Emerging experimental methods to study the thermodynamics of biomolecular condensate formation.

The formation of biomolecular condensates in vivo is increasingly recognized to underlie a multitude of crucial cellular functions. Furthermore, the evolution of highly dynamic protein condensates into progressively less reversible assemblies is thought to be involved in a variety of disorders, from cancer over neurodegeneration to rare genetic disorders. There is an increasing need for efficient experimental methods to characterize the thermodynamics of condensate formation and that can be used in screening campaigns to identify and rationally design condensate modifying compounds. Theoretical advances in the field are also identifying the key parameters that need to be measured in order to obtain a comprehensive understanding of the underlying interactions and driving forces. Here, we review recent progress in the development of efficient and quantitative experimental methods to study the driving forces behind and the temporal evolution of biomolecular condensates.

Taylor Dispersion-Induced Phase Separation for the Efficient Characterisation of Protein Condensate Formation.

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid-liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion-induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL-3 and Ddx4. Uniquely accessible to this method, we find an unexpected re-entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α-synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS-modifying compounds can be screened highly efficiently.

An Overview of Pulses Production in India: Retrospect and Prospects of the Future Food with an Application of Hybrid Models.

Forecasts are valuable to countries to make informed business decisions and develop data-driven strategies. The production of pulses is an integral part of agricultural diversification initiatives because it offers promising economic opportunities to reduce rural poverty and unemployment in developing countries. Pulses are the cheapest source of protein needed for human health. India's pulses production guidelines must be based on accurate and best forecast models. Comparing classical statistical and machine learning models based on different scientific data series is the subject of high-level research today. This study focused on the forecasting behaviour of pulses production for India, Karnataka, Madhya Pradesh, Maharashtra, Rajasthan and Uttar Pradesh. The data series was split into a training dataset (1950-2014) and a testing dataset (2015-2019) for model building and validation purposes, respectively. ARIMA, NNAR and hybrid models were used and compared on training and validation datasets based on goodness of fit (RMSE, MAE and MASE). This research demonstrates that due to the diverse agricultural conditions across different provinces in India, there is no single model that can accurately predict pulse production in all regions. This study's highest accuracy model is ARIMA. ARIMA outperforms NNAR, a machine learning model. Pulse production in India, Rajasthan, and Madhya Pradesh will expand by 26.11%, 12.62%, and 0.51% from 2020 to 2030, whereas it would decline by - 6.5%, - 6.21%, and - 6.76 per cent in Karnataka, Maharashtra, and Uttar Pradesh, respectively. The current forecast results could allow policymakers to develop more aggressive food security and sustainability plans and better Indian pulses production policies in the future.

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.

Glycosaminoglycans have variable effects on α-synuclein aggregation and differentially affect the activities of the resulting amyloid fibrils.

Parkinson's disease is mainly a sporadic disorder in which both environmental and cellular factors play a major role in the initiation of this disease. Glycosaminoglycans (GAG) are integral components of the extracellular matrix and are known to influence amyloid aggregation of several proteins, including α-synuclein (α-Syn). However, the mechanism by which different GAGs and related biological polymers influence protein aggregation and the structure and intercellular spread of these aggregates remains elusive. In this study, we used three different GAGs and related charged polymers to establish their role in α-Syn aggregation and associated biological activities of these aggregates. Heparin, a representative GAG, affected α-Syn aggregation in a concentration-dependent manner, whereas biphasic α-Syn aggregation kinetics was observed in the presence of chondroitin sulfate B. Of note, as indicated by 2D NMR analysis, different GAGs uniquely modulated α-Syn aggregation because of the diversity of their interactions with soluble α-Syn. Moreover, subtle differences in the GAG backbone structure and charge density significantly altered the properties of the resulting amyloid fibrils. Each GAG/polymer facilitated the formation of morphologically and structurally distinct α-Syn amyloids, which not only displayed variable levels of cytotoxicity but also exhibited an altered ability to internalize into cells. Our study supports the role of GAGs as key modulators in α-Syn amyloid formation, and their distinct activities may regulate amyloidogenesis depending on the type of GAG being up- or down-regulated in vivo.

Comparison of Kinetics, Toxicity, Oligomer Formation, and Membrane Binding Capacity of α-Synuclein Familial Mutations at the A53 Site, Including the Newly Discovered A53V Mutation.

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to late-onset PD. In this study, we characterized for the first time the biophysical properties of A53V, including the aggregation propensities, toxicity of aggregated species, and membrane binding capability, along with those of all familial mutations at the A53 position. Our data suggest that the A53V mutation accelerates fibrillation of α-Syn without affecting the overall morphology or cytotoxicity of fibrils compared to those of the wild-type (WT) protein. The aggregation propensity for A53 mutants is found to decrease in the following order: A53T > A53V > WT > A53E. In addition, a time course aggregation study reveals that the A53V mutant promotes early oligomerization similar to the case for the A53T mutation. It promotes the largest amount of oligomer formation immediately after dissolution, which is cytotoxic. Although in the presence of membrane-mimicking environments, the A53V mutation showed an extent of helix induction capacity similar to that of the WT protein, it exhibited less binding to lipid vesicles. The nuclear magnetic resonance study revealed unique chemical shift perturbations caused by the A53V mutation compared to those caused by other mutations at the A53 site. This study might help to establish the disease-causing mechanism of A53V in PD pathology.

Cytotoxic Oligomers and Fibrils Trapped in a Gel-like State of α-Synuclein Assemblies.

α-Synuclein (α-Syn) aggregation is associated with Parkinson's disease (PD) pathogenesis. In PD, the role of oligomers versus fibrils in neuronal cell death is debatable, but recent studies suggest oligomers are a proximate neurotoxin. Herein, we show that soluble α-Syn monomers undergo a transformation from a solution to a gel state on incubation at high concentration. Detailed characterization of the gel showed the coexistence of monomers, oligomers, and short fibrils. In vitro, the gel was highly cytotoxic to human neuroblastoma cells. The individual constituents of the gel are short-lived species but toxic to the cells. They comprise a structurally heterogeneous population of α-helical and ß-sheet-rich oligomers and short fibrils with the cross-ß motif. Given the recent evidence of the gel-like state of the protein associated with neurodegenerative diseases, the gel state of α-Syn in this study represents a mechanistic and structural model for the in vivo toxicity of α-Syn in PD.

Thermodynamic characterization of amyloid polymorphism by microfluidic transient incomplete separation.

Amyloid fibrils of proteins such as α-synuclein are a hallmark of neurodegenerative diseases and much research has focused on their kinetics and mechanisms of formation. The question as to the thermodynamic stability of such structures has received much less attention. Here, we newly utilize the principle of transient incomplete separation of species in laminar flow in combination with chemical depolymerization for the quantification of amyloid fibril stability. The relative concentrations of fibrils and monomer at equilibrium are determined through an in situ separation of these species based on their different diffusivity inside a microfluidic capillary. The method is highly sample economical, using much less than a microliter of sample per data point and its only requirement is the presence of aromatic residues (W, Y) because of its label-free nature, which makes it widely applicable. Using this method, we investigate the differences in thermodynamic stability between different fibril polymorphs of α-synuclein and quantify these differences for the first time. Importantly, we show that fibril formation can be under kinetic or thermodynamic control and that a change in solution conditions can both stabilise and destabilise amyloid fibrils. Taken together, our results establish the thermodynamic stability as a well-defined and key parameter that can contribute towards a better understanding of the physiological roles of amyloid fibril polymorphism.

Evaluation of management practices in rice-wheat cropping system using multicriteria decision-making methods in conservation agriculture.

In this study, we employed two multiple criteria decision-making (MCDM) methods, namely the Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) and the Analytic Hierarchic Process (AHP), to determine the best management choice for the cultivation of wheat with a regime of conservation agriculture (CA) practices. By combining alternative tillage approaches, such as reduced tillage and zero tillage, with the quantity of crop residues and fertilizer application, we were able to develop the regime of CA practices. The performance of the regimes compared to the conventional ones was then evaluated using conflicting parameters relating to energy use, economics, agronomy, plant protection, and soil science. TOPSIS assigned a grade to each alternative based on how close it was to the ideal solution and how far away it was from the negative ideal solution. However, employing AHP, we determined the weights of each of the main and sub-parameters used for this study using pairwise comparison. With TOPSIS, we found ZERO1 (0% residue + 100% NPK) followed by ZERO4 (50%residue + 100% NPK), and ZERO2 (100% residue + 50% NPK) were the best performing tillage-based alternatives. To best optimize the performance of wheat crops under various CA regimes, TOPSIS assisted the decision-makers in distinguishing the effects of the parameters on the outcome and identifying the potential for maneuvering the weak links. The outcomes of this investigation could be used to improve management techniques for wheat production with CA practices for upscaling among the farmers.

Spectrally Resolved FRET Microscopy of α-Synuclein Phase-Separated Liquid Droplets.

Liquid-liquid phase separation (LLPS) has emerged as an important phenomenon associated with formation of membraneless organelles. Recently, LLPS has been shown to act as nucleation centers for disease-associated protein aggregation and amyloid fibril formation. Phase-separated α-synuclein droplets gradually rigidify during the course of protein aggregation, and it is very challenging to understand the biomolecular interactions that lead to liquid-like to solid-like transition using conventional ensemble measurements. Here, we describe a spectrally-resolved fluorescence microscopy based Förster resonance energy transfer (FRET) imaging to probe interactions of α-synuclein in individual droplets during LLPS-mediated aggregation. By acquiring entire emission spectral profiles of individual droplets upon sequential excitation of acceptors and donors therein, this technique allows for the quantification of sensitized emission proportional to the extent of FRET, which enables interrogation of the evolution of local interactions of donor-/acceptor-labeled α-synuclein molecules within each droplet. The present study on single droplets is not only an important development for studying LLPS but can also be used to investigate self-assembly or aggregation in biomolecular systems and soft materials.

FRAP and FRET Investigation of α-Synuclein Fibrillization via Liquid-Liquid Phase Separation In Vitro and in HeLa Cells.

Liquid-liquid phase separation (LLPS) acts as an important biological phenomenon in membraneless organelle formation. These phase-separated bodies can also act as nucleation centers for disease-associated amyloid formation. Fluorescence recovery after photobleaching (FRAP) is a crucial technique to analyze the material property (liquid or solid) of protein LLPS. On the other hand, Förster resonance energy transfer (FRET) is used to understand the domain-specific involvement (intermolecular interactions) of protein molecules inside the phase-separated droplets. In this protocol, we delineate mechanisms of liquid-to-solid transition of α-synuclein LLPS by using in vitro and in cell FRAP as well as in vitro FRET techniques.

Mass photometric detection and quantification of nanoscale α-synuclein phase separation.

Protein liquid-liquid phase separation can lead to disease-related amyloid fibril formation. The mechanisms of conversion of monomeric protein into condensate droplets and of the latter into fibrils remain elusive. Here, using mass photometry, we demonstrate that the Parkinson's disease-related protein, α-synuclein, can form dynamic nanoscale clusters at physiologically relevant, sub-saturated concentrations. Nanoclusters nucleate in bulk solution and promote amyloid fibril formation of the dilute-phase monomers upon ageing. Their formation is instantaneous, even under conditions where macroscopic assemblies appear only after several days. The slow growth of the nanoclusters can be attributed to a kinetic barrier, probably due to an interfacial penalty from the charged C terminus of α-synuclein. Our findings reveal that α-synuclein phase separation occurs at much wider ranges of solution conditions than reported so far. Importantly, we establish mass photometry as a promising methodology to detect and quantify nanoscale precursors of phase separation. We also demonstrate its general applicability by probing the existence of nanoclusters of a non-amyloidogenic protein, Ddx4n1.

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.

Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids.

Synergistic-aggregation and cross-seeding by two different proteins/peptides in the amyloid aggregation are well evident in various neurological disorders including Alzheimer's disease. Here, we show co-storage of human Prolactin (PRL), which is associated with lactation in mammals, and neuropeptide galanin (GAL) as functional amyloids in secretory granules (SGs) of the female rat. Using a wide variety of biophysical studies, we show that irrespective of the difference in sequence and structure, both hormones facilitate their synergic aggregation to amyloid fibrils. Although each hormone possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation by PRL seeds and the inability of cross seeding by mixed fibrils suggest tight regulation of functional amyloid formation by these hormones for their efficient storage in SGs. Further, the faster release of functional hormones from mixed fibrils compared to the corresponding individual amyloid, suggests a novel mechanism of heterologous amyloid formation in functional amyloids of SGs in the pituitary.

The formation of plaques of proteins called 'amyloids' in the brain is one of the hallmark characteristics of both Alzheimer's and Parkinson's disease, but amyloids can form in many tissues and organs, often disrupting normal activity. A lot of the research into amyloids has focused on their role in disease, but it turns out that amyloids can also appear in healthy tissues. For example, some protein hormones form amyloids that act as storage depots, helping cells to release the hormone when it is needed. Normally, amyloids are made mostly of a single type of protein or protein fragment associated with a particular disease like Alzheimer's. Often, this type of amyloid promotes plaque formation in other proteins, which aggravates other diseases (for example, the amyloids that form in Alzheimer's can lead to Parkinson's disease or type II diabetes getting worse).The plaques start growing from small amyloid fragments called seeds. In mixed amyloids ­ amyloids made of two types of proteins ­ seeds made of one protein can trigger the formation of amyloids of the other protein. This raises the question, is this true for hormones? The body often releases more than one hormone at a time from the same tissue; for example, the pituitary gland releases prolactin and galanin simultaneously. However, these hormones have completely different structures, so whether they can form a mixed amyloid is unclear. To answer this question, Chatterjee et al. first determined that, within the pituitary gland of female rats, prolactin and galanin could be found together in the same cells, forming mixed amyloids. To understand out how this happens, Chatterjee et al. tried seeding new amyloids using either prolactin or galanin. This revealed that only prolactin seeds were able to trigger the formation of galanin amyloids. Chatterjee et al. also found that the mixed amyloids could release the hormones faster than amyloids made from either protein alone. Together, these results suggest that the collaboration between these two proteins may help maintain hormone balance in the body. Problems with hormone storage and release lead to various human diseases, including prolactinoma. Understanding amyloid storage depots could reveal new ways to control hormone levels. Further research could also help to explain more about well-studied diseases linked to amyloids, like Alzheimer's.

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation.

Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

Effect of Disease-Associated P123H and V70M Mutations on ß-Synuclein Fibrillation.

Synucleinopathies are a class of neurodegenerative diseases, including Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA). The common pathological hallmark of synucleinopathies is the filamentous α-synuclein (α-Syn) aggregates along with membrane components in cytoplasmic inclusions in the brain. ß-Synuclein (ß-Syn), an isoform of α-Syn, inhibits α-Syn aggregation and prevents its neurotoxicity, suggesting the neuroprotective nature of ß-Syn. However, this notion changed with the discovery of disease-associated ß-Syn mutations, V70M and P123H, in patients with DLB. It is still unclear how these missense mutations alter the structural and amyloidogenic properties of ß-Syn, leading to neurodegeneration. Here, we characterized the biophysical properties and investigated the effect of mutations on ß-Syn fibrillation under different conditions. V70M and P123H show high membrane binding affinity compared to wild-type ß-Syn, suggesting their potential role in membrane interactions. ß-Syn and its mutants do not aggregate under normal physiological conditions; however, the proteins undergo self-polymerization in a slightly acidic microenvironment and/or in the presence of an inducer, forming long unbranched amyloid fibrils similar to α-Syn. Strikingly, V70M and P123H mutants exhibit accelerated fibrillation compared to native ß-Syn under these conditions. NMR study further revealed that these point mutations induce local perturbations at the site of mutation in ß-Syn. Overall, our data provide insight into the biophysical properties of disease-associated ß-Syn mutations and demonstrate that these mutants make the native protein more susceptible to aggregation in an altered microenvironment.

α-Synuclein aggregation nucleates through liquid-liquid phase separation.

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

PNPLA3-I148M: a problem of plenty in non-alcoholic fatty liver disease.

Fatty liver disease (FLD) affects more than one-third of the population in the western world and an increasing number of children in the United States. It is a leading cause of obesity and liver transplantation. Mechanistic insights into the causes of FLD are urgently needed since no therapeutic intervention has proven to be effective. A sequence variation in patatin like phospholipase domain-containing protein 3 (PNPLA3), rs 738409, is strongly associated with the progression of fatty liver disease. The resulting mutant causes a substitution of isoleucine to methionine at position 148. The underlying mechanism of this disease remains unsolved although several studies have illuminated key insights into its pathogenesis. This review highlights the progress in our understanding of PNPLA3 function in lipid droplet dynamics and explores possible therapeutic interventions to ameliorate this human health hazard.