Researchers from the Institute for Bioengineering of Catalonia (IBEC), located at the Barcelona Science Park, together with the Centre for Genomic Regulation (CRG) and the Wellcome Sanger Institute, have created the first map revealing how mutations affect a protein in its transition state—an ephemeral and difficult-to-study phase. This is an analysis on an unprecedented scale: they examined more than 140,000 versions of the Aβ42 peptide, which forms harmful plaques in the brain. The study, published in the scientific journal Science Advances, opens new avenues for preventing Alzheimer’s disease and suggests a method applicable to the study of other proteins involved in different pathologies.

This new large-scale study has mapped the first molecular events that drive the formation of harmful amyloid protein aggregates found in Alzheimer’s disease, pointing towards a new potential therapeutic target.

“The approach we used in this study opens the door to revealing the structures of other protein transition states, including those implicated in other neurodegenerative diseases. The scale at which we analysed the amyloid peptides was unprecedented – it’s something that hasn’t been done before, and we have shown it’s a powerful new method to take forward”, said Professor Ben Lehner, co-senior author, Head of Generative and Synthetic Genomics at the Wellcome Sanger Institute and ICREA Research Professor at the CRG. “We hope this takes us one step closer to developing treatments against Alzheimer’s disease and other neurodegenerative conditions” adds Lehner.

Understanding the origins of neurodegeneration

More than 55 million people worldwide suffer from dementia, and between 60% and 70% of cases are Alzheimer’s disease. Although current treatments can alleviate some symptoms, they do not stop or reverse the disease.

More than 55 million people worldwide suffer from dementia, and between 60% and 70% of cases are Alzheimer’s disease. Although current treatments can alleviate some symptoms, they do not stop or reverse the disease.

One of the key characteristics of Alzheimer’s is the accumulation of the beta-amyloid (Aβ) peptide in the brain. This peptide is a short chain of amino acids that, under normal conditions, is naturally produced and cleared. But in the case of Alzheimer’s, these molecules begin to cluster and stick together, forming elongated structures known as amyloid fibrils. Over time, these fibrils accumulate into plaques that disrupt the functioning of neurons and contribute to cognitive decline. For these fibrils to form, the Aβ peptides must pass through a highly unstable intermediate state, called the “transition state”, which requires energy and lasts for only a very short time. Due to this instability, this initial step does not occur in most people, but when it does, it can trigger the pathological aggregation process.

Understanding exactly how this transition occurs is key to developing treatments that prevent plaque formation before the disease begins. However, its fleeting nature makes it very difficult to study using traditional techniques, posing a significant challenge for Alzheimer’s research.

“Our method of study is crucial to understand the first events in the process of protein aggregation that leads to dementia, but it also offers a powerful framework to dissect the key initiating steps of many biological reactions, not just those we’ve studied so far. I look forward to seeing all the ways in which this strategy will be employed in the future”, stated Dr Benedetta Bolognesi, co-senior author and Group Leader of the Protein Phase Transitions in Health and Disease group at IBEC.

A large-scale analysis

The researchers used a combination of three techniques in order to handle large amounts of information about Aβ42 at the same time. The team used massively parallel DNA synthesis to study how changing amino acids in Aβ affects the amount of energy needed to form a fibril, and genetically engineered yeast cells to measure this rate of reaction. They then used machine learning, a type of artificial intelligence, to analyse the results and generate a complete energy landscape of amyloid beta aggregation reaction, showing the effect of all possible mutations in this protein on how fast fibrils are formed.

These techniques enabled the researchers to conduct the study at a large scale: “We measured the effect of more than 140,000 Aβ42 mutations and could apply neural networks, a type of machine learning, to extract, for each of them, the energy that drives the process of pathological aggregation.”, detailed Dr Mireia Seuma, co-first author formerly at IBEC and CRG, and now Senior Scientist at ALLOX, located at Barcelona Science Park. This scale has not been achieved before and helps improve the quality and accuracy of the models developed in the study. “Mutations, and the interactions between them, made it possible for us to “draw a portrait” of the transition state of the AB42 aggregation reaction. This is the key conformation driving the aggregation reaction, and it is extremely challenging (if not impossible) to study by classical biophysical methods”, adds Seuma.

The researchers discovered that only a few key interactions between specific parts of the amyloid protein had a strong influence on the speed of fibril formation. They found that the Aβ42 aggregation reaction begins at the end of the protein, known as the C-terminal region, one of the hydrophobic cores of the protein – the tightly packed water-repellent region of the peptide. As it is here where the peptide starts aggregating into a fibril, the researchers suggest that it is the interactions in the C-terminal region that need to be prevented to protect against and treat Alzheimer’s disease.

This is the first large-scale map of how mutations influence a protein’s behaviour in the notoriously difficult to study transition state. By identifying the interactions that drive the formation of amyloid fibrils, the team believes that preventing the formation of this transition state could pave the way for new therapeutic strategies, offering hope for future Alzheimer’s treatments.

» Article of reference: Anna Arutyunyan, Mireia Seuma, Andre J. Faure, Benedetta Bolognesi, Ben Lehner. Massively parallel genetic perturbation suggests the energetic structure of an amyloid-β transition state. Science Advances (2025). doi: 10.1126/sciadv.adv1422

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