Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.
The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.
Waelter
S
S
Max-Planck-Institut für Molekulare Genetik, D-14195 Berlin (Dahlem), Germany. wanker@molgen.mpg.de
Boeddrich
A
A
Lurz
R
R
Scherzinger
E
E
Lueder
G
G
Lehrach
H
H
Wanker
E E
EE
D048948:14-3-3 Proteins; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D005091:Exons; D006360:Heat-Shock Proteins; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000378:metabolism; D015151:Immunoblotting; D002479:Inclusion Bodies / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D046988:Proteasome Endopeptidase Complex; D011506:Proteins; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051843:Synucleins; D000074019:T-Cell Intracellular Antigen-1; D019076:Transgenes; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D051844:alpha-Synuclein
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; C086055:HTT protein, human; D006360:Heat-Shock Proteins; D000071058:Huntingtin Protein; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D039102:Poly(A)-Binding Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497604:SNCA protein, human; D051843:Synucleins; D000074019:T-Cell Intracellular Antigen-1; C071558:TIA1 protein, human; D014746:Vimentin; D051844:alpha-Synuclein; C067713:lactacystin; D014446:Tyrosine 3-Monooxygenase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine