Recent Advances in Co-Condensation and Co-Aggregation of Amyloid Proteins Linked to Neurodegenerative Diseases.

Liquid–Liquid Phase Separation and Its Mechanistic Role in Pathological Protein Aggregation

Protein liquid-liquid phase separation (LLPS) has been associated with protein amyloid aggregation. Amyloid aggregation of tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases. This protocol provides steps to prepare tau condensates via LLPS, so that researchers can further study its driving forces and its relationship with tau amyloid aggregation.

Emerging roles of O-glycosylation in regulating protein aggregation, phase separation, and functions

Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors.IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.

Neurodegenerative diseases such as dementia and Alzheimer's disease are caused by liquid-liquid phase separation (LLPS) proteins. LLPS is a phenomenon in which a dense liquid phase of proteins is formed in a liquid phase in which proteins are dispersed at a low concentration. The concentrated proteins enable highly efficient chemical reactions, but at the same time, there is a risk of forming insoluble aggregates that cause diseases. In fact, neurodegenerative disease-related proteins form insoluble aggregates, which cause great damage to nerves, resulting in memory and motor disorders. Drug discovery requires the design of drug candidates that can strongly bind to the intrinsically disordered region of a phase-separated protein and control the phase-separated state. This paper mainly introduces our research on peptide design that binds to phase-separated proteins. For peptide drug discovery, it is necessary to efficiently search for drug candidates among a huge number of peptides. As an efficient search method for peptides that control phase-separated proteins, we searched for amino acids that can control liquid-liquid phase separation, and devised a method for designing peptides containing effective amino acids. It was demonstrated that this method can be used to control the LLPS and solid aggregate formation of the neurodegenerative disease-related protein FUS. Furthermore, we devised a method for rationally designing a peptide that binds complementarily to the intrinsically disordered region of the target, and demonstrated the functional control of the cancer disease-related protein p53. Finally, we discuss the possibility of peptide drug discovery for disease-related LLPS proteins.

Many proteins perform their functions within membraneless organelles, where they form a liquid-like condensed state, also known as droplet state. The FuzDrop method predicts the probability of spontaneous liquid–liquid phase separation of proteins and provides a sequence-based score to identify the regions that promote this process. Furthermore, the FuzDrop method estimates the propensity of conversion of proteins to the amyloid state, and identifies aggregation hot-spots, which can drive the irreversible maturation of the liquid-like droplet state. These predictions can also identify mutations that can induce formation of amyloid aggregates, including those implicated in human diseases. To facilitate the interpretation of the predictions, the droplet-promoting and aggregation-promoting regions can be visualized on protein structures generated by AlphaFold. The FuzDrop server (https://fuzdrop.bio.unipd.it) thus offers insights into the complex behavior of proteins in their condensed states and facilitates the understanding of the functional relationships of proteins.

The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43

Liquid–liquid phase separation (LLPS), an emerging biophysical phenomenon, can sequester molecules to implement physiological and pathological functions. LLPS implements the assembly of numerous membraneless chambers, including stress granules and P‐bodies, containing RNA and protein. RNA–RNA and RNA–protein interactions play a critical role in LLPS. Scaffolding proteins, through multivalent interactions and external factors, support protein–RNA interaction networks to form condensates involved in a variety of diseases, particularly neurodegenerative diseases and cancer. Modulating LLPS phenomenon in multiple pathogenic proteins for the treatment of neurodegenerative diseases and cancer could present a promising direction, though recent advances in this area are limited. Here, we summarize in detail the complexity of LLPS in constructing signaling pathways and highlight the role of LLPS in neurodegenerative diseases and cancers. We also explore RNA modifications on LLPS to alter diseases progression because these modifications can influence LLPS of certain proteins or the formation of stress granules, and discuss the possibility of proper manipulation of LLPS process to restore cellular homeostasis or develop therapeutic drugs for the eradication of diseases. This review attempts to discuss potential therapeutic opportunities by elaborating on the connection between LLPS, RNA modification, and their roles in diseases.

Amyloid is traditionally viewed as a consequence of protein misfolding and aggregation and is most notorious for its association with debilitating and chronic human diseases. However, a growing list of examples of "functional amyloid" challenges this bad reputation and indicates that many organisms can employ the biophysical properties of amyloid for their benefit. Because of developments in the structural studies of amyloid, a clearer picture is emerging about what defines amyloid structure and the properties that unite functional and pathological amyloids. Here, we review various amyloids and place them within the framework of the latest structural models.

Raman spectroscopy and imaging of protein droplet formation and aggregation.

The aggregation and deposition of amyloid proteins are considered key contributors to the pathogenesis of various diseases. In recent years, a variety of nanoparticles have been employed to modulate amyloid protein aggregation. Among them, silica nanoparticles (SNs) have emerged as particularly promising due to the excellent biocompatibility of silicon-based materials and their favorable physicochemical properties, offering significant therapeutic potential. This review summarizes recent studies on the use of SNs to regulate the aggregation of amyloid proteins. The effects of different types and surface-modified SNs on the aggregation behavior of various amyloid proteins are systematically compared, aiming to provide valuable insights for future research and potential therapeutic applications.

A high-throughput method for exploring the parameter space of protein liquid-liquid phase separation

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

The 2021 FASEB Virtual Science Research Conference on Protein Aggregation: Function, Dysfunction, and Disease, June 23-25, 2021.

Protein liquid-liquid phase separation (LLPS) plays a crucial role in mediating dynamic assembly of different membraneless organelles such as stress granules (SGs). Dysregulation of dynamic protein LLPS leads to aberrant phase transition and amyloid aggregation which is closely associated with neurodegenerative diseases. In this study, we found that three types of graphene quantum dots (GQDs) exhibit potent activity in preventing SG formation and promoting SG disassembly. We next demonstrate that GQDs can directly interact with the SGs-containing protein fused in sarcoma (FUS), inhibit and reverse FUS LLPS, and prevent its abnormal phase transition. Moreover, GQDs display superior activity in preventing amyloid aggregation of FUS and disaggregating preformed FUS fibrils. Mechanistic study further demonstrates that GQDs with different edge-site exhibit distinct binding affinity to FUS monomers and fibrils, which accounts for their distinct activities in modulating FUS LLPS and fibrillation. Our work reveals the potent capability of GQDs in modulating SG assembly, protein LLPS, and fibrillation and sheds light on rational design of GQDs as effective modulators of protein LLPS for therapeutics application.