A Multiple Hit Model for the Diverse Pathogenic Effects Specific to Huntingtin Aggregate Stage in Cells
Huntington's disease is a fatal neurodegenerative disorder characterized by the aggregation of polyglutamine-expanded huntingtin into oligomers and fibrils. How protein aggregation leads to cellular dysfunction is not well understood. To address this question, we combined in-cell single molecule fluorescence spectroscopy with quantitative proteomics to define how huntingtin engages in aberrant protein interactions during its progressive aggregation. We find that huntingtin interacts preferentially with key components of distinct cellular processes as aggregation proceeds from soluble oligomers to end-stage inclusions. The aberrant interactions of soluble oligomers are highly enriched on RNA-binding proteins and with proteins functioning in ribosome biogenesis, translation, transcription, and vesicle transport. A significant characteristic of these interactors is the presence of extended low-complexity sequence regions. Compared to the soluble aggregates, the interactome of insoluble inclusions is significantly less complex and is enriched in protein quality control components. Our results suggest a 'multiple hit' model for polyglutamine interactions in pathogenesis, with detrimental effects on cell function occurring in an aggregation stage-dependent manner.
Sample Processing Protocol
The SILAC-MS experiments were performed as previously described. N2a cells were labeled in custom medium supplemented with light (L), medium (M) or heavy (H) arginine and lysine isotopes. For the total proteome analysis, cell lysates were adjusted to equal protein concentration and mixed at a 1:1:1 ratio. For analysis of the HttEx1SOL interactome, equal numbers of differentiated cells induced to express Q18, Q64, or Q150+ for 24 or 48 hr were lysed in 1 % Triton X-100 in PBS supplemented with Complete Protease Inhibitors. Cellular debris and polyQ inclusions (when present) were removed by centrifugation (5000 x g, 5 min). eGFP-tagged HttEx1 and its interactors were isolated from the supernatant fraction with anti-GFP MicroBeads. Bound proteins (HttEx1SOL) were eluted, adjusted to equal amounts of eGFP-tagged HttEx1 (determined by quantitative immunoblotting against the eGFP-tag), mixed at a 1:1:1 ratio and prepared for LC-MS/MS.
Data Processing Protocol
SILAC-MS spectra were analyzed using MaxQuant version 126.96.36.199. Ratios (Q150+/Q18 and Q64/Q18) normalized by the amount of purified eGFP-tag on HttEx1 were used to determine the fold-change of HttEx1SOL interactors. The Q18 interactome was analyzed using uninduced (without Q18 expression) cells as the background control. For the analysis of cellular proteome changes, MaxQuant normalized ratios were used to determine the fold-change.
Kim YE, Hosp F, Frottin F, Ge H, Mann M, Hayer-Hartl M, Hartl FU. Soluble Oligomers of PolyQ-Expanded Huntingtin Target a Multiplicity of Key Cellular Factors. Mol Cell. 2016 Sep 15;63(6):951-64 PubMed: 27570076