Expression of extended polyglutamine sequentially activates initiator and effector caspases.
To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.
Miyashita
T
T
Department of Genetics, National Children's Medical Research Center, 3-35-31 Taishido, Setagayaku, Tokyo, 154-8509, Japan. tmiyashita@nch.go.jp
Matsui
J
J
Ohtsuka
Y
Y
Mami
U
U
Fujishima
S
S
Okamura-Oho
Y
Y
Inoue
T
T
Yamada
M
M
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000209:etiology / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D010051:Ovarian Neoplasms; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D008164:Luminescent Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D049452:Green Fluorescent Proteins; C097188:polyglutamine; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases