Aggregated proteins can cause diseases such as Alzheimer’s and Parkinson’s. Using cryo-electron microscopy (cryo-EM), Daniel Southworth and his team at the University of Michigan, in collaboration with Jim Shorter at the University of Pennsylvania, discovered how yeast solves the problem of protein aggregates. They observed on an atomic level how a protein called Hsp104 recognizes and unfolds proteins, thereby dissolving harmful protein aggregates (Science 357 273).
Many organisms have adapted to the threat of aggregated proteins by producing so called disaggregases. Now for the first time, researchers have observed the molecular mechanism of such a disaggregase.
The Hsp104 complex consists of six subunits arranged in a spiral with a central channel through which the aggregated protein is pulled. By switching between two conformations, one subunit after the other releases its grip on the target protein and grabs it again further up the chain. Every switch pulls the aggregated protein further through the channel, thereby unfolding it. The cooperative action of six subunits powered by adenosine triphosphate (ATP) hydrolysis provides enough force to even dissolve stable aggregates.
Like many other disaggregases, Hsp104 belongs to a group of proteins powered by ATP. These proteins fulfil diverse functions by changing conformation upon ATP hydrolysis. When ATP is hydrolysed to ADP, energy is released that allows the protein to move to another conformation. In the case of Hsp104, two parts move like little arms to release the target protein and grab it again further down the chain.
In order to observe this fast process, in their study Southworth and colleagues used a molecule that is similar to ATP but is hydrolysed more slowly. This gave them time to observe the unfolding process using cryo-EM.
The low temperature in cryo-EM makes it possible to take images of protein complexes while minimizing radiation damage that results from the necessary electron beam. The researchers collected images of protein complexes at random angles and averaged these to yield a three-dimensional structure. Resolution down to 4Å, at which it is possible to see amino acid side chains, allowed Southworth and his team to observe the mechanism at great detail.
When proteins avoid water
Ideally, a protein assumes its intended three-dimensional structure, called the native state, after being synthesized. By moving to the protein centre, this process allows hydrophobic regions of the protein to avoid contact with water. If the hydrophobic regions end up on the surface instead, for example due to high temperature, they will bind to neighbouring proteins to avoid water. As a result, aggregates form. This process is irreversible because the proteins are in a low-energy state that makes them very stable so that they cannot refold without energy input and the help of other proteins such as disaggregases.
Large aggregates of proteins disrupt normal cellular processes especially in nerve cells. Apart from Alzheimer’s and Parkinson’s, there are many other amyloidoses, diseases caused by protein aggregates, including amyotrophic lateral sclerosis (ALS), famous from the ice bucket challenge, and bovine spongiform encephalopathy, also known as mad cow disease.
In their study, which was published in Science on 21 July, the authors used casein, a protein commonly found in milk that readily aggregates, as a model substrate protein. Their assumption is that the mechanism of disaggregases on casein is the same as for physiological substrates. The discovery of this general mechanism might help to find drugs and therapies against aggregated proteins.
