Chaperone networks in protein disaggregation and prion propagation
Introduction
Proteins are the most versatile and structurally complex biological macromolecules, which are involved in almost every biological process. To be functional proteins must adopt a defined three-dimensional structure, termed the “native fold”. The energy barriers separating native and non-native conformations are usually small. Therefore native proteins are at permanent risk of unfolding. This holds especially true under environmental stress conditions, including e.g. heat or oxidative stress, which causes massive protein misfolding. A cellular quality control system composed of chaperones and proteases counteracts the accumulation of non-native conformers by either refolding or degrading them. However, when the generation of misfolded proteins exceeds the refolding or proteolytic capacity of a cell, protein aggregates accumulate. Protein aggregates are often linked to cellular dysfunction and fitness reduction and are associated with numerous diseases including neurodegeneration.
Protein aggregates display a remarkable structural variability, as seen in electron microscopy, depending on the nature of the involved protein and the stress conditions causing protein aggregation (Kawai-Noma et al., 2010, Wang et al., 2010). Heat-shock induced protein aggregates display a rather disordered or amorphous structure, potentially caused by the co-aggregation of a large diversity of differently misfolded protein species (Tyedmers et al., 2010). In contrast, a more restricted set of proteins forms amyloid-like aggregates in cells. Here, protein aggregation represents a more organized, sequence-specific process that leads to formation of well-structured cross β-sheet rich fibers where the intermolecular β-sheets run perpendicular to the fiber axis (Chiti and Dobson, 2006). Such amyloid-like structures also differ in their physicochemical properties compared to amorphous aggregates as they are SDS-insoluble and specifically bind dyes such as Congo Red. A subgroup of amyloidogenic proteins are prions, representing protein based heritable genetic elements composed of aggregated fibers, which self-perpetuate alterations in protein conformation and function. Amyloid-forming prion domains of yeast proteins are enriched in glutamine and asparagine residues and drive the sequence specific aggregation into β-sheet rich fibers. Oligopeptide repeats, located within prion domains, are additionally required for the replication and stable inheritance of some fibrils (Alberti et al., 2009, Osherovich et al., 2004).
All of the structurally diverse aggregates identified are substrates for the cellular protein quality control network. In bacteria, fungi, plants and mitochondria of eukaryotes the processing of disordered as well as highly structured protein aggregates is mainly achieved by the Hsp70 chaperone system and the AAA+ chaperone Hsp100, including the Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB family members, which constitute a bichaperone system that solubilizes and refolds aggregated proteins (Glover and Lindquist, 1998, Goloubinoff et al., 1999). Here we review how the bichaperone systems of E. coli and S. cerevisiae process structurally diverse protein aggregates and what the consequences for the aggregated protein species are.
Section snippets
Hsp104 and ClpB: threading machines for protein disaggregation
Hsp104 and ClpB belong to the AAA+ protein superfamily, which comprise a multitude of oligomeric ATPases associated with various cellular activities engaged in the remodeling of macromolecules. AAA+ proteins share the AAA domain, which is defined by a region of ∼230 amino acids in length, compromising conserved Walker A and Walker B motifs for nucleotide binding and hydrolysis (Wendler et al., 2012). In addition to ATP hydrolysis, AAA domains also drive protein oligomerization, usually into
Hsp70 involvement in protein folding at multiple levels
Hsp70 chaperones are conserved in all kingdoms of life and assist a variety of cellular processes including de novo protein folding, refolding of misfolded and aggregated proteins but also protein degradation and transport of proteins across membranes (Hartl et al., 2011). Hsp70s are composed of an N-terminal ATPase domain and a C-terminal substrate-binding domain, which are separated by a conserved linker region. Hsp70 binds to short exposed peptide segments enriched in hydrophobic residues
Hsp70 and Hsp104/ClpB in protein disaggregation: isolated vs. cooperative activities
While it is well documented that the cooperation of Hsp70 and Hsp104/ClpB is mandatory for the solubilization of many aggregates, disaggregation activities have also been reported for the individual chaperones alone. Pioneering in vitro work from Zylicz and coworkers demonstrated that the E. coli DnaK chaperone machinery (KJE) rescues heat denatured RNA-polymerase (Skowyra et al., 1990). This disaggregation activity was also documented for other aggregated substrates and KJE seems particular
Formation and propagation of yeast prion aggregates
Hsp104 function is not restricted to reverse stress-induced protein aggregation but is also required for the stable inheritance of prion aggregates. Prion aggregates result from the specific conversion of soluble proteins into ordered amyloids. Their propagation involves several steps, starting from prion fibril growth via incorporation of soluble monomers, followed by the generation of seed templates (propagons) by fibril fragmentation and their delivery to daughter cells during cell division (
Hsp104 is a key component for prion propagation
Hsp104 was the first identified cellular factor required for prion propagation. Yeast cells lacking Hsp104 were originally shown to be deficient in maintaining Sup35 prion aggregates (Chernoff et al., 1995). Further studies revealed that Hsp104 is essential for the propagation of all known yeast prions (Tuite et al., 2011). In vitro low Hsp104 levels can accelerate prion fiber assembly of Sup35 and Ure2, suggesting that it facilitates the conversion to the prion conformation (Shorter and
Old partners joining the prion cycle: interplay of Hsp40/Hsp70 and Hsp104 in prion propagation
Historically Hsp104 was viewed as the central key player in prion propagation. Meanwhile, mounting evidence indicates that the Hsp70 chaperone machinery plays an equivalent crucial role in prion maintenance (Fig. 2). Ssb and Ssa and its co-chaperones Ydj1, Sis1 and Sse1 associate with different prions including Sup35 and Rnq1 (Allen et al., 2005, Bagriantsev et al., 2008, Sondheimer et al., 2001). Isolation of Sup35 fibrils from yeast cell lysates even revealed almost quantitative
Acknowledgments
This work was supported by grants from the BMBF (Agenet) and the Deutsche Forschungsgemeinschaft (Bu617/17-1 and TY93/1-1) to B.B., A.M. and J.T., respectively. J.W. was supported by the Network Aging Research Heidelberg. We apologize to all those whose important work could not be cited owing to space limitations.
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