Stability of the ABCD1 Protein with a Missense Mutation: A Novel Approach to Finding Therapeutic Compounds for X-Linked Adrenoleukodystrophy

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.

Aberrant DNA Methylation of ABC Transporters in Cancer

“…The human ABCD1 gene is found on chromosome Xq28 and contains ten exons. Mutations in ABCD1 cause X-linked adrenoleukodystrophy, a progressive neurodegenerative disease, characterized by the accumulation of very long chain fatty acids in plasma and tissues [ 107 ].…”

Peroxisomal membrane protein degradation in yeast

“…Here, Doa10p was suggested to play a major role in Pex15Δ30 degradation while Msp1p deals only with a fraction of Pex15Δ30 that is mistargeted to mitochondria. Because the authors followed the steady protein levels and not the rate of Pex15Δ30 turnover, it is plausible that in cells lacking Msp1p or Doa10p, Pex15Δ30 degradation is inhibited to a similar extent, suggesting that Msp1p and Doa10p act in the same pathway as proposed by (Matsumoto et al, 2019). Nevertheless, it is clear that Pex15Δ30 degradation is mediated by the ubiquitin proteasome system (UPS) which involves Msp1p, Doa10p and Cdc48p complex.…”

“…Until now neither the mechanism of instability nor the proteases responsible for degradation of the mutant proteins have not been fully characterized. Recently, Takahashi et al, reported that the degradation of mutant ALDP protein was partially inhibited by treatment with MG132, suggesting that these ALDP mutants are degraded via the proteasome , Interestingly, a subset of unstable ALDP mutant proteins in patient cells became stable under low-temperature culture conditions and exhibited proper peroxisomal localization (Morita et al, 2019) while under these conditions patient cells displayed increased residual β-oxidation activity (Zhang et al, 2011). Some of the ALDP mutants which were rapidly degraded in vivo still possessed ATP hydrolysis activity in vitro , indicating that mutant forms of ALDP retain a certain degree of functionality.…”

“…To date only four PMPs-Pex3p (yeast), Pex13p (yeast and plants), Pex15p (yeast) and ALDP (humans) have been observed to undergo degradation by the proteasome Morita et al, 2019;Pan et al, 2016;. Significantly, the role for ubiquitin in Pex3p, Pex13p and Pex15p turnover has been established however, the ubiquitination machinery (i.e) E2s and E3s involved in ALDP degradation have not been identified yet.…”

“…As a result, very-long chain fatty acids are not transported to-and metabolized within-the peroxisomes but instead accumulate in the brain and other tissues. (Engelen et al, 2014;Morita et al, 2019).…”