Modulating huntingtin half-life alters polyglutamine-dependent aggregate formation and cell toxicity.
A common finding among the expanded polyglutamine disorders is intracellular protein aggregates. Although the precise role of these aggregates in the disease process is unclear, they are generally ubiquitinated, implicating the ubiquitin-proteasome pathway in neuronal degeneration. To investigate the mechanism of aggregate formation, we have developed a cell culture model to express huntingtin designed to have an altered degradation rate through the ubiquitin-dependent N-end rule pathway. We fused the first 171 amino acids of huntingtin, containing either a pathogenic or normal polyglutamine tract, to the enhanced green fluorescent protein (EGFP). The half-life of soluble huntingtin-EGFP was dependent on the degradation signal and the polyglutamine tract length. However, once huntingtin-EGFP with a pathogenic tract had aggregated, the protein was extremely stable. Huntingtin-EGFP with a pathogenic glutamine tract and a shorter half-life displayed a delayed onset of aggregate formation and was more toxic to transfected cells. These data suggest that rapid clearance through the ubiquitin-proteasome pathway slows aggregate formation, yet increases cellular toxicity. Polyglutamine-induced neurotoxicity may therefore be triggered by non-aggregated protein, and aggregate formation itself may be a cellular defense mechanism.
Kaytor
Michael D
MD
Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA. kaytor001@umn.edu
Wilkinson
Keith D
KD
Warren
Stephen T
ST
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D010455:Peptides / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D013997:Time Factors; D014162:Transfection; D019680:Trinucleotide Repeat Expansion / Q000235:genetics; D025801:Ubiquitin / Q000378:metabolism
C086055:HTT protein, human; D000071058:Huntingtin Protein; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; C097188:polyglutamine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex