Ultrastructure of nuclear aggregates formed by expressing an expanded polyglutamine

• Published in: Biochemical and biophysical research communications 2002-06, Vol.294 (2), p.429-440
• Main Authors: Hazeki, Noriko, Tsukamoto, Tadashi, Yazawa, Ikuru, Koyama, Minami, Hattori, Shunji, Someki, Iori, Iwatsubo, Takeshi, Nakamura, Koichiro, Goto, Jun, Kanazawa, Ichiro
• Format: Article
• Language: English
• Subjects: Aggregate; Amino Acids - analysis; Animals; Cell Line; Cell Nucleus - chemistry; Cell Nucleus - metabolism; Cell Nucleus - ultrastructure; Cell Nucleus Structures - chemistry; Cell Nucleus Structures - metabolism; Cell Nucleus Structures - ultrastructure; Flow Cytometry; Green Fluorescent Proteins; Heterogeneous nuclear ribonucleoproteins; Histone; Histones - biosynthesis; Humans; Huntingtin Protein; Huntington Disease - genetics; Huntington's disease; Luminescent Proteins - genetics; Macromolecular Substances; Mice; Nerve Tissue Proteins - genetics; Neuroblastoma - metabolism; Neuroblastoma - ultrastructure; Nuclear Localization Signals - genetics; Nuclear Proteins - genetics; Peptides - genetics; Peptides - metabolism; Polyglutamine; Protein Binding - physiology; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Ribonucleoproteins - biosynthesis; Transfection; Trinucleotide Repeat Expansion - physiology; Ubiquitin; Ubiquitin - biosynthesis
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/S0006-291X(02)00498-9

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.