ALADINI482S causes selective failure of nuclear protein import and hypersensitivity to oxidative stress in triple A syndrome.

[triple a syndrome]

Triple A syndrome is an autosomal recessive neuroendocrinological disease caused by mutations in a gene that encodes 546 amino acid residues . The encoded protein is the nucleoporin ALADIN , a component of nuclear pore complex (NPC ). We identified a mutant ALADIN (I 482 S ) that fails to target NPC and investigated the consequences of mistargeting using cultured fibroblasts (I 482 Sf ) from a patient with triple A syndrome . ALADIN (I 482 S ) affected a karyopherin-alpha /beta -mediated import pathway and decreased nuclear accumulations of aprataxin (APTX ), a repair protein for DNA single -strand breaks (SSBs ), and of DNA ligase I in I 482 Sf . This decrease was restored by wild-type ALADIN . ALADIN (I 482 S ) had no effect on imports of M 9 /kap-beta 2 , BIB /kap-beta 3 , histone H 1 /importin 7 , the ubiquitin conjugating enzyme UbcM 2 /importin 11 , or the spliceosome protein U 1 A , indicating that ALADIN (I 482 S ) selectively impaired transport of discrete import complexes through NPC . Cell survival assay showed hypersensitivity of I 482 Sf to l-buthionine-(S ,R )-sulfoximine (BSO ), a glutathione-depleting agent . BSO decreased nuclear APTX and ligase I levels in I 482 Sf and normal control fibroblasts , but increased SSBs only in I 482 Sf . These observations implied that I 482 Sf are hypersensitive to BSO and no longer sufficiently repair SSBs . Consistent with this notion , I 482 Sf transfected with both APTX and ligase I had increased resistance to BSO , whereas I 482 Sf transfected with LacZ vector remained hypersensitive to BSO . We propose that oxidative stress aggravates nuclear import failure , which is already compromised in patient cells . Consequent DNA damage , beyond the limited capacity of DNA repair proteins , i .e ., APTX and ligase I , may participate in triggering cell death .