Protein Droplets Research Articles

Effects of oil-water interfacial properties on protein structuring and droplet deformation in high moisture meat analogues containing oil

Adding oil to high moisture meat analogues (HMMA) can increase juiciness, and can be achieved by incorporating emulsion droplets during extrusion. Since these droplets can coalesce when subjected to high shear, selecting appropriate emulsion stabilisers is important. For several commercial plant-protein emulsion stabilisers, it was investigated how oil-water interfacial mechanical properties affect droplet deformation and protein structuring in extrusion of HMMAs. Emulsions with 10 wt% or 15 wt% oil, stabilised by potato protein isolates (POPI-1 (Rich in patatin) and POPI-2 (rich in protease inhibitor)) and pea protein isolate PPI, were used to make extrudates with 5.7 wt% and 8.5 wt% oil, respectively. In 8.5 wt%-extrudates, POPI-2 had the most oil leakage from the cooling die, while PPI had the smallest amount despite having softer and more stretchable interfaces. Blade-cutting tests showed the highest maximum force for 8.5 wt%-extrudates with POPI-1, likely because POPI-1 formed the stiffest interfaces. Tensile stress testing showed the largest fracture strain in 8.5% wt%-extrudates with PPI, corresponding to its longer wedge length. Multiphoton excitation microscopy was used to visualise the extrudates protein structure and oil droplets. This showed that droplets near the surface of the extrudate were less deformed than droplets in the centre. There were only small differences between protein stabilisers regarding oil droplet deformation, indicating droplet deformation was dominated by deformation of the protein matrix. The O/W interfacial properties significantly affected oil leakage, cutting force, and tensile strength of extrudates. These results are important to consider when designing emulsions for HMMAs processed by extruders.

Phase-separated super-enhancers confer an innate radioresistance on genomic DNA.

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.

Raman spectroscopy in the study of amyloid formation and phase separation.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, share a common pathological feature of amyloid structure accumulation. However, the structure-function relationship between these well-ordered, β-sheet-rich, filamentous protein deposits and disease etiology remains to be defined. Recently, an emerging hypothesis has linked phase separation, a process involved in the formation of protein condensates, to amyloid formation, suggesting that liquid protein droplets serve as loci for amyloid initiation. To elucidate how these processes contribute to disease progression, tools that can directly report on protein secondary structural changes are needed. Here, we review recent studies that have demonstrated Raman spectroscopy as a powerful vibrational technique for interrogating amyloid structures; one that offers sensitivity from the global secondary structural level to specific residues. This probe-free technique is further enhanced via coupling to a microscope, which affords structural data with spatial resolution, known as Raman spectral imaging (RSI). In vitro and in cellulo applications of RSI are discussed, highlighting studies of protein droplet aging, cellular internalization of fibrils, and Raman imaging of intracellular water. Collectively, utilization of the myriad Raman spectroscopic methods will contribute to a deeper understanding of protein conformational dynamics in the complex cellular milieu and offer potential clinical diagnostic capabilities for protein misfolding and aggregation processes in disease states.

GENESIS CGDYN: large-scale coarse-grained MD simulation with dynamic load balancing for heterogeneous biomolecular systems.

Residue-level coarse-grained (CG) molecular dynamics (MD) simulation is widely used to investigate slow biological processes that involve multiple proteins, nucleic acids, and their complexes. Biomolecules in a large simulation system are distributed non-uniformly, limiting computational efficiency with conventional methods. Here, we develop a hierarchical domain decomposition scheme with dynamic load balancing for heterogeneous biomolecular systems to keep computational efficiency even after drastic changes in particle distribution. These schemes are applied to the dynamics of intrinsically disordered protein (IDP) droplets. During the fusion of two droplets, we find that the changes in droplet shape correlate with the mixing of IDP chains. Additionally, we simulate large systems with multiple IDP droplets, achieving simulation sizes comparable to those observed in microscopy. In our MD simulations, we directly observe Ostwald ripening, a phenomenon where small droplets dissolve and their molecules redeposit into larger droplets. These methods have been implemented in CGDYN of the GENESIS software, offering a tool for investigating mesoscopic biological processes using the residue-level CG models.

Kinetic trapping organizes actin filaments within liquid-like protein droplets

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

Entropy Tug-of-War Determines Solvent Effects in the Liquid-Liquid Phase Separation of a Globular Protein.

Liquid-liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-Crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and those that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically. A recently developed THz-calorimetry method enables quantitative comparison of the experimental and computational entropy changes of the released water molecules. The strong correlation mutually validates the two approaches and opens the way to a detailed atomic-level understanding of the different driving forces underlying the LLPS.

N-acetyl amino acid amide solubility in aqueous 1,6-hexanediol solutions: Insights into the protein droplet deformation mechanism

Proteinaceous liquid droplets, generated by liquid–liquid phase separation, function as membraneless compartments that are essential for diverse biological functions. Studies addressing droplet generation have used 1,6-hexanediol (1,6-HD) as a droplet-discerning agent owing to its capacity to induce droplet deformation. Despite the empirical utility of 1,6-HD, the mechanism underlying 1,6-HD-induced droplet deformation remains unknown. In this study, the solubilities of N-acetyl amino acid amides, which correspond to proteinogenic amino acid residues, were examined in the presence of 1,6-HD at 25 °C. Other solvents included ethanol, 1-propanol, and amides. Remarkably, 1,6-HD effectively solubilized hydrophobic species (particularly aromatic species) and exhibited reduced efficacy in solubilizing hydrophilic species and peptide bond moieties. These solubilizing effects are reflected in changes in protein solubility and structure. Specifically, 1,6-HD primarily targets the hydrophobic regions of a protein, increasing protein solubility without causing substantial structural changes. This solubilization mechanism is essential for elucidating the role of 1,6-HD as a droplet-discerning agent and recognizing its potential limitations.

Akt1 is involved in HCV release by promoting endoplasmic reticulum-to-endosome transition of infectious virions

AimsHepatitis C virus (HCV) relies on the viral and host factors to complete its life cycle. It has evolved to profit from Akt activation at some stage in its life cycle through various mechanisms, notably by activating lipogenesis, which is crucial for infectious virions production. Materials and methodsBy employing an Akt-specific inhibitor, the impact of Akt on intracellular and extracellular infectivity was investigated. To ascertain the role of Akt in the HCV life cycle, the two-part cell culture-derived HCV infection protocol utilizing Akt1 small interfering RNAs (siRNAs) was implemented. The impact of Akt1 on intracellular HCV transition was determined using membrane flotation assay and proximity ligation assay coupled with Anti-Rab7 immunoprecipitation and immunofluorescence. Key findingsAkt1 silencing reduced infectious virions release to a degree comparable to that of ApoE, a host component involved in the HCV assembly and release, suggesting Akt1 was critical in the late stage of the HCV life cycle. Extracellular infectivity of HCV was inhibited by brefeldin A, and the inhibitory effect was augmented by Akt1 silencing and partially restored by ectopic Akt1 expression. Immunofluorescence revealed that Akt1 inhibition suppressed the interaction between HCV core protein and lipid droplet. Akt1 silencing impeded the transition of HCV from the endoplasmic reticulum to the endosome and hence inhibited the secretion of HCV infectious virions from the late endosome. SignificanceOur study demonstrates that Akt1 has an impact on the lipogenesis pathway and plays a critical role in the assembly and secretion of infectious HCV.

NAGPKin: Nucleation-and-growth parameters from the kinetics of protein phase separation.

The assembly of biomolecular condensate in eukaryotic cells and the accumulation of amyloid deposits in neurons are processes involving the nucleation and growth (NAG) of new protein phases. To therapeutically target protein phase separation, drug candidates are tested in in vitro assays that monitor the increase in the mass or size of the new phase. Limited mechanistic insight is, however, provided if empirical or untestable kinetic models are fitted to these progress curves. Here we present the web server NAGPKin that quantifies NAG rates using mass-based or size-based progress curves as the input data. A report is generated containing the fitted NAG parameters and elucidating the phase separation mechanisms at play. The NAG parameters can be used to predict particle size distributions of, for example, protein droplets formed by liquid-liquid phase separation (LLPS) or amyloid fibrils formed by protein aggregation. Because minimal intervention is required from the user, NAGPKin is a good platform for standardized reporting of LLPS and protein self-assembly data. NAGPKin is useful for drug discovery as well as for fundamental studies on protein phase separation. NAGPKin is freely available (no login required) at https://nagpkin.i3s.up.pt.

Self-assembly of oat proteins into various colloidal states as function of the NaCl concentration and pH

Oats are increasingly used in a range of liquid and semi-solid foods. Insights into the behavior of native oat proteins in aqueous media are crucially important to understand their functionality, but are sorely lacking. Here, for the first time, the impact of varying NaCl concentration and pH on the colloidal state of native oat proteins was systematically studied. Non heat-treated oat groats were used as raw material to produce oat protein isolates (OPI). Upon addition of varying concentrations of NaCl to a native OPI solution, oat proteins self-assembled into different colloidal states. Most notably, at <50 mM NaCl, oat proteins formed protein aggregates with a fractal dimension of 1.8, while at 50–300 mM NaCl, oat proteins microphase-separated to form concentrated protein droplets of ≥1 μm. To allow assessing the impact of pH systematically, titration experiments were performed to establish the relation between pH and protein charge density (α). Upon changing the charge density of a native OPI solution, protein microphase separation was found to occur in the region α = −155 (pH 8.2) to α = −118 (pH 7.1). At lower charge densities (and thus pH values), this phenomenon was no longer observed, but protein aggregation occurred, and pronouncedly so at the protein iso-ionic point (α = 0, pH 5.0). In conclusion, oat proteins self-assemble into different types of colloidal structures at varying NaCl concentration and pH. This could have important implications for the properties of oat proteins in a variety of foods and should be investigated further.

Oxidative regulation of TDP-43 self-association by a β-to-α conformational switch

An evolutionarily conserved region of the TDP-43 low-complexity domain (LCD) twenty residues in length can adopt either an α-helical or β-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-β structure. Self-association can be monitored via the formation of phase-separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the LCD. Oxidation disassembles the cross-β structure, thus eliminating both self-association and phase separation. Here, we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate β-strand-mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent and that exposure to lipids enhances the β-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen-bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of LCDs.

Thermodynamic forces from protein and water govern condensate formation of an intrinsically disordered protein domain

Liquid-liquid phase separation (LLPS) can drive a multitude of cellular processes by compartmentalizing biological cells via the formation of dense liquid biomolecular condensates, which can function as membraneless organelles. Despite its importance, the molecular-level understanding of the underlying thermodynamics of this process remains incomplete. In this study, we use atomistic molecular dynamics simulations of the low complexity domain (LCD) of human fused in sarcoma (FUS) protein to investigate the contributions of water and protein molecules to the free energy changes that govern LLPS. Both protein and water components are found to have comparably sizeable thermodynamic contributions to the formation of FUS condensates. Moreover, we quantify the counteracting effects of water molecules that are released into the bulk upon condensate formation and the waters retained within the protein droplets. Among the various factors considered, solvation entropy and protein interaction enthalpy are identified as the most important contributions, while solvation enthalpy and protein entropy changes are smaller. These results provide detailed molecular insights on the intricate thermodynamic interplay between protein- and solvation-related forces underlying the formation of biomolecular condensates.

Regulating the lipid droplet interface based on milk fat globule membrane and milk proteins to improve lipid digestion of model infant formula emulsion

The interfacial properties of lipid droplets play a crucial role in lipid digestion. In this study, milk fat globule membrane (MFGM) was used to improve the lipid droplet interface of model infant formula emulsions (IFEs). We focused on the effects of the addition sequence of MFGM and milk proteins commonly used in infant formula (IFE1: Milk proteins added before homogenization, MFGM added after homogenization; IFE2: MFGM added before homogenization, milk proteins added after homogenization; IFE3: MFGM and milk proteins added before homogenization) on lipid droplets' interfacial compositions, structure, and lipid digestion behaviors and compared with human milk (HM) and commercial infant formula (IF). The results showed that the interfacial protein load was significantly lower (P < 0.05), and the interfacial phospholipid load was significantly higher (P < 0.05) in IFE2 compared to IFE1, IFE3, and IF. The lipid droplet structure of IFE2 is closer to that of HM. MFGM fragments-covered lipid droplets can be observed in IFE2. In contrast, the lipid droplets in IFE1, IFE3, and IF were covered by casein micelles, and MFGM fragments were free in the serum phase. After digestion, the lipolysis degree of IFE2 (91.7 ± 1.51%) was comparable to that of HM (94.98 ± 1.08%) and significantly higher than other samples (P < 0.05). During digestion, IFE1 and IFE3 formed thicker and denser protein knit networks compared to IFE2. The lipid droplets in IFE1 and IFE3 were embedded in protein networks, thereby inhibiting lipid digestion. These results indicated that changes in the interfacial composition of lipid droplets improved lipid digestion by mitigating the aggregate and coalescence of protein and lipid droplets.

Detection of Fibril Nucleation in Micrometer-Sized Protein Condensates and Suppression of Sup35NM Fibril Nucleation by Liquid-Liquid Phase Separation.

Elucidating the link between amyloid fibril formation and liquid-liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective.

Infectious Bursal Disease Virus Assembly Causes Endoplasmic Reticulum Stress and Lipid Droplet Accumulation.

Gumboro illness is caused by the highly contagious immunosuppressive infectious bursal disease virus (IBDV), which affects the poultry industry globally. We have previously shown that IBDV hijacks the endocytic pathway to construct viral replication complexes on endosomes linked to the Golgi complex (GC). Then, analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b, the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), and its substrate, the small GTPase ADP-ribosylation factor 1 (ARF1), for IBDV replication. In the current work, we focused on elucidating the IBDV assembly sites. We show that viral assembly occurs within single-membrane compartments closely associated with endoplasmic reticulum (ER) membranes, though we failed to elucidate the exact nature of the virus-wrapping membranes. Additionally, we show that IBDV infection promotes the stress of the ER, characterized by an accumulation of the chaperone binding protein (BiP) and lipid droplets (LDs) in the host cells. Overall, our results represent further original data showing the interplay between IBDV and the secretory pathway, making a substantial contribution to the field of birnaviruses-host cell interactions.

Fabrication of grape seed proanthocyanidin-loaded W/O/W emulsion gels stabilized by polyglycerol polyricinoleate and whey protein isolate with konjac glucomannan: Structure, stability, and in vitro digestion

In this work, the effects of konjac glucomannan (KGM) concentrations on microstructure, gel properties, stability and digestibility of water-in-oil-in-water emulsion gels stabilized by polyglycerol polyricinoleate and whey protein isolate were investigated. Visual appearance indicated that a non-layered double emulsion gel was formed when KGM increased to 0.75%. Emulsion gels with 1.5% KGM showed the highest encapsulation, freeze-thaw and photochemical stability due to the formation of the smallest droplets, which were supported by microscopic observations. Moreover, the addition of KGM improved water holding capacity, rheological and texture properties of emulsion gels. Particularly, at 1.5% or 1.75% KGM, color and potential of hydrogen showed the most stable level after 14 days of storage. During in vitro digestion, KGM delayed the hydrolysis of protein and oil droplets, and then improved the bioavailability of grape seed proanthocyanidin. These results promoted the application of KGM in emulsion gels and the encapsulation of nutraceuticals.

Coagulation of model infant formulae: Impact on their in vitro dynamic gastric digestion

The rheological properties and the in vitro dynamic gastric digestion behaviours of model infant formulae containing various ratios of casein to whey protein (0:100, 40:60, 60:40 and 80:20) were investigated using a rheometer and an infant human gastric simulator. The pH profiles and the viscoelastic moduli were determined during acidification by glucono-δ-lactone and at different pepsin concentrations (0, 0.3, 1.0, 1.7 and 2.5 U/mL). The infant formula containing whey protein only formed a weak gel at about pH 5. In the presence of pepsin, all casein-containing infant formulae formed gels at pH ∼6.0 and only low pepsin activity (>0.3 U/mL) was required for the gelation. The strength of the gel/coagulum was dependent mainly on the casein content. Coagulation of protein and flocculation of oil droplets, induced by both pepsin proteolysis and the low pH under gastric conditions, were observed in the infant formulae during dynamic digestion in the human gastric simulator. Confocal images showed that the infant formula containing whey protein only formed smaller flocs of aggregated protein and oil droplets, which may have led to their observed faster protein digestion, than the infant formulae containing a combination of casein and whey protein. The casein-dominant infant formulae had greater aggregation than the whey-protein-dominant infant formulae during gastric digestion, which led to their lower rate of casein digestion. These results suggest that the extent of coagulation and the gastric digestion of the proteins in infant formulae can be controlled by the rational selection of the protein components and the ratio of casein to whey protein, such that different nutritional requirements of infants can be met.

Sleeve gastrectomy decreased hepatic lipid accumulation by inducing autophagy via AMPK/mTOR pathway

This study was designed to investigate the roles of autophagy in the attenuation of hepatic lipid accumulation after sleeve gastrectomy (SG). Thirty-two rats were divided into normal control, obesity group, sham group, and SG group. Then serum glucagon-like polypeptide-1 (GLP-1) and lipid accumulation were determined, followed by measuring the activity of autophagy based on immunohistochemistry (IHC) and Western blot analysis. Our data showed significant decrease in the lipid accumulation after SG compared with sham group. GLP-1 and autophagy showed significant increase in rats underwent SG compared with the sham group (P < 0.05). In vitro experiments were conducted to analyze the roles of GLP-1 in autophagy. We knock-downed the expression of Beclin-1 in HepG2, and then analyzed the expression of autophagy-related protein (i.e. LC3BII and LC3BI) and lipid droplet accumulation. In HepG2 cells, GLP-1 analog reduced lipid accumulation by activating autophagy through modulating the AMPK/mTOR signaling pathway. All these concluded that SG decreased hepatic lipid accumulation by inducing autophagy through modulating AMPK/mTOR pathway.

Labellar Structure of the Maxillaria splendens Alliance (Orchidaceae: Maxillariinae) Indicates Floral Polyphenols as a Reward for Stingless Bees

Several studies have reported stingless Meliponini bees gathering hairs from the labella of Maxillaria spp., including M. ochroleuca, a member of the M. splendens alliance. Such hairs usually contain food materials and are thought to have nutritional value. The papillose labella of representatives of the Maxillaria splendens alliance, however, bear scattered, simple 1-5-celled uniseriate trichomes (hairs) that lack food materials. By contrast, here, as well as polyphenolic compounds, typical labellar papillae usually contain small quantities of starch, protein, and minute droplets of lipid, the last probably involved in the production of fragrance. Towards the labellum apex occur elevated groups of papillae that lack food materials, but contain volatile compounds, probably fragrance precursors. In the past, the terms 'trichomes' or 'hairs' and 'papillae' have been used interchangeably, causing some confusion. Since the trichomes, however, unlike the papillae, are easily detachable and can fragment, it is most likely they, not the papillae, that have previously been observed being collected by bees, but their poor food content indicates that they do not function as food-hairs. Even so, our field observations of M. ochroleuca reveal that stingless bees scrape polyphenol-rich labellar tissue and possibly use this material to produce a resinous, complex, heterogeneous substance commonly referred to as 'bee glue', used for nest construction and repair.

Soluble protein particles produced directly from mung bean flour by simple coacervation

Poor solubility is a common characteristic of many plant protein ingredients which often hampers product formulation. We exploit simple coacervation, or liquid-liquid phase separation, of plant proteins in flours, to formulate plant proteins as powders consisting of submicron colloids with good solubility and dispersibility. We consider the specific case of mung bean flour, but the approach is more general. First, we study the influence of pH on the formation of submicron protein droplets (“coacervates”) after alkaline protein extraction from mung bean flour. Next, the proteins in droplets were heat-set into colloidal protein microgels, and the morphology of the colloids was assessed by scanning electron microscopy. The mung bean protein colloids have an intrinsic viscosity much lower than typical food thickeners, indicating the dense nature of the particles. After spray-drying, they maintain a good dispersibility even close to the isoelectric point. Heat-induced gelation of redispersed protein particles resulted in gels with moduli much less than those of commercial mung bean protein concentrates at equivalent protein concentration. Hence, the main impact of the pretreatment is on dispersibility and heat stability, with relevance to formulations such as plant-protein based beverages.