Ultrastructure of nuclear aggregates formed by expressing an expanded polyglutamine.
Intranuclear inclusions have been observed in the brains of patients affected with Huntington's disease (HD). Neuro 2A cells that transiently expressed HD exon 1 bearing 74 glutamine repeats linked to the green fluorescent protein (GFP) and the nuclear localization sequence (NLS) contained aggregates in nuclei. The aggregates were purified by fractionation with centrifugation followed by fluorescence-activated cell sorting (FACS). Heat treatment of the aggregate in an SDS sample buffer caused the dense aggregate cores to disappear and generated a basket-like structure composed of fibrils. Biochemical analysis of the aggregates revealed that the HD exon 1-GFP fusion protein was the major component. The heterogeneous nuclear ribonucleoproteins F and H, histones and ubiquitin were found to be associated with the aggregates. Our observations suggest that the N-terminal fragment of huntingtin may organize the skeletal structure of the aggregates and may disturb normal cellular functions by trapping other proteins within the aggregates.
Hazeki
Noriko
N
CREST, Japan Science and Technology Corporation, Tokyo, Japan. nhazeki-tky@umin.ac.jp
Tsukamoto
Tadashi
T
Yazawa
Ikuru
I
Koyama
Minami
M
Hattori
Shunji
S
Someki
Iori
I
Iwatsubo
Takeshi
T
Nakamura
Koichiro
K
Goto
Jun
J
Kanazawa
Ichiro
I
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D022003:Cell Nucleus Structures / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006657:Histones / Q000096:biosynthesis; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D009447:Neuroblastoma / Q000378:metabolism / Q000648:ultrastructure*; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012261:Ribonucleoproteins / Q000096:biosynthesis; D014162:Transfection; D019680:Trinucleotide Repeat Expansion / Q000502:physiology*; D025801:Ubiquitin / Q000096:biosynthesis
D000596:Amino Acids; C086055:HTT protein, human; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006657:Histones; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; C097188:polyglutamine