Protein phase transitions in health and disease

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


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. Liquid condensates, for example, are formed by proteins containing intrinsically disordered regions, through a process of liquid de-mixing in the cytoplasm. The self-assembly of these proteins 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.

Tracking the interactions of toxic and non-toxic engineered sequences with chaperones and known yeast prions.

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 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 effect 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 high-throughput strategies to measure in vivo the effect of mutations on the physical state the proteins acquire upon mutation (diffuse, liquid de-mixed, insoluble). We couple these approaches to a high-resolution investigation of the mechanisms underlying toxicity, which we normally carry out by standard biophysical and biochemical methods.  Overall, we aim at generating exhaustive datasets that will give insights into the specific conformations and mechanisms leading to toxicity.

Toxic and non-toxic Amyloid-Beta fibrillar structures imaged by AFM

Currently we are focusing mainly on classical amyloids, such as the amyloid-beta peptide, the main component of the plaques found in Alzheimer’s disease patients, but we are also exploring a less characterised part of the proteome:  prion-like domains. Prion-like domains are intrinsically disordered domains able to populate multiple physical states and to take part in membrane-less organelles.

The fitness effects of amino acid substitutions in a 16 aa human protein domain expressed in S. cerevisiae, measured by deep mutational scanning.

Importantly, prion-like domains are frequently mutated in a number of neurodegenerative conditions. Pathological mutations affect the equilibria among different states in ways we cannot yet fully understand or predict.  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.







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





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







  • Ben Lehner
    CRG, Barcelona
  • Sofia Giorgetti
    University of Pavia, Italy
  • Xavier Salvatella
    IRB Barcelona
  • Ina Vorberg
    DZNE Bonn, Germany
  • Broder Schmidt
    University of Stanford





Investigadors duen a terme milers de mutacions per comprendre millor l’esclerosi lateral amiotròfica

Investigadors de l’IBEC i el CRG a Barcelona utilitzen una tècnica denominada ‘mutagènesi d’alt rendiment’ per estudiar l’esclerosi lateral amiotròfica (ELA) i n’obtenen resultats inesperats.

Aquests resultats van demostrar que l’agregació de TDP-43, no només no és perjudicial sinó que, en realitat, protegeix les cèl·lules, fet que canvia el que se sabia sobre l’ELA i obre la porta a enfocaments terapèutics totalment nous.

L’esclerosi lateral amiotròfica (ELA) és una malaltia devastadora i incurable del sistema nerviós que afecta les cèl·lules nervioses del cervell i la medul·la espinal i que provoca pèrdua de control muscular i, habitualment, la mort al cap de pocs anys de ser diagnosticada. En l’ELA, com en altres malalties neurodegeneratives, fa temps que es consideren certs agregats de proteïnes com a trets distintius patològics, tot i que encara no se sap amb claredat la causa real de la malaltia. De fet, la reducció de l’agregació d’aquestes proteïnes no ha tingut èxit com a

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