Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.

Paclitaxel-induced peripheral neuropathy (PIPN) is one of the serious adverse events associated with paclitaxel-based cancer treatments. A recent case study showed that the antiplatelet agent clopidogrel inhibits paclitaxel metabolism via cytochrome P450 (CYP) 2C8, resulting in severe PIPN. The aim of this study was to determine the impact of clopidogrel as a risk factor for the development of PIPN, using a retrospective cohort study. Data from paclitaxel-treated patients with or without clopidogrel and low-dose aspirin treatment were retrieved from medical charts. A total of 161 adult patients were included in this study: 135 were controls, 9 were clopidogrel-treated and 17 were aspirin-treated. The clopidogrel group had a greater proportion of males and a higher rate of comorbidities, such as diabetes mellitus and dyslipidemia, than the control group. However, patient characteristics were similar between the clopidogrel and aspirin groups. Severe PIPN was diagnosed in 3 (2.2%) and 2 (22.2%) patients in the control and clopidogrel groups, respectively (odds ratio: 12.0; p = 0.031). No patients in the aspirin group presented with severe neuropathy. These pilot data suggest that concomitant treatment with clopidogrel leads to a greater risk of PIPN. The avoidance of concomitant clopidogrel use may be effective in reducing clopidogrel-associated PIPN.

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem. Biophys. Res. Commun. 288, 321-327]. While analyzing the mutant cells, we found a novel Pex5p isoform (Pex5pM), which was shorter by seven amino acids than Pex5pL and longer by 30 amino acids than Pex5pS. Similar levels of mRNA syntheses for the PEX5 gene were observed in both the wild type and mutant cells, but the protein levels of Pex5p isoforms were markedly reduced in the mutant cells cultured at 37 degrees C and only slightly discernible at 30 degrees C, suggesting that they could be rapidly degraded. Furthermore, we characterized the peroxisomal localization of thiolase and acyl-CoA oxidase (Aox) in SK32 cells. The proteins in the organelle fraction were protected from proteinase K-digestion in the mutant cells, indicating that they were translocated inside peroxisomes. However, the conversion of Aox from component A to components B and C was completely prevented at both 30 and 37 degrees C, and the precursor form of thiolase was partially processed to the mature one in a temperature-sensitive manner. Transformed SK32 cells stably expressing one of the wild type Pex5p isoforms were isolated, and then the maturation steps for thiolase and Aox were examined. Pex5pM and S restored the processing of the two enzymes, but Pex5pL did not. In addition, Pex5pL prevented the maturation of thiolase observed at 30 degrees C. These results indicate that (i) the novel Pex5pM is functional and (ii) a seven amino acids-insertion, which is present in the L isoform but absent in the M isoform, plays some role in the process of maturation of thiolase and Aox.

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