ALADINI482S causes selective failure of nuclear protein import and hypersensitivity to oxidative stress in 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(I482S) that fails to target NPC and investigated the consequences of mistargeting using cultured fibroblasts (I482Sf) from a patient with triple A syndrome. ALADIN(I482S) 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 I482Sf. This decrease was restored by wild-type ALADIN. ALADIN(I482S) had no effect on imports of M9/kap-beta2, BIB/kap-beta3, histone H1/importin 7, the ubiquitin conjugating enzyme UbcM2/importin 11, or the spliceosome protein U1A, indicating that ALADIN(I482S) selectively impaired transport of discrete import complexes through NPC. Cell survival assay showed hypersensitivity of I482Sf to l-buthionine-(S,R)-sulfoximine (BSO), a glutathione-depleting agent. BSO decreased nuclear APTX and ligase I levels in I482Sf and normal control fibroblasts, but increased SSBs only in I482Sf. These observations implied that I482Sf are hypersensitive to BSO and no longer sufficiently repair SSBs. Consistent with this notion, I482Sf transfected with both APTX and ligase I had increased resistance to BSO, whereas I482Sf 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.
Hirano
Makito
M
Department of Neurology, Nara Medical University, 840 Shijo-cho, Kashihara 634-8522, Japan.
Furiya
Yoshiko
Y
Asai
Hirohide
H
Yasui
Akira
A
Ueno
Satoshi
S
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000328:Adult; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000378:metabolism; D004700:Endocrine System Diseases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009419:Nerve Tissue Proteins; D009422:Nervous System Diseases / Q000235:genetics / Q000378:metabolism*; D022022:Nuclear Pore / Q000737:chemistry / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D018384:Oxidative Stress / Q000235:genetics*; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D013577:Syndrome
C416506:AAAS protein, human; D009419:Nerve Tissue Proteins; D028861:Nuclear Pore Complex Proteins; D011506:Proteins; D019328:Buthionine Sulfoximine; D045643:DNA Repair Enzymes