ReviewAberrant liquid-liquid phase separation and amyloid aggregation of proteins related to neurodegenerative diseases
Introduction
The cell is the basic building block of living organisms, which is traditionally considered as a complex entity made up of membrane-bound, subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, and others, which have distinct chemical environments needed for controlling particularly defined functions necessary for typical performance of the cell. Despite possessing various membrane-encapsulated subcellular organelles, cells commonly contain compartmentalized consolidated structures, membrane-less organelles (MLOs) that have specific sets of molecules, such as proteins and nucleic acids [1], [2], [3]. The existence of some MLOs, such as the nucleolus, P granules [4], Cajal bodies, nuclear speckles, stress granules, and promyelocytic leukemia (PML) nuclear bodies has been known for a number of years [2], [3], [5], [6]. Though these functional subcellular components vary in content, cellular distribution, and mechanism of action [7], almost all have key characteristics, such as a membrane-less spherical form influenced by surface tension, liquid-like dynamic characteristics, and an underlying molecular mechanism of assembly [4], [8]. An acceptable encompassing term for these cellular bodies is “biomolecular condensate,” which refers to their ability to strongly concentrate and condense specific ensembles of biomolecules in distinct cellular locations [9], [10].
The fact that most of these MLOs or biomolecular condensates are rapidly formed by condensation of protein molecules via liquid-liquid phase separation (LLPS) within a biological system is linked to their recent re-discovery [11], [12]. In this context, depending on the peculiarities of the environment, such as temperature, ionic strength, the presence of specific ions and/or small molecules, as well as concentration of specific proteins and nucleic acids, which are the flexible constituents with multiple valency, the protein-rich condensed droplets and a liquid phase depleted in these proteins can evolve either reversibly or irreversibly [5]. Such multiphase complex condensates assembled via unique pathways, also known as biomolecule coacervates, also contribute to the colloidal structure of the cell, which was mentioned in the book “The Physical Basis of Life” by E. Wilson: “No idea of current biology holds more commitment for the physico-chemical understanding of critical concepts than just the cell as a colloidal system, and life mostly as complex of numerous chemical reactions in the matter of this system” [13].
Numerous studies have previously reported compartmentalization of biomolecules at certain locations where they accurately accomplish important functions in various cellular processes. It has been proposed that these membrane-less assemblies are created via LLPS; i.e., a reversible de-mixing process leading to the formation of two distinct liquid phases with different concentrations of solutes [4], [14], [15], [16], [17]. Because of the lack of their own membranes, MLOs can easily interchange their constituents with the neighboring environment [18], [19], [20]. Most of the biomolecular condensates have similar features: they are highly dynamic, fluid, and spherical in shape, yet deforming on physical interaction, fusing, and eventually reverting to the spherical shape [4].
Among the illustrative examples of biomolecules involved in the phenomenon of phase separation is Crystallin (water-soluble structural protein found in the lens), one of the first proteins, for which the phenomena of phase separation was initially observed [21]. Later, it was also revealed that phase separation can reversibly happen in solutions of human hemoglobin at temperatures close to physiological conditions [22]. It turns out that these highly complex liquid condensates can aid in the condensation of hemoglobin polymers, a process that occurs in sickle cell anemia. Currently, the list of proteins known to undergo phase transitions includes multiple intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) [23] which often contain prion-like domains (PrLDs) [24] and low-complexity domains (LCDs) [25]; i.e., regions with reduced amino acid alphabet [26]. These IDRs and LCDs have repetitive sequence elements that do not have fixed structures but can exist as highly dynamic conformational ensembles [6]. This forms the basis of weak multivalent sticky intermolecular interactions [25], [27], [28]. As a result, proteins can interact with proteins and other biopolymers in a number of homotypic and heterotypic ways [6], [29], [30], [31].
Most neurodegenerative conditions are associated with protein aggregation. Toxicity appears to occur when proteins misfold and assemble to the aggregates and/or oligomers. To maintain proper folding of cellular proteins and to clear aggregated or otherwise damaged proteins, the cell has evolved a variety of mechanisms, which are collectively known as protein homeostasis (proteostasis) [32], [33], [34], [35], [36]. Protein aggregation, caused by protein misfolding and a failure of cellular clearing systems, is a frequent feature among several neurodegenerative disorders. Therefore, the presence of protein aggregates represents an important feature for diagnosing a variety of human diseases, the most common of which are neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). The key findings in the field of biomolecular condensate in recent times have linked the aberrant LLPS mechanisms to protein aggregation in neuropathology, increasing both our biophysical understanding of LLPS and our comprehension of the pathophysiology. Indeed, pathogenic aggregates contain an increase in the number of proteins which could undergo the mechanism of LLPS physiologically. As a result, it has been proposed that LLPS may promote amyloid aggregation [37], [38].
Here, we will explore the most current findings about the LLPS, such as the molecular and physical factors that drive LLPS and influence the formation of MLOs and biomolecular condensates, functional abilities of these subcellular bodies, and their consequences for biological systems as well as diseases, with an emphasis on neurological disorders. Emerging evidence suggests a link between LLPS and disorders associated with amyloidogenesis [6], [39]. Unlike LLPS, which is an adaptively bidirectional process leading to the formation of liquid droplets within the liquid cytosol or nucleosol, amyloid formation is an immutable liquid-to-solid transition, which is often irreversible. This change could be caused by the biopolymer entanglement or enhanced protein interactions leading to amyloid-like fibril development, as seen in several protein condensates linked to neurological diseases such as α-synuclein (α-Syn), TAR DNA-binding protein 43 (TDP-43), the RNA-binding protein fused in sarcoma (FUS), Microtubule associated protein tau (Tau), and heterogeneous nuclear ribonucleoprotein (hnRNPA1) [6], [39], [40], [41], [42].
Because the smaller oligomers, but not the larger fibrillar proteins, show most toxicity in these disorders, LLPS and the amyloid oligomeric protein formation are most likely two aspects of similar molecules. We will examine the roles of LLPS in nucleation of α-Syn, FUS, TDP-43, and Tau aggregation and fibrillation. We identify common features that potentially underpin the propensity of proteins to undergo LLPS, phase transition to solid phase, and maturation to pathologic aggregation under physiological or genetic changes, despite the evident structural and functional distinctions (Fig. 1).
Section snippets
Evolution of the LLPS idea
Edmund Becher Wilsonin stated in 1899 that the cytoplasm (the core mass of the cell) looks and acts like a collection of chemically suspended droplets [13] (see Fig. 2 for the abbreviated timeline of the development in LLPS and MLO). Researchers did not recognize that LLPS may underpin biogenesis and architecture of numerous MLOs until 2009, when Clifford P. Brangwynne and Anthony A. Hyman discovered that the P granules show liquid-like behavior and also that the location is controlled via
LLPS and multivalent interactions
Two different forms of multivalent interactions (Fig. 3) have been shown to lead to LLPS resulting in the formation of MLOs. The first one includes complex intracellular nucleic acid-nucleic acid, protein-protein, and protein-nucleic acid interactions, whereas another form encompasses a multitude of weak, momentary, multiple-phase connections between IDRs, including the π–π interactions, cation–anion interactions, dipole–dipole interactions, and π–cation interactions [45], [46], [54].
Modulatory regions and multivalent pliable linkers are drivers of LLPS
The determinants involved in LLPS have only recently began to be explored [6], [47], [55]. The heteropolymeric nature of proteins opens up a multitude of molecular possibilities regulating LLPS. In particular, the interaction within protein and between proteins and nucleic acids (most often, RNA) have been found to have contribution to LLPS mechanisms [45], [46]. In fact, some of the protein molecules associated with cell signaling in biological system had multiple regions separated by pliable
LLPS are driven by IDRs
The polypeptides containing a significant number of folded components are known as structured proteins. On the other hand, intrinsically disorganized polypeptides have no determined secondary and/or tertiary structures during their lifetime. Remarkably, over 44 % of mammalian polypeptide-encoding genes translate for protein molecules containing structured as well as unstructured fragments, known as intrinsically disorderly regions (IDRs) [56], [57], [58], [59]. Contribution of these IDRs in
Protein roles in the LLPS mechanisms are decided by electrostatic interactions (positive and negative charge, polar side chain, and cation-π and π-π patterns)
Previously reported studies suggested that the positive and negative charge moieties are widely distributed within the protein amino acid sequences. The remarkable behavior demonstrated by proteins containing clusters of oppositely charged residues is their capability to undergo LLPS via the mechanism of the wide-scale electrostatic attraction. Behavior of such polyampholyte entities was shown to depend on the patterning of the oppositely charged residues within the amino acid sequence [75].
Role of prion-like domains, RNA-binding motifs, and RNA intermolecular interactions in LLPS
Prions are infectious proteins with established capability to be transmitted between cells and organisms [87]. Such proteins can exist in functional and infectious forms and contain regions that can adopt the amyloid fold in the infectious conformation. An amyloid is made up of stacking β-sheets that can guide transition of native protein to the amyloid structure [88]. Many PLDs are employed in RNA binding and metabolic activities, and they are estimated to be found in 70 % of all human
Role of PTMs (post translational modifications) in LLPS
The PTMs are the protein chemical modifications (Fig. 1) that are incorporated into a polypeptide chain after its biosynthesis. Such chemical alterations of a protein molecule expand the spectrum of amino acid configurations and characteristics, resulting in a diversity of protein shapes and activities. Routine targets of these modifications are known to be phosphorylation of tyrosine, serine, and threonine, as well as methylation of lysine and arginine. Although the significance of PTMs for
Roles of protein secondary structure in LLPS and liquid-to-solid phase transition
The fact that the maturation path from a liquid-like droplet to a semi-crystalline or solid-like fibrillar assembly might vary based only on the sequence makeup of the protein represents an important finding. With time, numerous proteins which generate extremely mobile liquid condensates turn increasingly viscoelastic but also inflexible, finally forming a gel-like form, which is not able to interchange their components with the external environment [6], [39], [42]. Misfolding mechanisms that
Physiological role of LLPS in cell biology
Phase separation is a relatively new idea, explaining assembly of proteins and RNA within a cell with incipient collective component features that has just evolved. The reversible process of a homogeneous fluid rupturing into two separate liquid state, known as LLPS, was introduced as a potential propel mechanism for intracellular self-assembling and compartmentalization. The perceptible fusion, drenching, and adaptive interchange of interior material of stress granules [112], P-granules [4],
Roles of LLPS in neuropathologies
Protein misfolding and aggregate formation are responsible for the pathogenesis of a vast range of neurological disorders in humans. The molecular chaperons found in the cell are highly efficacious in either refolding or degrading these misfolded proteins. In a suitable physico-chemical state (or under appropriate conditions), many proteins can produce the amyloid fibrils [148]. Numerous proteins capable of the amyloidosis mediation under physiological conditions exhibit ample solvent
Methods utilized in study and identification of LLPS
Different methods have been utilized to observe and characterize the LLPS process in the cytoplasm as well as the nucleus of the cell. Some of these methods are briefly outlined below. First of all, there are several computational tools available to predict the intrinsic disorder status of various specific proteins and their potential to undergo LLPS. For example, D2P2 (https://d2p2.pro/search) [230] is a library that gathers information about disorder status of a query protein, localization of
Conclusions and future perspectives
In recent years, a new concept of processes involved in organization of biological system has developed, which became a fascinating field of research in cellular biology. Despite certain advances, our knowledge of LLPS remains limited. The formation of membrane-less organelles (MLOs) or biomolecular condensates through the LLPS mechanism is of great importance in human health and disease. Many of these biomolecular condensates have unique functions, often accelerating and simplifying
Acknowledgement
The authors are highly thankful for the facilities obtained at AMU Aligarh. We are grateful to DBT for generous research support to this project vide file no. BT/PR32907/MED/122/227/2019.
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