ALADINI482S causes selective failure of nuclear protein import and hypersensitivity to oxidative stress in triple A syndrome Makito Hirano*, Yoshiko Furiya*, Hirohide Asai*, Akira Yasui†, and Satoshi Ueno*‡ *Department of Neurology, Nara Medical University, 840 Shijo-cho, Kashihara 634-8522, Japan; and †Department of Molecular Genetics, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575, Japan Edited by William S. Sly, Saint Louis University School of Medicine, St. Louis, MO, and approved December 21, 2005 (received for review July 4, 2005) Triple A syndrome is an autosomal recessive neuroendocrinological the receptor (3). Although these findings have improved our disease caused by mutations in a gene that encodes 546 amino acid understanding of the biology of nucleocytoplasmic transport, it residues. The encoded protein is the nucleoporin ALADIN, a com- remains unclear which level of disruption results in cell dysfunction ponent of nuclear pore complex (NPC). We identified a mutant leading to human disease. Cronshaw et al. (4) have recently shown ALADINI482S that fails to target NPC and investigated the conse- that most disease-associated mutant ALADINs are predominantly quences of mistargeting using cultured fibroblasts (I482Sf) from a localized in the cytoplasm and not correctly targeted to NPCs. patient with triple A syndrome. ALADINI482S affected a karyo- However, further investigations are required to clarify how mistar- pherin-␣͞␤-mediated import pathway and decreased nuclear ac- geting participates in the pathogenesis of disease. This point re- cumulations of aprataxin (APTX), a repair protein for DNA single- mains unclear primarily because ALADINQ15K causes triple A strand breaks (SSBs), and of DNA ligase I in I482Sf. This decrease syndrome in homozygotes, despite the fact that it precisely localizes was restored by wild-type ALADIN. ALADINI482S had no effect on to NPC (4). imports of M9͞kap-␤2, BIB͞kap-␤3, histone H1͞importin 7, the Recent experimental and clinical studies have shed light on the ubiquitin conjugating enzyme UbcM2͞importin 11, or the spliceo- causative relationship between neurodegeneration and defects in some protein U1A, indicating that ALADINI482S selectively impaired repair of DNA damage. Exposure of adult motor neurons in transport of discrete import complexes through NPC. Cell survival suspension to oxidizing agents can induce both DNA single-strand assay showed hypersensitivity of I482Sf to L-buthionine-(S,R)- breaks (SSBs) and neurodegeneration (5). Reactive oxygen species sulfoximine (BSO), a glutathione-depleting agent. BSO decreased (ROS) induce nuclear loss of DNA repair proteins (Ku 70 and Ku nuclear APTX and ligase I levels in I482Sf and normal control 80), leading to apoptosis in mammalian cells (6). Aprataxin fibroblasts, but increased SSBs only in I482Sf. These observations (APTX), an SSB repair protein, deserves particular attention for several reasons. APTX-defective cells are hypersensitive to geno- implied that I482Sf are hypersensitive to BSO and no longer toxic chemicals that produce SSBs (7). Mutated APTX leads to the sufficiently repair SSBs. Consistent with this notion, I482Sf trans- development of autosomal recessive cerebellar ataxia (7–9). X-ray fected with both APTX and ligase I had increased resistance to BSO, repair cross-complementing group 1 (XRCC1), a molecular scaf- whereas I482Sf transfected with LacZ vector remained hypersen- fold of the DNA repair complex for SSBs, interacts with APTX (9). sitive to BSO. We propose that oxidative stress aggravates nuclear We have focused on DNA ligase I and chromatin assembly factor import failure, which is already compromised in patient cells. 1 (CAF1) p150 protein, two factors that participate in repairing Consequent DNA damage, beyond the limited capacity of DNA ROS-induced SSBs (10). Androgen receptor (AR) regulates gene repair proteins, i.e., APTX and ligase I, may participate in triggering transcription, and its mutant causes a human neurodegenerative cell death. disease, spinal, and bulbar muscular atrophy (11). In biological and medical contexts, we decided to analyze nuclear accumulations of aprataxin ͉ DNA ligase I ͉ nuclear pore complex the above proteins (APTX, XRCC1, ligase I, CAF1, and AR) to test the ability of cells to respond to oxidative stress and to repair riple A syndrome is a neuroendocrinological disease with an DNA damage. Tautosomal recessive mode of inheritance (1). In addition to the We report a mutant ALADINI482S that failed to target NPC in clinical triad of adrenocorticotropic hormone-resistant adrenal a pair of Japanese siblings with triple A syndrome. Here we provide failure that occasionally leads to life-threatening hypoglycemia, information on the consequences of ALADIN mistargeting in achalasia, and alacrima, triple A syndrome is commonly associated patient fibroblasts, termed I482Sf. We show that selective nuclear with progressive neurological impairments involving the cranial import failure was the primary defect caused by disease-associated nerves, autonomic nervous system, and motor neurons. Causative mutant ALADIN, which increased the susceptibility of cells to mutations were recently identified in a gene that encodes 546 amino oxidative stress and led to the accumulation of damaged DNA, acid residues, possessing a molecular mass of 60 kDa (2). The leading to cell death. encoded protein, termed ALADIN, is a component of nuclear pore Results complex (NPC). ALADIN was the first nucleoporin linked to I482S human inheritable disease (2). Vertebrate NPC comprises large ALADIN in Patients. Sequence analysis of the ALADIN gene multiprotein complexes (125 MDa) consisting of 80–100 different revealed that the two siblings with triple A syndrome were ho- nucleoporins. The NPC embedded in the nuclear envelope contains an aqueous channel and acts as the sole gatekeeper controlling the Conflict of interest statement: No conflicts declared. exchange of macromolecules Ͼ40–60 kDa between the cytoplasm and nucleus (3). To overcome this barrier, proteins destined for This paper was submitted directly (Track II) to the PNAS office. nuclear import have specific signals that are recognized by a Abbreviations: NPC, nuclear pore complex; NLS, nuclear localization signal; SSB, single- strand break; XRCC1, X-ray repair cross-complementing group 1; CAF1, chromatin assembly structurally related family of soluble transport receptor proteins, factor 1; APTX, aprataxin; AR, androgen receptor; GFPx2, GFP–GFP; BSO, L-buthionine- collectively termed karyopherin (kap). Typically, nuclear localiza- (S,R)-sulfoximine; GSH, glutathione; DEC, decylubiquinone; VA, all-transretinol; ROS, re- tion signal (NLS) containing cargo is bound by transport receptors. active oxygen species; 8-oxoG, 7,8-dihydro-8-oxoguanine. After the cargo-transport receptor complex is translocated into the ‡To whom correspondence should be addressed. E-mail: [email protected]. nucleus through NPC, RanGTP induces release of the cargo from © 2006 by The National Academy of Sciences of the USA 2298–2303 ͉ PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505598103 Downloaded by guest on September 27, 2021 Fig. 1. ALADINI482S mislocalizes from nuclear pore complex (NPC) to cyto- plasm in transfected HeLa cells. The localization of GFP–ALADIN is shown in the left column (green), and NPCs immunostained with monoclonal antibody mAb414 are shown in the middle column (red). The distribution of wild-type (wt) ALADIN and ALADINQ15K overlapped with that of NPC, whereas ALADINI482S and ALADIN312del localized to the cytoplasm, rather than to NPC in the right column (green͞red). mozygous and their mother was heterozygous for a T-to-G trans- MEDICAL SCIENCES version in exon 16, resulting in a substitution from phylogenetically conserved isoleucine to serine at codon 482, ALADINI482S. This mutation was identified in blood DNA samples from the three family members, as well as in skin fibroblast DNA from the female Fig. 2. ALADINI482S affects nuclear import pathways in I482S fibroblasts patient. I482Sf and age- and sex-matched normal control fibroblasts (I482Sf). (A) The kap-␣͞␤-mediated nuclear imports of GFPx2–NLSAPTX and were primarily cultured for this study. GFPx2–NLSSV40 were decreased in I482Sf. GFPx2–NLSXRCC1(239–266) showed iden- tical distributions in both I482Sf and normal control fibroblasts. stNLSXRCC1(239– ALADINI482S Mislocalized from NPC to Cytoplasm. HeLa cells were 403) was highly efficient for nuclear import in both I482Sf and normal control ͞ ␤ ͞ ␤ transfected with GFP–wild-type (wt) and GFP–mutant ALADINs fibroblasts. I482Sf showed normal nuclear imports of M9 kap- 2, BIB kap- 3, histone H1͞importin 7, and UbcM2͞importin 11 complexes and the spliceo- and then fixed and stained with monoclonal antibody mAb414 as an some protein U1A. (B) The above data expressed in a bar graph format NPC marker. mAb414 recognizes nucleoporins (Nup 358, Nup214, (mean Ϯ SD). *, P Ͻ 0.05, unpaired Student’s t test as compared with control Nup153, and Nup62) located at the cytoplasmic or nuclear faces of cells. NPC. GFP–wtALADIN and GFP–ALADINQ15K localized to NPC, whereas GFP–ALADINI482S and GFP–ALADIN312del mis- localized to the cytoplasm (Fig. 1). Although the findings for The nuclear import of GFPx2-NLSXRCC1(239–266) (mediated by ALADINI482S were consistent with those for most other ALADIN kap-␣͞␤) was identical in I482Sf and control cells. An additional mutants as reported by Cronshaw et al. (4), ALADINI482S was