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.

Role of NH2-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: common features in eukaryotic organisms.

In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1-3 possesses the NH2-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH2-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH2-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH2-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH2-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH2-terminal H0 motif in organelle targeting is widely conserved in living organisms.

22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting oxidoreductases.

Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3ß-ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH(2)) formation as a dominant fluorescent product. Basing on LC-MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3ß-ol were deduced to be steroidal co-products of NBD-NH(2), indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors.