Protein misfolding and amyloid nucleation through liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is an emerging phenomenon in cell physiology and diseases. The weak multivalent interaction prerequisite for LLPS is believed to be facilitated through intrinsically disordered regions, which are prevalent in neurodegenerative disease-associated proteins. These aggregation-prone proteins also exhibit an inherent property for phase separation, resulting in protein-rich liquid-like droplets. The very high local protein concentration in the water-deficient confined microenvironment not only drives the viscoelastic transition from the liquid to solid-like state but also most often nucleate amyloid fibril formation. Indeed, protein misfolding, oligomerization, and amyloid aggregation are observed to be initiated from the LLPS of various neurodegeneration-related proteins. Moreover, in these cases, neurodegeneration-promoting genetic and environmental factors play a direct role in amyloid aggregation preceded by the phase separation. These cumulative recent observations ignite the possibility of LLPS being a prominent nucleation mechanism associated with aberrant protein aggregation. The present review elaborates on the nucleation mechanism of the amyloid aggregation pathway and the possible early molecular events associated with amyloid-related protein phase separation. It also summarizes the recent advancement in understanding the aberrant phase transition of major proteins contributing to neurodegeneration focusing on the common disease-associated factors. Overall, this review proposes a generic LLPS-mediated multistep nucleation mechanism for amyloid aggregation and its implication in neurodegeneration.

Modulating α-Synuclein Liquid-Liquid Phase Separation.

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.

Liquid-liquid Phase Separation of α-Synuclein: A New Mechanistic Insight for α-Synuclein Aggregation Associated with Parkinson's Disease Pathogenesis.

Aberrant aggregation of the misfolded presynaptic protein, α-Synuclein (α-Syn) into Lewy body (LB) and Lewy neuritis (LN) is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Numerous studies have suggested that prefibrillar and fibrillar species of the misfolded α-Syn aggregates are responsible for cell death in PD pathogenesis. However, the precise molecular events during α-Syn aggregation, especially in the early stages, remain elusive. Emerging evidence has demonstrated that liquid-liquid phase separation (LLPS) of α-Syn occurs in the nucleation step of α-Syn aggregation, which offers an alternate non-canonical aggregation pathway in the crowded microenvironment. The liquid-like α-Syn droplets gradually undergo an irreversible liquid-to-solid phase transition into amyloid-like hydrogel entrapping oligomers and fibrils. This new mechanism of α-Syn LLPS and gel formation might represent the molecular basis of cellular toxicity associated with PD. This review aims to demonstrate the recent development of α-Syn LLPS, the underlying mechanism along with the microscopic events of aberrant phase transition. This review further discusses how several intrinsic and extrinsic factors regulate the thermodynamics and kinetics of α-Syn LLPS and co-LLPS with other proteins, which might explain the pathophysiology of α-Syn in various neurodegenerative diseases.

Intermolecular interactions underlie protein/peptide phase separation irrespective of sequence and structure at crowded milieu.

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological phenomenon underlying the sequestration of macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides but with distinct phase regimes irrespective of their sequence and structure. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study in the presence and absence of molecular crowder, polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase regimes and intermolecular interactions. We further demonstrated that electrostatic, hydrophobic, and H-bonding or a combination of such intermolecular interactions plays a crucial role in individual protein/peptide LLPS.

Phase separation and other forms of α-Synuclein self-assemblies.

α-Synuclein (α-Syn) is a natively unstructured protein, which self-assembles into higher-order aggregates possessing serious pathophysiological implications. α-Syn aberrantly self-assembles into protein aggregates, which have been widely implicated in Parkinson's disease (PD) pathogenesis and other synucleinopathies. The self-assembly of α-Syn involves the structural conversion of soluble monomeric protein into oligomeric intermediates and eventually fibrillar aggregates of amyloids with cross-ß-sheet rich conformation. These aggregated α-Syn species majorly constitute the intraneuronal inclusions, which is a hallmark of PD neuropathology. Self-assembly/aggregation of α-Syn is not a single-state conversion process as unfolded protein can access multiple conformational states through the formation of metastable, transient pre-fibrillar intermediate species. Recent studies have indicated that soluble oligomers are the potential neurotoxic species responsible for cell death in PD pathogenesis. The heterogeneous and transient nature of oligomers formed during the early stage of aggregation pathway limit their detailed study in understanding the structure-toxicity relationship. Moreover, the precise molecular events occurring in the early stage of α-Syn aggregation process majorly remain unsolved. Recently, liquid-liquid phase separation (LLPS) of α-Syn has been designated as an alternate nucleation mechanism, which occurs in the early lag phase of the aggregation pathway leading to the formation of dynamic supramolecular assemblies. The stronger self-association among the protein molecules triggers the irreversible liquid-to-solid transition of these supramolecular assemblies into the amyloid-like hydrogel, which may serve as a reservoir entrapping toxic oligomeric intermediates and fibrils. This review strives to provide insights into different modes of α-Syn self-assemblies including LLPS-mediated self-assembly and its recent advancements.

Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum.