Protein phase transitions in health and disease



Benedetta Bolognesi | Junior Group Leader
Marta Badia Graset | PhD Student
Mireia Seuma Areñas | PhD Student
Trinidad Sanmartín | Laboratory Technician

About

What we study

Our lab aims at understanding how protein sequences can become toxic upon mutation. We are particularly interested in amino acid sequences that can adopt different conformations and undergo a process of self-assembly which results in distinct physical states.

The concept of protein aggregation has mainly been associated to the formation of insoluble amyloid fibrils, best known for their implication in the pathogenesis of a number of neurodegenerative conditions, such as Parkinson’s disease or Amyotrophic Lateral Sclerosis. However, examples of functional amyloid are also widespread, especially across bacteria and fungi. Recently, it has become clear that proteins can assemble also into a more dynamic and reversible state through a process of liquid de-mixing.

Liquid condensates are frequently formed by proteins containing intrinsically disordered regions.The self-assembly of these protein regions results in a distinct liquid phase and it’s key to the formation of many membrane-less organelles, hence contributing to the organisation of the intracellular space. However, also for proteins undergoing liquid de-mixing, the balance between function and dysfunction is far from clear. It is also unknown if, in vivo, liquid de-mixed states are precursors of insoluble amyloid-like states, and to which extent proteins are structured once in the liquid state.

Map of the effect of mutations on toxicity of the TDP-43 Prion-like Domain.

How we do it

In order to understand how mutations affect these delicate equilibria and to elucidate when and why a sequence becomes toxic for the cell, our lab integrates experimental and computational approaches in different model systems. Recently, we developed a Deep Mutational Scanning (DMS) strategy that allows to quantify the toxicity of thousands of mutations in a disordered protein sequence . The idea behind this type of approach is that by portraying the full landscape of the effects of mutations in a specific protein domain we can reach a more systematic and comprehensive understanding of the determinants of toxicity. Besides developing high-throughput methods to measure the toxicity of thousands of mutations in parallel, we are also interested in developing similar strategies to measure in vivo the effect of mutations on the physical state the proteins acquire upon mutation (diffuse, liquid de-mixed, insoluble) and on their ability to nucleate amyloid fibrils. Overall, we aim at generating exhaustive datasets that will give insights into the specific conformations and mechanisms leading to toxicity.

We focus on classical amyloids, such as the amyloid-beta peptide, the main component of the plaques found in Alzheimer’s disease patients, but also on functional yeast prions and on a less characterised part of the human proteome: prion-like domains. Just like most disordered protein regions, prion-like domains are particularly difficult to study in vitro. In this perspective, in vivo approaches such as the ones we develop, can provide a unique opportunity to investigate these sequences in a systematic way.

Average effect of mutations on nucleation, visualised on the cross-section of an amyloid-beta fibril (PDB:5KK3)

Projects

National projects
PRIOMUT Escaneado exhaustivo de mutaciones en un dominio priónico para entender la toxicidad inducida por proteínas (2019-2021) MICIU, Retos investigación: Proyectos I+D Benedetta Bolognesi

 

 

 

Publications

Badia, M., Bolognesi, B., (2021). Assembling the right type of switch: Protein condensation to signal cell death Current Opinion in Cell Biology 69, 55-61

Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival.

Keywords: Amyloid, Cell death, Deep mutagenesis, LLPS, RNA-binding proteins


Seuma, M., Faure, A., Badia, M., Lehner, B., Bolognesi, B., (2021). The genetic landscape for amyloid beta fibril nucleation accurately discriminates familial Alzheimer’s disease mutations eLife 10, e63364

Plaques of the amyloid beta (Aß) peptide are a pathological hallmark of Alzheimer’s disease (AD), the most common form of dementia. Mutations in Aß also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of Aß. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in Aß, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in Aß.


Bolognesi, Benedetta, Faure, Andre J., Seuma, Mireia, Schmiedel, Jörrn M., Tartaglia, Gian Gaetano, Lehner, Ben, (2019). The mutational landscape of a prion-like domain Nature Communications 10, (1), 4162

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

Keywords: Computational biology and bioinformatics, Genomics, Mechanisms of disease, Neurodegeneration, Systems biology


Bolognesi, Benedetta, Lehner, Ben, (2018). Reaching the limit eLife 7, e39804

How many copies of a protein can be made before it becomes toxic to the cell?

Keywords: Protein burden, Overexpression, Glycolysis


 

 

Equipment

  • Thermo MaxQ 8000

 

Collaborations

  • Ben Lehner
    CRG, Barcelona
  • Sofia Giorgetti
    University of Pavia, Italy
  • Xavier Salvatella
    IRB Barcelona
  • Priyanka Narayan
    NIDDK-NIH, Washington D.C
  • Broder Schmidt
    University of Stanford

 

News/Jobs

IBEC receives a visit from the Mayor of Barcelona interested in our research against Covid19

The Mayor of Barcelona, Ada Colau, visited IBEC facilities last Friday to learn, by our Director and a group of researchers, how bioengineering can help find solutions to health problems such as COVID19, cancer, or degenerative diseases.

When in early 2020, more than 200 scientists gathered in La Pedrera in Barcelona to discuss the present and future of bioengineering, no one imagined that the world would experience the first pandemic of the 21st century and that science would take on more importance than ever.

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Researchers perform thousands of mutations to understand amyotrophic lateral sclerosis

Researchers from IBEC and CRG in Barcelona use a technique called high-throughput mutagenesis to study Amyotrophic Lateral Sclerosis (ALS), with unexpected results.

Results showed that aggregation of TDP-43 is not harmful but actually protects cells, changing our understanding of ALS and opening the door to radically new therapeutic approaches.

Amyotrophic lateral sclerosis (ALS) is a devastating and incurable nervous system disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control and normally death within a few years of diagnosis. In ALS, like in other neurodegenerative diseases, specific protein aggregates have long been recognized as the pathological hallmarks, but it is not clear whether they represent the actual cause of the disease. Indeed, alleviating aggregation has repeatedly failed as a therapeutic strategy when trying to treat neurodegenerative diseases such as Alzheimer’s disease.

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New IBEC group creates ‘fitness heatmaps’ of gene mutations

The start of the autumn semester finds a new face in IBEC’s research community, with Dr. Benedetta Bolognesi joining the institute as junior group leader.

Benedetta has come from Barcelona’s Centre for Genomic Regulation, where she was a postdoc in Ben Lehner’s and Gian Gaetano Tartaglia’s groups. At IBEC she will launch and lead the Protein Phase Transitions in Health and Disease group.

During her postdoc, Benedetta focused on why certain genes are toxic when over-expressed. She found that, in some cases, they cause toxicity because the proteins they code for end up forming a different liquid phase in the cytoplasm.

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