Formation of crystal-like protein aggregates leads to disease

Disease and treatment 2. apr 2021 3 min Professor Daniel E. Otzen Written by Morten Busch

Misfolding of proteins can lead to the formation of insoluble amyloid fibrils and harmful protein aggregates in one or more organs. This is best known from various neurodegenerative disorders such as Parkinson’s, Alzheimer’s and Huntington’s as well as amyloidosis, which damages the liver and heart. A new study provides new understanding of these protein aggregates and may eventually contribute to developing new strategies for prevention and treatment.

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Amyloidosis starts with fatigue, diarrhoea and weight loss and moves on to intestinal and skin bleeding and oedema. In severe cases, it gradually worsens within a few years and results in organ failure and death. Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are the most common outcome of the accumulation of complex and insoluble protein aggregates called amyloid fibrils, but they accumulate throughout the body. Researchers have had difficulty understanding why and when this happens but now have a clearer idea.

“Correct protein folding is crucial for proper protein function. The new study helps us to identify the link between normal folding and misfolding in a simple way by showing that the difference between folding and the creation of amyloid fibrils is whether the supersaturation barrier between proper and improper protein folds works or has broken down. By explaining the accumulation of amyloid fibrils through a relatively straightforward mechanism, we now have a simple framework to view the problem. Hopefully, new prevention and treatment methods can be developed to combat potentially harmful protein aggregates,” explains co-author Daniel Otzen, Professor at the Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, Aarhus University.

Like supercooled seawater

Led by Yuji Goto of Osaka University, the researchers tested how amyloids form from various proteins through heating and ultrasonication, which laboratories use to destroy cell walls to examine the contents. The tests examine which factors determine whether the proteins ended up in a folded, unfolded or amyloid state, with the latter often associated with neurodegenerative disorders.

“The existing hypothesis about protein folding, known as Anfinsen’s dogma or the thermodynamic hypothesis, states that the native structure of a protein represents a free energy minimum. When the amyloid fibrils were first discovered, it was thought that this dogma did not apply to small globular proteins, because the misfolded state found in amyloid fibrils apparently has a higher free energy and therefore should not be the most stable state for the protein to adopt,” says Daniel Otzen.

This is where supersaturation comes in. Supersaturation is a fundamental natural phenomenon that determines the phase transition of substances. A well-known example is supercooling of seawater, which remains liquid even if it is colder than 0°C. However, all the liquid immediately crystallizes and forms large flakes of ice simultaneously if one ice crystal is added to the solution.

“We observed the same phenomenon in our experiment with a supersaturated protein solution, which remains liquid until we change the temperature or use ultrasonic agitation, after which they precipitate. Depending on the type of protein, they crystallize into small ordered protein crystals or form larger, sometimes amorphous aggregates,” explains Daniel Otzen.

With the new experiments, the researchers have succeeded in dividing proteins into three main transition types – S, A and B – with different reaction patterns when the salt concentration or temperature changes.

“But regardless of the types of proteins, the supersaturation barrier is what keeps protein folding and amyloid fibril formation apart, and aggregation therefore occurs when this barrier breaks down,” says Daniel Otzen.

Also provides direct answers

The new study may prove to be key to one of the hottest fields in biology right now. Liquid–liquid phase separation is a theory of how cells can separate certain proteins through various types of liquid phases – without membranes. Although the fact that these membrane-free organelles are essential for a cell to function has been accepted for some time, an increasing number of studies show that liquid–liquid phase separation may also play a role in disease.

“This is especially interesting in relation to the many proteins that form the destructive aggregates in neurodegenerative disorders such as Alzheimer’s, amyotrophic lateral sclerosis and Parkinson’s because they are very prone to this phase separation. Although many studies raise new questions or even muddy the waters, this new study is illuminating and creates clarity, because suddenly many pieces slot into place in understanding how the harmful processes happen,” explains Daniel Otzen.

The new study supports and thus also explains well the strong association between liquid–liquid phase separation and the pathogenic process in these neurodegenerative disorders. This may benefit the process towards improving treatment. However, although there may well be a long way to go, the new study has provided even more direct answers for other disorders.

“In neurodegenerative disorders such as Parkinson’s, for example, small soluble protein aggregates do the damage. However, diseases such as familial transthyretin amyloidosis deposit large insoluble fibrils of transthyretin in the lungs and heart and cause irreparable damage. For these diseases, the new study directly tells us what happens physiologically and thus also very directly suggests what can be done about it,” says Daniel Otzen.

Breakdown of supersaturation barrier links protein folding to amyloid formation” has been published in Communications Biology. In 2017, the Novo Nordisk Foundation awarded a grant to Daniel Otzen for the project Specific Binders Crossing the Blood–brain Barrier to Diagnose and Combat Parkinson’s Disease.

Our research activities fall within 3 main areas: membrane protein folding, protein-detergent interactions and protein fibrillation. In all cases, we...

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