Single-molecule Analyses of the Dynamics of Heat Shock Protein 104 (Hsp104) and Protein Aggregates

Abstract

Background:The process of disaggregation by heat shock protein 104 (Hsp104) and heat shock protein 70/40 (Hsp70/40) has not been elucidated. Results: We developed several methods to investigate the dynamics of Hsp104 at single-molecule levels. Conclusion: Statistical analyses revealed that Hsp70/40 affected the dynamics of Hsp104. Significance: Single-molecule approaches are a unique way to unravel the functional mechanisms of disaggregases.

Adenosine diphosphate restricts the protein remodeling activity of the Hsp104 chaperone to Hsp70 assisted disaggregation

“…Hsp70 not only facilitates disaggregation by the M-domain driven derepression of Hsp104 but also acts in the phase preceding translocation of the substrate by Hsp104. The upstream role of Hsp70 in disaggregation involves initial aggregate remodeling ( Zietkiewicz et al, 2006 ) and targeting Hsp104 to aggregates ( Acebrón et al, 2009 ; Okuda et al, 2015 ; Seyffer et al, 2012 ). Therefore, we asked whether the latter function of Hsp70 enables disaggregation in the presence of ADP.…”

“…Hsp70 interacts with the M-domain of Hsp104 and facilitates Hsp104-mediated protein renaturation at several stages of the process ( Miot et al, 2011 ; Rosenzweig et al, 2013 ; Schlee et al, 2004 ). Firstly, Hsp70 targets Hsp104 to aggregates and stabilizes the Hsp104-aggregate complexes ( Acebrón et al, 2009 ; Okuda et al, 2015 ; Winkler et al, 2012 ). Secondly, Hsp70 activates Hsp104 by affecting the position of the M-domain against the surface of NBD1 ( Haslberger et al, 2007 ; Lee et al, 2013 ; Lipińska et al, 2013 ; Oguchi et al, 2012 ; Seyffer et al, 2012 ; Sielaff and Tsai, 2010 ).…”

Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding

“…GroEL was prepared by hydrophobic interaction chromatography and size exclusion chromatography as previously reported 40 . Yeast Ydj1 was purified by anion exchange chromatography and hydroxyapatite chromatography according to the previous report 41 . Hexahistidine-tagged yeast Ssa1 was expressed in S .…”

Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

“…Single-molecule techniques offer an exciting avenue by which the complex interactions between Hsps, co-chaperones and clients can be probed in exquisite detail. While to-date the majority of singlemolecule studies have looked at the function of individual Hsps, a small number of studies have examined the effect of multiple components and cochaperones on protein folding and chaperone function [133,[144][145][146]158,159]. Notably, many studies that investigate the function of Hsp70 invariably examine the role of its co-chaperone Hsp40 in its chaperone function.…”

“…To date, the majority of single-molecule chaperone studies have investigated the molecular function of the Hsp70/40, Hsp60 and Hsp90 classes of Hsp, while Hsp100 disaggregases have been studied less intensively. This was addressed recently by Okuda et al [146], who for the first time attempted to observe the dynamic interactions between an aggregated client and the yeast Hsp104 machinery using singlemolecule fluorescence microscopy. In this work, a variant of TIRF microscopy, termed highly inclined and laminated optical sheet (HILO) microscopy, was used to illuminate further into the sample volume (N 150 nm) to observe larger luciferase aggregates.…”