Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity

Abstract

Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein.Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.Supplementary material available online at

Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

“…However, it remained unclear whether expanded polyQ stretches embedded in native polyQ protein sequences like mHtt-exon1 are also inefficiently degraded by proteasomes in living cells. In the case of remaining polyQ stretches as partial cleavage products of incomplete proteolysis, these fragments would accumulate within the cell and start to aggregate, as previously shown in living cells expressing pure expanded polyQ peptides without flanking sequences (23).…”

“…The Htt-exon1-25Q-GFP construct was kindly provided by R. Kopito (Stanford University). GFP-Ub, GFP-Ub-Q112, and Ub-Q112 were generated as described previously (23). Ub-G76V-GFP was a kindly provided by N. Dantuma (Karolinska Institute, Stockholm).…”

“…Furthermore, levels of soluble and aggregated mHtt increase upon pro-teasomal inhibition in cell culture and HD mouse brain material (16 -18). Because IBs recruit proteins, including ubiquitin (Ub), many types of chaperones, and whole proteasomes, this suggests that cells attempt to clear the aggregation-prone mHtt protein by the proteasomal pathway (17,18,23,24).…”

“…3D). To prove that this particular antibody is able to detect pure polyQ tracts independent of the flanking Htt sequences by Western blot analysis, we expressed the constructs GFP-Ub-Q112 and, as a control, GFP-Ub in Neuro-2a cells and analyzed the polyQ peptides generated after N-terminal GFP-Ub hydrolysis by cellular deubiquitinases (23). The specific polyQ antibody 3B5H10 is able to detect polyQ peptides with a size of ϳ30 kDa (arrow) and higher molecular species, which may represent polyQ oligomers (asterisks) (Fig.…”

Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation

“…However, it remains unclear whether these nuclear structures are storage depots for misfolded proteins or are actively engaged in degrading misfolded proteins. Previous studies reported that N/Q-rich proteins cannot be degraded efficiently by the proteasome (74)(75)(76), unless proteasomal activity is increased (77). Therefore, we speculate that D. discoideum has evolved mechanisms to efficiently degrade and prevent the aggregation of prion-like proteins.…”