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Structural studies of liquid-phase protein systems related to neurodegenerative diseases
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As doenças neurodegenerativas são condições devastadoras que impõem um peso significativo na sociedade e nos sistemas de saúde. Entre elas estão as doenças de neurónios motores, como a esclerose lateral amiotrófica (ELA) e a atrofia muscular espinhal (AME). No entanto, em 2023, não existem terapias eficazes para tratar estas doenças debilitantes. Na última década, evidências têm revelado uma conexão entre a separação de fase líquido-líquido (SFLL) de proteínas e a etiologia da neurodegeneração. O desenvolvimento de abordagens terapêuticas requer a compreensão dos detalhes moleculares que regulam a SFLL de proteínas na saúde e em doenças. Nesta tese, utilizando uma combinação de metodologias de ressonância magnética nuclear (RMN), técnicas microscópicas e ensaios de turbidez, visámos desvendar a base estrutural e a relevância fisiológica da SFLL de proteínas, e investigar os mecanismos patológicos que podem contribuir para a progressão da ELA e AME. Especificamente, estudámos a proteína relacionada com ELA, fused in sarcoma (FUS), e a interação entre a proteína small nucleolar arginine-rich (Gar1) e a proteína survival motor neuron (SMN) na AME. Os nossos resultados demonstram que a separação de fase da FUS em resposta ao choque térmico a frio pode envolver um mecanismo estrutural baseado na desnaturação a frio da proteína. A baixas temperaturas, observámos que a FUS sofre uma desnaturação reversível que resulta na exposição de resíduos hidrofóbicos. É importante destacar que a desnaturação a frio da FUS depende da estabilidade do seu domínio globular zinc-finger, mediada por iões Zn2+. Estas descobertas esclarecem como o stress persistente em resposta ao frio e a desregulação dos níveis de Zn2+ podem levar à agregação patológica de FUS encontrada na ELA. Adicionalmente, a nossa investigação revela que a Gar1 sofre separação de fase na presença de RNA, um processo modulado pela sua interação com a proteína SMN. Estruturalmente, fornecemos evidências de que o domínio Tudor da SMN interage com a Gar1 através dos seus loops conservados, e que esta interação é perturbada pela mutação E134K observada na AME. Esta perturbação afeta a modulação da SMN Tudor nas propriedades materiais e na morfologia dos condensados de Gar1. Estas descobertas destacam um potencial mecanismo que relaciona SFLL e AME, baseado na perturbação da interação entre a SMN e a Gar1. Os nossos resultados fornecem informações valiosas sobre mecanismos estruturais que podem subjazer à SFLL em ELA e AME.
Neurodegenerative diseases are devastating disorders that place a significant burden on society and healthcare systems. Among them are motor neuron disorders such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). However, as of 2023, there are no effective therapies to treat these debilitating diseases. Over the last decade, emerging evidence has uncovered a compelling connection between protein liquid-liquid phase separation (LLPS) and the etiology of neurodegeneration. The development of therapeutic approaches requires the comprehension of the molecular details governing protein LLPS in health and disease. In this thesis, using a combination of nuclear magnetic resonance (NMR) methodologies, microscopic techniques and phase separation assays, we aimed to uncover the structural basis and physiological relevance of protein LLPS, and investigate the pathological mechanisms that may contribute to ALS and SMA disease progression. Specifically, we focused on the ALS-related fused in sarcoma (FUS) protein, and on the interplay between the small nucleolar arginine-glycine rich 1 protein (Gar1) and the survival motor neuron (SMN) in SMA. Our results demonstrate that hydrophobic-driven FUS phase separation in cold shock stress might entail a structural mechanism based on protein cold denaturation. At low temperature, we found that FUS undergoes reversible unfolding, which results in the exposure of hydrophobic residues. Importantly, we demonstrate that FUS cold denaturation relies on the stability of its folded zinc-finger domain by the coordination of Zn2+ ions. These findings shed light on how persistent cold stress and dysregulation of Zn2+ levels could lead to FUS pathological aggregation found in ALS. Furthermore, our research reveals that Gar1 undergoes phase separation in the presence of RNA, a process modulated by its interaction with the SMN protein. Structurally, we provided strong evidence that the Tudor domain from SMN interacts with Gar1 through its conserved loops, and that this interaction is disrupted by the SMA-related disease mutation E134K on the Tudor domain. This disruption affects the ability of SMN Tudor to modulate the morphology and material properties of the Gar1 condensates. These findings highlight a potential LLPS-related mechanism in SMA, rooted on the disruption of the SMN and Gar1 interaction. Collectively, our research findings provide valuable insights into the structural mechanisms that may underlie LLPS in ALS and SMA pathogenesis.
Neurodegenerative diseases are devastating disorders that place a significant burden on society and healthcare systems. Among them are motor neuron disorders such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). However, as of 2023, there are no effective therapies to treat these debilitating diseases. Over the last decade, emerging evidence has uncovered a compelling connection between protein liquid-liquid phase separation (LLPS) and the etiology of neurodegeneration. The development of therapeutic approaches requires the comprehension of the molecular details governing protein LLPS in health and disease. In this thesis, using a combination of nuclear magnetic resonance (NMR) methodologies, microscopic techniques and phase separation assays, we aimed to uncover the structural basis and physiological relevance of protein LLPS, and investigate the pathological mechanisms that may contribute to ALS and SMA disease progression. Specifically, we focused on the ALS-related fused in sarcoma (FUS) protein, and on the interplay between the small nucleolar arginine-glycine rich 1 protein (Gar1) and the survival motor neuron (SMN) in SMA. Our results demonstrate that hydrophobic-driven FUS phase separation in cold shock stress might entail a structural mechanism based on protein cold denaturation. At low temperature, we found that FUS undergoes reversible unfolding, which results in the exposure of hydrophobic residues. Importantly, we demonstrate that FUS cold denaturation relies on the stability of its folded zinc-finger domain by the coordination of Zn2+ ions. These findings shed light on how persistent cold stress and dysregulation of Zn2+ levels could lead to FUS pathological aggregation found in ALS. Furthermore, our research reveals that Gar1 undergoes phase separation in the presence of RNA, a process modulated by its interaction with the SMN protein. Structurally, we provided strong evidence that the Tudor domain from SMN interacts with Gar1 through its conserved loops, and that this interaction is disrupted by the SMA-related disease mutation E134K on the Tudor domain. This disruption affects the ability of SMN Tudor to modulate the morphology and material properties of the Gar1 condensates. These findings highlight a potential LLPS-related mechanism in SMA, rooted on the disruption of the SMN and Gar1 interaction. Collectively, our research findings provide valuable insights into the structural mechanisms that may underlie LLPS in ALS and SMA pathogenesis.
Descrição
Palavras-chave
Neurodegenerative diseases biomolecular condensates LLPS NMR