RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils

Abstract

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

“…In this model, the Rvbs would primarily play a role in regulating subunit stoichiometry. In the second alternative model, the ATPase stimulation caused by Ino80INS may reflect the Rvbs’ potential role for remodeling protein complexes, as Rvb1 and Rvb2 have been hypothesized to be involved in disaggregation of amyloid fibrils (Zaarur et al, 2015) and chromosome decondensation (Magalska et al, 2014). Importantly, our observation that protein, not nucleic acid, is a robust activator of Rvb1/Rvb2 helps exclude models involving a helicase-like role of the Rvbs in vivo.…”

Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo

“…The ability of PrP repeats to functionally substitute for those of Sup35 in [ PSI + ] maintenance suggests that they could play a similar role in mammals. Recent studies have identified potential candidates for this function, including the AAA+ ATPase RuvbL [102] or the combination of Hsp70, DNAJB1, and Hsp110 [103], although their contributions to mammalian prion propagation have not yet been addressed.…”

Chemical Cross-linking Mass Spectrometry for Profiling Protein Structures and Protein-Protein Interactions

“…First and foremost, cross-linking can take a snap-shot of real-time dynamic interaction networks on the scale of the entire interactome in vitro and in vivo [8][9][10]. Also, cross-linking can not only identify the direct interacting partners but also determine the interaction sites at the same time.…”

“…GPMAW is suitable for small-scale analysis. xQuest can be used for large proteome-wide cross-linking studies [8,9].…”

“…An isotopically tagged linker substitutes one or more atoms in the cross-linker with heavy stable isotopes, usually deuterium or 13 C. Peptides cross-linked with a mixture of heavy and light crosslinking reagents generate unique doublet peaks in the mass spectra, which not only facilitates MS detection of cross-linked peptides and but also enables targeted MS/MS of cross-linked peptides. Isotopically tagged cross-linkers have been used to investigate complex protein interaction networks at large scale [8,9].…”