Abnormal accumulation of aggregated α-synuclein (α-Syn) is seen in a variety of neurodegenerative diseases, including Parkinson’s disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB), Parkinson’s disease dementia (PDD), and even subsets of Alzheimer’s disease (AD) showing Lewy-body-like pathology. These synucleinopathies exhibit differences in their clinical and pathological representations, reminiscent of prion disorders. Emerging evidence suggests that α-Syn self-assembles and polymerizes into conformationally diverse polymorphs in vitro and in vivo, similar to prions. These α-Syn polymorphs arising from the same precursor protein may exhibit strain-specific biochemical properties and the ability to induce distinct pathological phenotypes upon their inoculation in animal models. In this review, we discuss clinical and pathological variability in synucleinopathies and several aspects of α-Syn fibril polymorphism, including the existence of high-resolution molecular structures and brain-derived strains. The current review sheds light on the recent advances in delineating the structure–pathogenic relationship of α-Syn and how diverse α-Syn molecular polymorphs contribute to the existing clinical heterogeneity in synucleinopathies.

Structural and Functional Insights into α-Synuclein Fibril Polymorphism

Liquid-liquid phase separation (LLPS) has received significant attention in recent biological studies. It refers to a phenomenon that biomolecule exceeds the solubility, condensates and separates itself from solution in liquid like droplets formation. Our understanding of it has also changed from memebraneless organelles to compartmentalization, muti-functional crucibles, and reaction regulators. Although this phenomenon has been employed for a variety of biological processes, recent studies mainly focus on its physiological significance, and the comprehensive research of the underlying physical mechanism is limited. The characteristics of side chains of amino acids and the interaction tendency of proteins function importantly in regulating LLPS thus should be pay more attention on. In addition, the importance of post-translational modifications (PTMs) has been underestimated, despite their abundance and crucial functions in maintaining the electrostatic balance. In this review, we first introduce the driving forces and protein secondary structures involved in LLPS and their different physical functions in cell life processes. Subsequently, we summarize the existing reports on PTM regulation related to LLPS and analyze the underlying basic principles, hoping to find some common relations between LLPS and PTM. Finally, we speculate several unreported PTMs that may have a significant impact on phase separation basing on the findings. 

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Post-translational modifications in liquid-liquid phase separation: a comprehensive review

Abstract Condensed states of proteins, including liquid-like membraneless organelles and solid-like aggregates, contribute in fundamental ways to the organisation and function of the cell. Perturbations of these states can lead to a variety of diseases through mechanisms that we are now beginning to understand. We define protein condensation diseases as conditions caused by the disruption of the normal behaviour of the condensed states of proteins. We analyze the problem of the identification of targets for pharmacological interventions for these diseases and explore opportunities for the regulation of the formation and organisation of aberrant condensed states of proteins. 

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Protein condensation diseases: therapeutic opportunities

Liquid-liquid phase separation of α-Synuclein: A new mechanistic insight for α-Synuclein aggregation associated with Parkinson's disease pathogenesis.

Biomolecular condensates present in cells can fundamentally affect the aggregation of amyloidogenic proteins and play a role in the regulation of this process. While liquid-liquid phase separation of amyloidogenic proteins by themselves can act as an alternative nucleation pathway, interaction of partly disordered aggregation-prone proteins with pre-existing condensates that act as localization centers could be a far more general mechanism of altering their aggregation behavior. Here, we show that so-called host biomolecular condensates can both accelerate and slow down amyloid formation. We study the amyloidogenic protein α-synuclein and two truncated α-synuclein variants in the presence of three types of condensates composed of non-aggregating peptides, RNA or ATP. Our results demonstrate that condensates can dramatically speed up amyloid formation when proteins localize to their interface. However, condensates can also significantly suppress aggregation by sequestering and stabilizing amyloidogenic proteins, thereby providing living cells with a possible protection mechanism against amyloid formation.

Biomolecular condensates can both accelerate and suppress aggregation of α-synuclein

Liquid-liquid phase separation (LLPS) is a crucial phenomenon for the formation of functional membraneless organelles. However, LLPS is also responsible for protein aggregation in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease (PD). Recently, several reports, including ours, have shown that α-synuclein (α-Syn) undergoes LLPS and a subsequent liquid-to-solid phase transition, which leads to amyloid fibril formation. However, how the environmental (and experimental) parameters modulate the α-Syn LLPS remains elusive. Here, we show that in vitro α-Syn LLPS is strongly dependent on the presence of salts, which allows charge neutralization at both terminal segments of protein and therefore promotes hydrophobic interactions supportive for LLPS. Using various purification methods and experimental conditions, we showed, depending upon conditions, α-Syn undergoes either spontaneous (instantaneous) or delayed LLPS. Furthermore, we delineate that the kinetics of liquid droplet formation (i.e., the critical concentration and critical time) is relative and can be modulated by the salt/counterion concentration, pH, presence of surface, PD-associated multivalent cations, and N-terminal acetylation, which are all known to regulate α-Syn aggregation in vitro. Together, our observations suggest that α-Syn LLPS and subsequent liquid-to-solid phase transition could be pathological, which can be triggered only under disease-associated conditions (high critical concentration and/or conditions promoting α-Syn self-assembly). This study will significantly improve our understanding of the molecular mechanisms of α-Syn LLPS and the liquid-to-solid transition.

Modulating α-Synuclein Liquid-Liquid Phase Separation.

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses – SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV – using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

https://www.biorxiv.org/content/biorxiv/early/2023/06/01/2023.05.31.543022.full.pdf

Eric Kummerant

Papers

Modulating α-Synuclein Liquid-Liquid Phase Separation.

Universal features of Nsp1-mediated translational shutdown by coronaviruses