Protein aggregation is an aberrant cellular process that has become a focal point of biological and medical research in recent years. It has been identified as the principle underlying a growing number of amyloidogenic and related protein folding diseases. The strong correlation between clinical patterns and the appearance of protein deposits documents that protein aggregation poses a severe threat to any living organism. Cells contain molecular chaperones, a dedicated set of proteins that prevent and combat aggregation. Among them is class of molecular motor proteins with the unique ability to reverse protein aggregation. These disaggregases
use the energy provided by ATP hydrolysis to actively dissolve protein aggregates by extracting individual polypeptide chains for their subsequent renaturation. Hsp104 from yeast has become a model system to investigate catalyzed disaggregation. Like other disaggregases, it forms barrel-shaped hexamers containing a total of 12 AAA+ domains. Our long-term goal
is to understand the mechanism of catalyzed protein disaggregation on a molecular level.
The Molecular Chaperone Hsp104
In yeast, the recovery from heat stress depends mainly on the activity of a single protein called Hsp104. Hsp104 is able to resolubilize protein aggregates which have formed in the yeast cytoplasm during exposure to high temperature. This process is assisted by a number of other proteins, and based on our studies we suggest the following mechanism (see Figure below): Hsp104 docks on the surface of an aggregate and "grabs" a part of a polypeptide that is trapped in the aggregate. It then starts to pull this molecule into its central channel. Since the channel is too narrow to allow the passage of the aggregate, the interaction between the trapped polypeptide and the rest of the aggregate is disrupted step by step. This process is called threading and eventually results in the release of an individual polypeptide chain from the channel exit. In essence, Hsp104 represents a cellular machine which converts chemical energy (ATP) into a mechanical pulling force that extracts polypeptide chains from aggregates.
Model for catalyzed protein disaggregation. (1) Hsp104 binds to the surface of an aggregate. (2) An exposed loop of a polypeptide chain (bold red) is inserted into the central channel of the chaperone. (3) By continuous rounds of ATP hydrolysis, the polypeptide is pulled further into the channel, resulting in its eventual release (4) from the aggregate. The disaggregation process is assisted by other proteins such as Hsp70 and Hsp40, which have been omitted for the sake of clarity.
Over the years, we have developed a toolbox of biochemical and biophysical methods we can use to resolve and analyze key steps of the disaggregation reaction, such as ATP binding and hydrolysis or polypeptide binding and release. We have also built up a large library of mutant Hsp104 proteins that allows us to identify the structural entities in Hsp104 responsible for individual steps in the disaggregation process such as polypeptide binding. In particular, we are interested in
- How does Hsp104 distinguish between aggregates and a healthy protein? After all, threading of healthy proteins may cause damage to the cell.
- How is the action between the 12 AAA modules of Hsp104 coordinated?
- How is protein disaggregation by Hsp104 connected to other cellular pathways such as protein synthesis and degradation? Preliminary results indicate that Hsp104 is embedded in a whole network of processes required to keep proteins in the cell active (protein homeostasis).
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