Stability of the ABCD1 Protein with a Missense Mutation: A Novel Approach to Finding Therapeutic Compounds for X-Linked Adrenoleukodystrophy
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.
KeywordsABCD1 Bortezomib Green fluorescent protein Missense mutation X-linked adrenoleukodystrophy
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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