Recovery of mutant ABCD1-GFP by the treatment of positive compounds. CHO/mutABCD1-GFP(A616T) cells were incubated in the medium containing each 19 positive compounds (5 μM) that were selected by measuring fluorescent intensity. After the 2-day incubation, cells were prepared for both immunofluorescence and immunoblot analysis as in Fig. 2. Among the 19 positive compounds, only 4 drugs (doxorubicin, idarubicin, aclarubicin, or bortezomib) showed the recovery of mutant ABCD1-GFPs (TIFF 1521 kb)
Suppl. Fig. 7
Effect of doxorubicin on the gene expression in CHO/mutABCD1-GFP and CHO/GFP-SKL. CHO/mutABCD1-GFP (A616T) cells and CHO/GFP-SKL cells were incubated in the presence or absence of doxorubicin (5 μM) for 48 h. After the incubation, total RNA was extracted and reverse transcribed into cDNA. The cDNA was used as template for real-time PCR using specific primer. The result shows the arbitral unit based on 18SrRNA (TIFF 1521 kb)
Suppl. Fig. 8
Effect of bortezomib on the recovery of mutant ABCD1 protein in X-ALD fibroblasts. X-ALD fibroblasts (A616T) were treated with bortezomib at the concentration of up to 100 nM for 2 days (a) or with bortezomib at 50 nM for 0, 3, 6, 12, 24, or 48 h (b). After the incubation, the total cellular protein (150 μg protein/lane) was separated by SDS-PAGE followed by immunoblotting using anti-ABCD1 or anti-β-actin antibodies (TIFF 1521 kb)
Suppl. Fig. 9
Cell viability of X-ALD fibroblasts in the presence of bortezomib. X-ALD fibroblasts (A616T) were cultured in the medium containing bortezomib (0, 5, 10, 20, 50, 100, 200, and 500 nM) for 2 days (closed bar) or 7 days (gray bar). After the incubation, cells were subjected to MTT assay. The values in the figure were indicated as percentage of the fibroblasts in the absence of bortezomib. Results are the means ± S.D.; n = 3 (TIFF 1521 kb)
Suppl. Table 1
Primer for mutation (DOCX 44 kb)
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Morita, M. et al. (2018). Stability of the ABCD1 Protein with a Missense Mutation: A Novel Approach to Finding Therapeutic Compounds for X-Linked Adrenoleukodystrophy. In: Morava, E., Baumgartner, M., Patterson, M., Rahman, S., Zschocke, J., Peters, V. (eds) JIMD Reports, Volume 44. JIMD Reports, vol 44. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8904_2018_118
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DOI: https://doi.org/10.1007/8904_2018_118
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