Abstract
Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.
Highlights
A group of human maladies termed conformational disorders (CD) result from misfolding and aggregation of different underlying disease proteins [1]
This form of Htt103Q contains an N-terminal FLAG tag, the first 17 amino acids encoded by HTT exon 1, a polyQ stretch of 103 amino acids, a C terminal GFP tag, and the SV40 nuclear localization signal (NLS)
Htt103QNLS lacks the proline-rich region that flanks the polyQ domain in full length Htt, so it has reduced aggregation propensity and is toxic in strains that contain [RNQ+] prions [6,7,8,35,36]
Summary
A group of human maladies termed conformational disorders (CD) result from misfolding and aggregation of different underlying disease proteins [1]. Whether the large amyloid-like assemblies detected in disease states are themselves causative or a protective cellular defense mechanism against an unknown toxic species is a topic of debate [3]. Protection against proteotoxicity in CD involves preventing entry of native proteins into the amyloidlike assembly pathway or, once entry has already occurred, driving the formation of a benign aggregated species. In both cases, protein quality control (PQC) factors such as the molecular chaperone Hsp act to prevent accumulation of toxic protein species
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