“Amyloid proteins form almost insoluble fibrils of a very regular, quasi-crystal beta-cross structure,” said Malgorzata Kotulska of the Department of Biomedical Engineering, Faculty of Fundamental Problems of Technology, Wroclaw University of Science and Technology, Poland. “In humans they are notoriously associated with Alzheimer’s, Parkinson’s, Huntington’s, and many other diseases, including type-2 diabetes. However, there is also a wide range of functional amyloids that are beneficially utilised by many organisms for a variety of functions.”
Kotulska and her colleagues and students are looking at both pathogenic (the bad) and functional (the good) amyloid proteins in humans and microorganisms, and specifically at their impact on neurodegenerative and other diseases.
Amyloids are a particular kind of protein. Their experimentally resolved high-resolution structures are still scarce, with only a few representing the functional amyloids. Structure modelling, even with the cutting-edge method, such as Alpha Fold, cannot provide answers, as shown by research done in the Kotulska’s group.
All amyloids, however, have well-packed structures reminding one of a zipper, difficult to get rid of, which can be very harmful to biological functions of cells. Chemical agents can destroy some but very few, and most of them cannot be used in the human organism.
She explained some of the history of research on amyloids noting that they were first observed as far back as the 17th century when it was found out that they could be dyed with iodide, and for a long time they were thought to be starch.
“Physicians didn’t know if they were the reason for or effect of a disease,” she said.
The first more detailed research on amyloids was done in 1906 by German psychiatrist Aloys Alzheimer who, during a post-mortem study, identified amyloid plaques in the brain of his patient and successfully identified the first case of Alzheimer’s disease, which then carried his name. Degeneration of the neurons in Alzheimer’s is specifically linked to the presence of amyloid fibrils.
Kotulska explained that the development of Alzheimer’s disease occurs as a cascade of events in which amyloid precursor protein is incorrectly cut creating Abeta 42 peptide and its amyloid plaques outside the neurons. They affect tau proteins, which start forming tangles inside the neurons. It leads to neuronal death due to the disrupted metabolic and immunological pathways. The original cutting may be dependent on genetic or environmental factors, and people may also have Abeta plaques without showing Alzheimer’s symptoms. It is the link to the tau proteins and the development of tangles that leads to the disease progression. But, of course, the cascade is also not that simple because it involves many other effects especially from the immune system. “In some people the immune system can control the plaques,” said Kotulska.
She explained some of the experimental, computational and machine-learning tools that have subsequently been used in amyloid studies. She also presented available bioinformatics methods for amyloid classification, prediction and interactions, along with information about some of the current databases of amyloid.
Kotulska pointed out that experimental studies of amyloid proteins are difficult, time-consuming, and very sensitive to subtle changes in conditions, including concentration, pH, temperature, solvent, the presence of other molecules and, even, the operator. Amyloids are hard to control and show unpredictable behaviours.
“Due to demanding experimental protocols, our understanding of amyloid formation or their interactions remains limited,” she said. The development of computational methods is essential, and more are becoming available. On the other hand, their development and evaluation also depends on the availability of experimental data.”
Despite the development of many different databases of amyloids in the last 15 years, the records proven by direct microscopic images of the fibres are up to about 2500 – which shows exactly how difficult this all is.
Kotulska’s research group and collaborators have produced, among others, AmyloGram which has proven to be an efficient predictor of amyloidogenic sequences in proteins, using random forests and n-gram analysis, with good accuracy in predicting short amyloid stretches. AmyloGraph, on the other hand, is a database based on meta-analyses – looking at amyloid:amyloid interactions, which was then used for development of PACT tool aiming to predict the unknown cross-talk outcomes. The bioinformatics studies are being supported by wet lab experiments carried out by the group members.
Focusing on the good guys
Kotulska also explained how work on the good or functional (non-pathogenic) amyloids is now seen as very important for further understanding diseases associated with amyloids – either in protecting against disease progression or interacting further with the pathogenic amyloids.
Functional amyloids are widespread in microorganisms like bacteria where they are often used for protection. The best known are CsgAs used in biofilms.
“This is interesting,” she said, “because if expressed by an organism they are evolutionary tailored, microorganisms are not killed by them and can handle them. We suspect they are different to pathological amyloids but don’t know where and why. However, right now we know very few of them.”
“Functional amyloids, expressed mostly by microorganisms including those from the human microbiome species, play important physiological roles for their host organisms, e.g. stabilising bacterial biofilms,” she said. “Many studies have shown that an amyloidogenic protein (or peptide) could affect the aggregation of another protein, which may result in amplification or attenuation of the fibril formation. Such cross interactions between pathological human amyloids and bacterial functional amyloids may be crucial for understanding the influence of microbial amyloids on several human diseases through the gut-brain axis. This effect has been demonstrated, for example, in Parkinson’s disease. Similarly, there has been research showing potential interactions between the aggregation of endogenous human proteins and the SARS-CoV-2 spike protein after its proteolysis, and their possible involvement in the microclots observed in patients with long-COVID symptoms.”
“We need to understand much more about the ‘cross talk’ between amyloids and what it means if they can interact,” said Kotulska. “There are good and bad amyloids but when they start talking, the good may not be so good anymore.”
Including Africa
Kotulska explained that geography and lifestyle shape the human gut microbiome and therefore susceptibility to certain diseases, triggered by microbiome-related proteins, may differ.
This means there is a need to include more global data. Thus far Africa has not been well represented in the datasets and Kotulska’s STIAS project therefore aims to compare populations in Europe and Africa looking at morbidity with amyloid-related disease. Thus far in her fellowship she has encountered datasets from the University of the Witwatersrand in South Africa and another one from Tunisia. Moreover, she mentioned an important work done by researchers at Stellenbosch University on COVID-related amyloids and resulting microclots.
“The complexity of a highly varied microbiome in different populations should reveal more nuances for a tentative link between amyloid-related diseases and functional amyloids expressed by the intestinal microbiota,” she said.
Michelle Galloway: Part-time media officer at STIAS
Photograph: SCPS Photography