Ask The Expert | Sandra Ribeiro
By Ana Filipa Castro | Joana Saavedra | Sofia Figueiredo | Vanessa Teixeira | Sára Varga – Members of the ESRs group
Since January 2021, Sandra Macedo-Ribeiro has been coordinating the Horizon 2020-funded Twinning project PhasAGE. We invited Sandra to comment on a few questions regarding her career development, the establishment of the PhasAGE consortium, and the challenges that will be experienced in the field of phase separation in human diseases at the national and European levels both during and after the conclusion of the project.
Sandra Macedo-Ribeiro holds a degree in Biochemistry from the University of Porto (Portugal) and a PhD in Chemistry from the Technical University of Munich (Germany). Sandra is a recognized expert in protein crystallography and leads the Biomolecular Structure and Function group at the Instituto of Molecular and Cell Biology (IBMC) and the Institute for Research and Innovation in Health (i3S) in Portugal.
Why did you choose a research career in the field of structural biology and why do you think it is so important to study the structure of proteins?
The different objects that we use in our everyday life have shapes, which are related to their function. As a child, being the granddaughter of a horologer, I was impressed with the amazing structures of the intricate gears of clocks. There, a series of accurately shaped moving wheels transmit motion to the minute and hour hands in mechanical timepieces, allowing us to count time. Likewise, proteins adopt a myriad of shapes that are intricately associated with how they interact with each other to fine-tune a multitude of cellular processes and regulate how cells communicate with each other and respond to external signals.
Protein crystallography still is the gold standard approach to determining the three-dimensional structures of globular proteins in atomic detail, and this was what initially determined the main topic of my PhD project. Protein structures are often represented as beautiful cartoons, but what is impressive is how the amino acid sequences fold into three-dimensional structures in such a way that a set of critical amino acids end up arranged in an optimal orientation to build, for example, a protease active site with exquisite specificity for the peptide sequences it can bind and cleave.
Knowing the protein structures in atomic detail is critical to understand how mutations – for example, associated with diseases – impact their function and interactions. Proteins are the targets of several molecules used to treat or ameliorate diseases, and high-resolution structural information is fundamental for the rational design of new drugs and for improving the specificity of various compounds. Finally, as we understand more about the rules that determine how a sequence folds into a three-dimensional structure, we can better predict the structures of proteins and design synthetic proteins for biotechnological and biomedical applications.
What hypothesis or discoveries led you to establishment of your research group at IBMC? Did you already find answers to the questions that you aimed to address at that time?
When I started my research group, I aimed to determine the structure of ataxin-3, the protein where expansion of a polyglutamine repeat of variable length leads to Machado-Joseph disease (a.k.a. Spinocerebellar Ataxia Type 3). The goal was to understand how polyglutamine expansion changed the structure of this protein triggering aggregation, a disease hallmark.
What we found out soon after starting the project was that ataxin-3 was very aggregation-prone, independently of the size of the polyglutamine tract. The protein contains a globular Josephin domain and a flexible region where the polyglutamine repeat is located. We were surprised to find that aggregation was triggered by the Josephin domain and that normal and polyglutamine-expanded ataxin-3 were able to assemble into amyloid-like fibrils under near-native conditions. This was very new and unexpected at the time, and we decided to study ataxin-3 aggregation to identify the first steps in this process that could be targeted by structure stabilizers and self-assembly inhibitors with neuroprotective properties. We made significant progress in understanding ataxin-3 aggregation mechanisms and identifying strategies to modulate them. We now understand that polyglutamine expansion does not induce major changes in the structure of ataxin-3, but it modifies its conformational dynamics and the exposure of the aggregation-prone sequences.
The aggregation propensity of the Josephin domain and the conformational plasticity of the C-terminal tail of ataxin-3 hampered the determination of its crystallographic structure, but the discoveries we made fostered my curiosity about the intriguing structural properties of polyglutamine repeats and intrinsically disordered regions and their roles in normal protein function and macromolecular interactions. This is a field that I am keen to continue to explore in my group.
The topic of liquid-liquid phase separation, biomolecular condensates and their roles in cell function is recent. How did you come up with the idea of establishing collaborations with all the partners of the consortium and submitting the PhasAGE project to a European grant funding agency?
I became interested in this topic during my participation in the COST action NGP-net coordinated by Silvio Tosatto, one of the partners of the current Twinning project. That COST action, where Salvador Ventura and Peter Tompa were also participants, focused on the study of non-globular proteins. This successful collaborative network joined computational and structural biology experts interested in intrinsically disordered proteins and protein aggregation. Naturally, we had a keen interest in understanding the properties and compositional features of the intrinsically disordered protein sequences that triggered droplet formation, a mechanism with important implications for locally clustering specific proteins and nucleic acids, allowing spatial and temporal localization of their activity in cells. This field has largely expanded and evolved in the last decade, but the impact of this research topic in Portuguese science was still limited. The PhasAGE project addresses this gap and aims to enhance the knowledge of the i3S community on the computational and experimental approaches to study liquid-liquid phase separation and macromolecular condensate assembly with implications for aging and age-related diseases.
Intrinsically disordered proteins (IDPs) are widely present in the human proteome and they have been associated with neurodegenerative and age-associated diseases. How can we get a better understanding of the function of the so simple yet so complex unstructured regions and unstructured proteins?
Contrary to globular proteins, where it is possible to infer function from the identification of sequence features and motifs in the amino acid sequences, attributing a function to the intrinsically disordered regions of proteins is a challenging task. NMR can be considered the gold standard approach to studying the local conformational features of IDPs. To better understand the function of IDPs we need to gather high-quality structural and functional experimental data on these regions/proteins. Currently, Silvio Tosatto’s lab heads a community effort aiming to manually curate intrinsically disordered regions for which there is experimental evidence. This information is compiled in the DisProt database where the experimentally validated IDP functions are accurately annotated to provide a detailed characterization of disorder functions, which might give us further clues for the pleiotropic roles of IDPs.
Translation of research findings to potential therapeutic solutions is of extreme importance. What is the contribution of the fundamental research of the PhasAGE project to the development of new therapeutic strategies that can be applied to treat neurodegenerative diseases?
With the PhasAGE project, we aim to identify new protein targets associated with neurodegenerative diseases, which undergo abnormal phase separation in aging cells. The investigation of the changes in composition and material properties of disease-related condensates will be key to designing strategies to avoid aberrant but not functional phase separation.
What do you want as a legacy of the PhasAGE project?
The PhasAGE project centers on the investigation of neurodegenerative diseases, but phase separation also has an impact on cancer and infectious diseases. I hope that as a result of the activities developed by PhasAGE, researchers at i3S are equipped with the knowledge and tools to investigate the role of phase separation in their topics of research. This is strategic to create at i3S an excellence hub for the study of phase separation, where early career researchers should be a major driving force.
More information about the PhasAGE research can be found here.
Some images were created with BioRender.com with free icons from the flaticon library.