Final Report Summary - FITNESS & EVOLUTION (Intracellular protein aggregation: fitness and evolution)
Protein aggregation is the self-assembly of polypeptide chains into insoluble deposits. Generally, the failure of a polypeptide to acquire or maintain a proper native functional conformation could lead to protein aggregation. The study of this event has become a dynamic scientific field principally because its association with numerous human diseases such as Alzheimer’s and Parkinson’s disease. In addition, aggregation is one of the most critical problems in protein recombinant expression in experimental investigations and large-scale protein production. Though potentially harmful, there is an increasing number of examples where cells exploit protein aggregation for crucial functional purposes such as scaffolding melanin in the skin or intracellular storage of hormones. These observations, therefore, suggest that during evolution, cells have been adapted to tolerate the presence of protein aggregates whereas proteins have been shaped to improve their function and stability. From a systemic point of view, the understanding of the determinants that modulate protein deposition is a necessary step for making progress in deciphering the organisation and regulation of the cell. Moreover, this knowledge is essential to develop new strategies to tackle the debilitating pathologies associated with protein aggregation and open new avenues to increase the solubility and functionality of recombinant proteins.
The synergy between experimental and computational studies has provided important insights in the understanding of protein aggregation process. The aggregation propensity of a polypeptide is just below the limit that allows proteins to remain soluble at the concentrations required for life20. Because of this tight equilibrium, aggregation event can result from even minor changes in the sequence or in the expression levels of otherwise harmless proteins21-23. Recent computational studies have reported the existence of a selective pressure to escape protein aggregation exerted on both the protein sequence and the gene expression levels. In addition, this pressure seems to differ based on protein structure, biological function, localization and abundance21-23. However, a direct experimental evidence demonstrating how natural selection shapes protein sequence and concentration in a live cell is still missing, principally due to the difficulties in reproducing evolutionary constraints and time-scales in the laboratory.
Against this backdrop, the present project has analyzed how protein aggregation is selected in a biological context employing yeast as a cellular model. The specific questions that we have answered are:
Q1. How protein aggregation affects cell fitness?
Q2. How protein aggregation is selected in a population?
Q3. How can this selection be modulated?
To answer these questions we have exploited a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. We have created a novel in vivo system that involves two main elements: an aggregation-prone tag and a functional protein. The functional protein is an enzyme that can catalyze two different reactions depending on the substrate provided. The Substrate1 can be transformed into a product essential for cell growth (Product1). The Substrate2 is transformed into a product that kills the cell (Product2). When the medium contains Product1 the cell can incorporate it and the enzymatic activity is not longer essential. As a result, in this model the protein aggregation could be (A) deleterious, (B) neutral or (C) beneficial for the cell fitness. By competing different strains in the same culture, we can simulate how protein aggregation is selected in a population. The data obtained from the competition experiments allows quantifying the selection coefficient associated to the protein aggregation. Due to the special properties of this model we can separately calculate three different fitness effects associated to protein aggregation. Accordingly, when the enzyme is not-essential we just measure misfolding stress, when the enzyme is essential we also measure a loss of function, and when the enzyme is toxic we measure a gain of a beneficial function.
Our results show that aggregation has a neutral effect on fitness and are the associated gain or loss of function that determined the final cell fitness. Our work also reveals that protein aggregation could increase population variability and survival expectancy of cells exposed to variable environments. These data suggest a tolerable and common protein aggregation process for which the cell has developed quality control mechanisms. Protein aggregation may not always entail a gain or loss of function and, in case of a non-essential protein could have low impact in cell fitness. Moreover, within the same genomic background, changes in the environment can modify the protein aggregation effects; enabling situations were aggregation is beneficial and others were is toxic. Overall, the present work supports that during evolution protein aggregation has been selected both for and against.
The synergy between experimental and computational studies has provided important insights in the understanding of protein aggregation process. The aggregation propensity of a polypeptide is just below the limit that allows proteins to remain soluble at the concentrations required for life20. Because of this tight equilibrium, aggregation event can result from even minor changes in the sequence or in the expression levels of otherwise harmless proteins21-23. Recent computational studies have reported the existence of a selective pressure to escape protein aggregation exerted on both the protein sequence and the gene expression levels. In addition, this pressure seems to differ based on protein structure, biological function, localization and abundance21-23. However, a direct experimental evidence demonstrating how natural selection shapes protein sequence and concentration in a live cell is still missing, principally due to the difficulties in reproducing evolutionary constraints and time-scales in the laboratory.
Against this backdrop, the present project has analyzed how protein aggregation is selected in a biological context employing yeast as a cellular model. The specific questions that we have answered are:
Q1. How protein aggregation affects cell fitness?
Q2. How protein aggregation is selected in a population?
Q3. How can this selection be modulated?
To answer these questions we have exploited a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. We have created a novel in vivo system that involves two main elements: an aggregation-prone tag and a functional protein. The functional protein is an enzyme that can catalyze two different reactions depending on the substrate provided. The Substrate1 can be transformed into a product essential for cell growth (Product1). The Substrate2 is transformed into a product that kills the cell (Product2). When the medium contains Product1 the cell can incorporate it and the enzymatic activity is not longer essential. As a result, in this model the protein aggregation could be (A) deleterious, (B) neutral or (C) beneficial for the cell fitness. By competing different strains in the same culture, we can simulate how protein aggregation is selected in a population. The data obtained from the competition experiments allows quantifying the selection coefficient associated to the protein aggregation. Due to the special properties of this model we can separately calculate three different fitness effects associated to protein aggregation. Accordingly, when the enzyme is not-essential we just measure misfolding stress, when the enzyme is essential we also measure a loss of function, and when the enzyme is toxic we measure a gain of a beneficial function.
Our results show that aggregation has a neutral effect on fitness and are the associated gain or loss of function that determined the final cell fitness. Our work also reveals that protein aggregation could increase population variability and survival expectancy of cells exposed to variable environments. These data suggest a tolerable and common protein aggregation process for which the cell has developed quality control mechanisms. Protein aggregation may not always entail a gain or loss of function and, in case of a non-essential protein could have low impact in cell fitness. Moreover, within the same genomic background, changes in the environment can modify the protein aggregation effects; enabling situations were aggregation is beneficial and others were is toxic. Overall, the present work supports that during evolution protein aggregation has been selected both for and against.
