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The determinants of cross-seeding of protein aggregation: a Multiple TANGO

Periodic Reporting for period 3 - MANGO (The determinants of cross-seeding of protein aggregation: a Multiple TANGO)

Reporting period: 2018-06-01 to 2019-11-30

Protein aggregation is a process by which proteins stick together in organised clumps in a self-recognition process. This molecular process is a unifying feature that connects many seemingly unrelated human pathologies such as diabetes, cataract, cancer and dementia, where each disease is characterised by the protein(s) that aggregate and the tissues where this happens. This gave rise to much scientific enquiry into the link between aggregation and pathology. In sharp contrast, protein aggregation also occurs commonly with food-born proteins during food preparation, and is employed by nature as a functional motif to construct highly stable biomaterials, such as spider silk and insect egg shell or for memory formation in mammalian brain cells. We have however still a very limited understanding on the biological effect of protein aggregates on biological systems, including the human body. We do know that aggregation seems to be associated with normal ageing, but we cannot tell the essential differences between functional, desired aggregation and unwanted and uncontrolled aggregation that occur in human pathology. And even in diseases where protein aggregates took centre-stage in the disease mechanism, such as the aggregation of the beta peptide in Alzheimer disease (AD), failure of therapeutic intervention targeting the aggregates to actually result in a benefit to patients, has led many to question if aggregates are not just consequences of some other, as of yet unknown, degenerative mechanism that would then be the primary cause of disease.

In the MANGO project, we study a reductionist hypothesis of the biological impact of protein aggregates: The research is based on the short stretch model of protein aggregation, which assumes that protein aggregates form because of short aggregation sequence fragments that are part of almost any naturally occurring protein sequence. When mutations or changes in the physiological context occur, these regions, which are normally buried inside the hydrophobic core of the natively folded protein, can be revealed, allowing them to interact with the same sequences on identical polypeptides. We speculate that the interactions these aggregation prone regions make with other proteins would be the basis of the biological effect that the aggregates have on the cells or tissues where they occur. The innovation of MANGO is to hypothesise that these short stretches that interact in the aggregate do not necessarily need to come from identical proteins, but could also occur in seemingly unrelated proteins. For example, if a protein exists in the human brain that shares an aggregation-prone region with the Alzheimer beta-peptide, that protein might interact with the beta-peptide aggregates in a way that affect its function and hence alters the state of the neurons where the interaction occurs.
One major objective of MANGO is to investigate this hypothesis in different disease contexts, trying to establish the role of this process in human protein aggregation pathologies.
The second major aim of MANGO is to study this with synthetic model systems, where use aggregation prone regions from proteins not known to be associated with protein aggregation in any disease context and we can study if the interaction between e.g. peptide bearing such aggregation prone regions and the protein from which the region was derived leads to loss of function, toxicity, etc. This will then hopefully feed back into the understanding of what is happening in the disease context.
For the first period, we made major breakthroughs in the synthetic aggregating peptides approach, learning several important lessons that will feed back into our work on disease-associated protein aggregation.

We showed that an aggregation prone fragment of a protein called “Vascular Endothelial Growth Factor Receptor” or VEGFR2 for short, forms amyloid fibrils that are structurally and biophysically indistinguishable from naturally occurring amyloids, functional or pathological. When these synthetic amyloids are added to cell expressing this protein, it induces the aggregation and functional inactivation of VEGFR2. Interestingly, this event is not toxic beyond the loss of function of this growth signalling molecule, showing that aggregation can be selectively toxic to cells who functionally rely on proteins that intrinsically can rely protein capable of interacting with the aggregates. In cells that do not express VEGFR2 or that express it, but functionally do not rely on it, the aggregation event itself appears to be physiologically neutral. Exploiting this idea, we employed the synthetic amyloid peptide in a melanoma tumour model in mouse in which the growth of the tumour depends strongly on the receptor because it mediates the growth of new vasculature, which the tumour needs to sustain it growth. Cutaneous malignant melanoma is the leading cause of skin cancer related deaths. Importantly, we could show that in mice treated with the synthetic amyloid that induces the aggregation of VEGFR2, tumor growth was significantly reduced compared to untreated animals. This suggests that synthetic amyloids may be part of cancer therapies of the future. (Gallardo et al. Science, 2016)

In collaboration with plant biotechnologist Eugenia Russinova, we demonstrated that protein aggregation in plants can generate valuable new traits by removing certain target proteins that limit e.g. plant growth or starch production. Again, we did not observe any toxic side effects from the presence of these aggregates, suggesting the loss of function of protein (co-)aggregate is a strong determinant of the effect of aggregates on their biological environment. (Betti, Plant Phys, 2016)

In the quest to understand what aggregation prone regions in proteins are available for aggregation reactions under conditions in which the protein is mostly folded, we coined the concept of critical aggregation prone regions. These are a minority of aggregation prone regions of proteins that reside in structurally unstable regions of the proteins, which local unfolding events can lead to their availability for aggregation. The corollary is that carefully design mutations in these aggregation prone regions can greatly reduce the aggregation propensity of these proteins. This is of special importance for the therapeutic use of proteins, such as monoclonal antibodies, the fastest growing market in human therapies, where aggregation is a factor that limits production and efficacy in patients. (Ganesan et al, Nature Communications, 2016) (Van der Kant, JMB, 2016)
By the end of the project, I hope to have unequivocally proven the importance of co-aggregation cascades in human pathology based in the first instance on some well worked-out high profile cases, but also on synthetic models that recapture essential features by design. Based on that I will use the knowledge generated to systematically analyse other aggregation pathologies for key co-aggregation or cross-seeding events. Also, I will explore the therapeutic potential of disrupting co-aggregation and test the utility of the synthetic variants as research tool for post-translational protein knockout.
Graphical representation of cells being invaded by small amyloid-like oligomers.