Effects Of Osmolytes Research Articles

Glycine betaine application improved seed cotton yield and economic returns under deficit irrigation

BackgroundDeficit irrigation exerts devastating effects on the productivity and economic returns of cotton crop, as well as carbon dioxide (CO2) emission from soil. Osmolytes play a significant role in facilitating the adaptation of cotton plants to abiotic stresses and improve productivity. MethodsThis study investigated the effects of different osmolytes (glycine betaine, ascorbic acid, salicylic acid 100 mg L−1 each) and deficit irrigation (50 %-I50, 75 %-I75, and 100 %-I100) on seed cotton yield, greenhouse gas emission (CO2-C), emission factor (EFs) and economic returns of cotton in Southern Anatolia, Türkiye. ResultsDeficit irrigation and osmolyte treatment, both separately and in combination, had a substantial impact on seed cotton yield, CO2-C emission and EFs. The lowest (3800 kg ha−1) and the highest (4746 kg ha−1) seed cotton yield was noted under I50, and I100 treatments, respectively. Similarly, no osmolyte application and application of glycine betaine resulted in the lowest (4097 kg ha−1) and the highest (4545 kg ha−1) seed cotton yield, respectively. The interactive effect indicated that application of glycine betaine and salicylic acid produced better yield than control treatment under all irrigation treatments. The lowest (1.55) and the highest (1.94 mg) CO2-C emission (mg CO2-C m−2 h−1) was recorded for I50, and I100 treatments respectively. Likewise, the lowest (1.52) and the highest (2.19) daily carbon emission were recorded for salicylic acid and glycine betaine application, respectively. The lowest and the highest EFs values were observed for glycine betaine and ascorbic acid application, respectively. Application of glycine betaine resulted in the highest economic returns under all irrigation treatments which was comparable to salicylic acid, whereas the lower economic returns were recorded for control treatment. ConclusionIt is concluded that application of glycine betaine can be used to improve seed cotton yield and economic returns under deficit irrigation. Similarly, glycine betaine proved helpful in reducing CO2-C emission under deficit irrigation compared to normal irrigation.

Modulation of Biomolecular Liquid-Liquid Phase Separation by Preferential Hydration and Interaction of Small Osmolytes with Proteins.

We examined the effects of trimethylamine N-oxide (TMAO) and urea (known osmolytes) on the liquid-liquid phase separation (LLPS) of fused in sarcoma (FUS) and three FUS-LLPS states: LLPS states at atmospheric pressure with low- and high-salt concentrations and a re-entrant LLPS state above 2 kbar. Temperature- and pressure-scan turbidity measurements revealed that TMAO and urea contributed to stabilizing and destabilizing LLPS, respectively. These results can be attributed to the excluded volume effect of TMAO (preferential hydration) and preferential interaction of urea with proteins. Additionally, TMAO counteracted the effects of equimolar urea on LLPS, a phenomenon not previously reported. The concept of the m-value for osmolyte-induced protein folding and unfolding can be applied to the osmolyte's effects on LLPS. In conclusion, biomolecular LLPS can be modulated by preferential hydration and the interaction of small osmolytes with proteins, thereby facilitating LLPS formation, even in extreme environments characterized by high-salt, high-urea, and high-pressure conditions.

Effect of Natural Osmolytes on Recombinant Tau Monomer: Propensity of Oligomerization and Aggregation.

The pathological misfolding and aggregation of the microtubule associated protein tau (MAPT), a full length Tau2N4R with 441aa, is considered the principal disease relevant constituent in tauopathies including Alzheimer's disease (AD) with an imbalanced ratio in 3R/4R isoforms. The exact cellular fluid composition, properties, and changes that coincide with tau misfolding, seed formation, and propagation events remain obscure. The proteostasis network, along with the associated osmolytes, is responsible for maintaining the presence of tau in its native structure or dealing with misfolding. In this study, for the first time, the roles of natural brain osmolytes are being investigated for their potential effects on regulating the conformational stability of the tau monomer (tauM) and its propensity to aggregate or disaggregate. Herein, the effects of physiological osmolytes myo-inositol, taurine, trimethyl amine oxide (TMAO), betaine, sorbitol, glycerophosphocholine (GPC), and citrulline on tau's aggregation state were investigated. The overall results indicate the ability of sorbitol and GPC to maintain the monomeric form and prevent aggregation of tau, whereas myo-inositol, taurine, TMAO, betaine, and citrulline promote tau aggregation to different degrees, as revealed by protein morphology in atomic force microscopy images. Biochemical and biophysical methods also revealed that tau proteins adopt different conformations under the influence of these osmolytes. TauM in the presence of all osmolytes expressed no toxicity when tested by a lactate dehydrogenase assay. Investigating the conformational stability of tau in the presence of osmolytes may provide a better understanding of the complex nature of tau aggregation in AD and the protective and/or chaotropic nature of osmolytes.

In situ analysis of osmolyte mechanisms of proteome thermal stabilization

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

Drought Stress Amelioration Attributes of Plant-Associated Microbiome on Agricultural Plants.

The future global food security depends on the availability of water for agriculture. Yet, the ongoing rise in nonagricultural uses for water, such as urban and industrial uses, and growing environmental quality concerns have increased pressure of irrigation water demand and posed danger to food security. Nevertheless, its severity and duration are predicted to rise shortly. Drought pressure causes stunted growth, severe damage to photosynthesis activity, loss in crop yield, reduced seed germination, and reduced nutrient intake by plants. To overcome the effects of a devastating drought on plants, it is essential to think about the causes, mechanisms of action, and long-term agronomy management and genetics. As a result, there is an urgent need for long-term medication to deal with the harmful effects of drought pressure. The review focuses on the adverse impact of drought on the plant, physiological, and biochemical aspects, and management measures to control the severity of drought conditions. This article reviews the role of genome editing (GE) technologies such as CRISPR 9 (CRISPR-Cas9) related spaces and short palindromic relapse between proteins in reducing the effects of phytohormones, osmolytes, external compounds, proteins, microbes (plant growth-promoting microorganism [PGPM]), approach omics, and drought on plants that support plant growth. This research is to examine the potential of using the microbiome associated with plants for drought resistance and sustainable agriculture. Researchers also advocate using a mix of biotechnology, agronomic, and advanced GE technologies to create drought-tolerant plant varieties.

Osmolyte-induced conformational stabilization of a hydrophobic polymer.

Elucidating the mechanistic role of osmolytes on conformations of hydrophobic prototypical macromolecules in principle is the stepping stone towards understanding the effect of osmolytes on proteins. Motivated by this, we use equilibrium simulations and umbrella sampling techniques to dissect the underlying mechanism of osmolyte-induced conformational stability of a hydrophobic polymer. Our results unveil a remarkable osmolyte-dependent conformational stabilization of the polymer. In an aqueous solution of 4 M choline chloride (ChCl), the polymer has an even more compact structure than in water. On the other hand, an aqueous solution of 8 M urea stabilizes the extended state of the polymer. Interestingly, the polymer adopts an intermediate hairpin conformation in a mixed osmolyte solution of 4 M ChCl and 8 M urea in water due to the interplay of ChCl and urea. Our simulations identify the relative accumulation of water and the hydrophilic part of choline or preferential binding of urea near the collapsed and the extended states, respectively. Analyses split out the enthalpic and entropic contributions to the overall free energy. This decides the stabilization of the preferred conformation in the chosen osmolyte solution. Our simulations show that in an aqueous solution of ChCl, the hairpin state is stabilized by entropy gain. In contrast, the enthalpic contribution stabilizes the hairpin state in mixed environments. However, a collapsed state is energetically not favored in the presence of urea. In brief, via employing an in silico approach, the current findings indicate the importance of osmolytes in stabilizing the conformational states of hydrophobic polymers.

Effects of osmolytes under crowding conditions on the properties of muscle glycogen phosphorylase b

The study of the relationship between the activity and stability of enzymes under crowding conditions in the presence of osmolytes is important for understanding the functioning of a living cell. The effect of osmolytes (trehalose and betaine) on the secondary and tertiary structure and activity of muscle glycogen phosphorylase b (Phb) under crowding conditions created by PEG 2000 and PEG 20000 was investigated using dynamic light scattering, differential scanning calorimetry, circular dichroism spectroscopy, fluorimetry and enzymatic activity assay. At 25 °C PEGs increased Phb activity, but PEG 20000 to a greater extent. Wherein, PEG 20000 significantly destabilized its tertiary and secondary structure, in contrast to PEG 2000. Trehalose removed the effects of PEGs on Phb, while betaine significantly reduced the activating effect of PEG 20000 without affecting the action of PEG 2000. Under heat stress at 48 °C, the protective effect of osmolytes under crowding conditions was more pronounced than at room temperature, and the Phb activity in the presence of osmolytes was higher in these conditions than in diluted solutions. These results provide important insights into the complex mechanism, by which osmolytes affect the structure and activity of Phb under crowding conditions.

Effect of Osmolytes on Photoassembly of Functionally Active Mn4CaO5 Cluster in Mn-Depleted Photosystem II Preparations Isolated from Spinach Leaves

The effect of osmolytes (trehalose, sucrose, betaine, D-glucose and hydroxyectoine) on the photoassembly of the functionally active inorganic core of the water-oxidizing complex (Mn4CaO5 cluster) in Mn-depleted PSII preparations (apo-WOC-PSII) was investigated. It was revealed that the efficiency of the photoassembly of the Mn4CaO5 cluster was considerably (three times) increased in the presence of 1 M disaccharides (trehalose or sucrose) in contrast to other osmolytes. It was found that the osmolytes (especially trehalose or sucrose) improved the redox interaction of exogenous Mn2+ with apo-WOC-PSII, enhanced the protective effect of Mn2+ against the photoinhibition of apo-WOC-PSII, protected CaCl2-treated PSII preparations against thermoinactivation, and stabilized the water-oxidizing complex and electron transport from QA to QB in native PSII preparations during heat treatment. It is suggested that the ability of osmolytes to enhance the efficiency of the photoassembly of a Mn4CaO5 cluster depends on their effect on the following key processes: the redox interaction of Mn2+ with apo-WOC-PSII; the stability of apo-WOC-PSII to photoinhibition during the photoactivation procedure; and the stabilization of both the newly assembled functionally active Mn4CaO5 cluster and the electron transfer from QA to QB.

A Synergistic View on Osmolyte's Role against Salt and Cold Stress in Biointerfaces.

We present our perspective on the role of osmolytes in mitigating abiotic stresses such as hypersalinity and sudden temperature changes. While the stabilizing effect of osmolytes on protein tertiary structures has been extensively studied, their direct impact on abiotic stress factors has eluded mainstream attention. Via highlighting a set of recent success stories of a joint venture of computer simulations and experimental measurements, we summarize the mechanistic insights into osmolytic action, particularly in the context of salt stress and combined cold-salt stress at the interface of biomolecular surfaces and saline environments. We stress the importance of chemical specificity in osmolytic activity, the interplay of differential osmolytic behaviors against heterogeneous salt stress, and the capability of osmolytes to adopt combined actions. Additionally, we discuss the potential of incorporating nanomaterial-based systems to enrich our understanding of osmolyte bioactions and facilitate their practical applications. We anticipate that this discourse will inspire interdisciplinary collaborations and motivate further investigations on osmolytes, ultimately broadening their applications in the fields of health and disease.

Modulation of protein-saccharide interactions by deep-sea osmolytes under high pressure stress

Deep-sea organisms must cope with high hydrostatic pressures (HHP) up to the kbar regime to control their biomolecular processes. To alleviate the adverse effects of HHP on protein stability most organisms use high amounts of osmolytes. Little is known about the effects of these high concentrations on ligand binding. We studied the effect of the deep-sea osmolytes trimethylamine-N-oxide, glycine, and glycine betaine on the binding between lysozyme and the tri-saccharide NAG3, employing experimental and theoretical tools to reveal the combined effect of osmolytes and HHP on the conformational dynamics, hydration changes, and thermodynamics of the binding process. Due to their different chemical makeup, these cosolutes modulate the protein-sugar interaction in different ways, leading to significant changes in the binding constant and its pressure dependence. These findings suggest that deep-sea organisms may down- and up-regulate reactions in response to HHP stress by altering the concentration and type of the intracellular osmolyte.

Temperature activated transient receptor potential ion channels from Antarctic fishes.

Antarctic notothenioid fishes (cryonotothenioids) live in waters that range between -1.86°C and an extreme maximum +4°C. Evidence suggests these fish sense temperature peripherally, but the molecular mechanism of temperature sensation in unknown. Previous work identified transient receptor potential (TRP) channels TRPA1b, TRPM4 and TRPV1a as the top candidates for temperature sensors. Here, cryonotothenioid TRPA1b and TRPV1a are characterized using Xenopus oocyte electrophysiology. TRPA1b and TRPV1a showed heat-evoked currents with Q10s of 11.1 ± 2.2 and 20.5 ± 2.4, respectively. Unexpectedly, heat activation occurred at a threshold of 22.9 ± 1.3°C for TRPA1b and 32.1 ± 0.6°C for TRPV1a. These fish have not experienced such temperatures for at least 15 Myr. Either (1) another molecular mechanism underlies temperature sensation, (2) these fishes do not sense temperatures below these thresholds despite having lethal limits as low as 5°C, or (3) native cellular conditions modify the TRP channels to function at relevant temperatures. The effects of osmolytes, pH, oxidation, phosphorylation, lipids and accessory proteins were tested. No conditions shifted the activity range of TRPV1a. Oxidation in combination with reduced cholesterol significantly dropped activation threshold of TRPA1b to 11.3 ± 2.3°C, it is hypothesized the effect may be due to lipid raft disruption.

Single-molecule analysis of osmolyte-mediated nanomechanical unfolding behavior of a protein domain

The small organic molecules, known as osmolytes being ubiquitously present in different cell types, affect protein folding, stability and aggregation. However, it is unknown how the osmolytes affect the nanomechanical unfolding behavior of protein domain. Here, we show the osmolyte-dependent mechanical unfolding properties of protein titin immunoglobulin-27 (I27) domain using an atomic force microscopy (AFM)-based single-molecule force spectroscopy. We found that amines and methylamines improved the mechanical stability of I27 domain, whereas polyols had no effect. Interestingly, glycine betaine (GB) or trimethylamine-N-oxide (TMAO) increased the average unfolding force of the protein domain. The kinetic parameters analyzed at single-molecule level reveal that stabilizing effect of osmolytes is due to a decrease in the unfolding rate constant of I27, which was confirmed by molecular dynamics simulations. Our study reveals different effects that diverse osmolytes have on the mechanical properties of the protein, and suggests the potential use of osmolytes in modulating the mechanical stability of proteins required for various nano-biotechnological applications.

Modulation of the conformation, fibrillation, and fibril morphologies of human brain α-, β-, and γ-synuclein proteins by the disaccharide chemical chaperone trehalose

Human α-, β-, and γ-synuclein (syn) are natively unfolded proteins present in the brain. Deposition of aggregated α-syn in Lewy bodies is associated with Parkinson's disease (PD) and γ-syn is known to be involved in both neurodegeneration and breast cancer. At physiological pH, while α-syn has the highest propensity for fibrillation followed by γ-syn, β-syn does not form any fibrils. Fibril formation in these proteins could be modulated by protein structure stabilizing osmolytes such as trehalose which has an exceptional stabilizing effect for globular proteins. We present a comprehensive study of the effect of trehalose on the conformation, aggregation, and fibril morphology of α-, β-, and γ-syn proteins. Rather than stabilizing the intrinsically disordered state of the synucleins, trehalose accelerates the rate of fibril formation by forming aggregation-competent partially folded intermediate structures. Fibril morphologies are also strongly dependent on the concentration of trehalose with ≤ 0.4M favoring the formation of mature fibrils in α-, and γ-syn with no effect on the fibrillation of β-syn. At ≥ 0.8M, trehalose promotes the formation of smaller aggregates that are more cytotoxic. Live cell imaging of preformed aggregates of a labeled A90C α-syn shows their rapid internalization into neural cells which could be useful in reducing the load of aggregated species of α-syn. The findings throw light on the differential effect of trehalose on the conformation and aggregation of disordered synuclein proteins with respect to globular proteins and could help in understanding the effect of osmolytes on intrinsically disordered proteins under cellular stress conditions.

#5607 EFFECTS OF COMMON OSMOLYTES ON THE EXPRESSION OF FOXP3 SPLICE VARIANTS IN HUMAN PBMCS

Abstract Background and Aims Regulatory T cells modulate autoimmune and allogenic responses in glomerulonephritis and transplantation. The two most abundant fork-head box P3 (FOXP3) splice variants, full-length FOXP3 (FOXP3-FL) and FOXP3 lacking exon 2 (FOXP3-Δ 2), confer suppressive ability to regulatory T cells. Some studies showed a weaker suppressive ability of FOXP3-Δ 2. Renal organic osmolytes, urea and Trimethylamine N-oxide (TMAO), play critical roles in the hyperosmotic renal environment with high concentrations of Na+ and glucose. However, the effects of these osmolytes on FOXP3 splice variants in peripheral blood mononuclear cells (PBMCs) remain unclear. The aim of this study was to evaluate the effects of common osmolytes on the expression of FOXP3 splice variants in human PBMCs. Method PBMCs were isolated from the blood of healthy donors using Ficoll density gradients and cultured in RPMI-1640 with or without additional osmolytes at 37°C. A part of PBMCs were activated with 25ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1ug/ml ionomycin (PMA+IONO). FOXP3-FL and FOXP3-Δ 2 mRNA were detected by qPCR after 4hrs’ incubation, using specific primers. The relative expression value was calculated by the formula of (1+E0)∧ Ct0(β-actin)/(1+E)∧ Ct (target gene). Values are given as the mean±S.E.M. Statistical significance was determined by One-Way ANOVA with Post Hoc Test, using Graphpad Prism 9. A P-value <0.05 was considered significant. Results The cell viabilities after 4 hours’ incubation were all above 90%. PMA+IONO significantly increased the relative expression of both FOXP3-Δ 2 (from 0.001051 to 0.001609, P = 0.0289, n = 5) and FOXP3-FL (from 0.001024 to 0.001684, P = 0.0101, n = 5) (Figure 1). However, increased concentrations of urea (from 5mM to 50mM) had no effect on either of the two FOXP3 splice variants (P > 0.05, n = 5, Figure 1). TMAO of 4um significantly reduced FOXP3-FL in the PMA+IONO-activated PBMCs from 0.001605 to 0.0007208 (P = 0.0051, Figure 2). However, TMAO didn’t affect FOXP3-FL expression in non-activated PBMCs and had little effect on FOXP3-Δ 2 expression either (P > 0.05, n = 4). On the other hand, compared with control group, the high concentration of glucose (25mM) significantly increased the expression of FOXP3-Δ 2 from 0.002815 to 0.003461 (P = 0.0375, n = 3) in non-activated PBMCs (Figure 2). The high concentration of NaCl (147mM) had no significant effect on either of the FOXP3 splice variants (P > 0.05, n = 3, Figure 2). Furthermore, the equal osmolarity of mannitol didn’t affect the mRNA expressions of FOXP3 splice variants, either (P > 0.05, n = 3, Figure 2). But compared with glucose group (0.003461), the expression of FOXP3-Δ 2 was significantly lower in mannitol group (0.002785) (P = 0.0479, n = 3, Figure 2). It suggested that the effect of high glucose in non-activated PBMCs was independent of the osmolarity change. Conclusion In isolated human PBMCs, PMA+IONO increases both FOXP3-Δ 2 and FOXP3-FL expressions. PMA+IONO-induced FOXP3-FL increase can be inhibited by TMAO. Urea and NaCl had little effect on FOXP3 splice expressions. High concentration of glucose increases FOXP3-Δ 2 mRNA in non-activated PBMCs, which was independent of the osmolarity change.

Effect of osmolytes on the EcoRI endonuclease: Insights into hydration and protein dynamics from molecular dynamics simulations

Osmolytes play an important role in cellular physiology by modulating the properties of proteins, including their molecular specificity. EcoRI is a model restriction enzyme whose specificity to DNA is altered in the presence of osmolytes. Here, we investigate the effect of two different osmolytes, glycerol and DMSO, on the dynamics and hydration of the EcoRI enzyme using molecular dynamics simulations. Our results show that the osmolytes, alter the essential dynamics of EcoRI. Particularly, we observe that the dynamics of the arm region of EcoRI which is involved in DNA binding is significantly altered. In addition, conformational free energy analyses reveals that the osmolytes bring about a change in the landscape similar to that of EcoRI bound to cognate DNA. We further observe that the hydration of the enzyme for each of the osmolyte is different, indicating that the mechanism of action of each of these osmolytes could be different. Further analyses of interfacial water dynamics using rotational autocorrelation function reveals that while the protein surface contributes to a slower tumbling motion of water, osmolytes, additionally contribute to the slowing of the angular motion of the water molecules. Entropy analysis also corroborates with this finding. We also find that the slowed rotational motion of interfacial waters in the presence of osmolytes contributes to a slowed relaxation of the hydrogen bonds between the interfacial waters and the functionally important residues in the protein. Taken together, our results show that osmolytes alter the dynamics of the protein by altering the dynamics of water. This altered dynamics, mediated by the changes in the water dynamics and hydrogen bonds with functionally important residues, may contribute to the altered specificity of EcoRI in the presence of osmolytes.

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

Analysis and comparison of amorphous & amyloid aggregates of the tumor suppressor p53.

Retention of a function of the tumor suppressor protein p53 is important in inhibiting tumorigenesis. In fact, p53 is mutated in around half of all cancers. The hotspot mutations are highly localized in the DNA-binding domain of p53 (p53-DBD), one of the domains in p53. The wild-type p53-DBD has been reported to be aggregation-prone and hotspot mutations increase the aggregation. Furthermore, it has been proposed that when p53 forms aggregates, the active wild-type p53 is incorporated into the aggregates, causing it to lose its function. p53 forms at least two types of aggregates - amorphous aggregates and amyloid aggregates - simultaneously. In this study, we hypothesized that there are factors that affect the formation of two types of p53 aggregates in the cell since intracellular p53 actually functions. We investigated the effects of intracellular osmolytes on p53 aggregation and function. The DNA binding and the degree of aggregation of p53-DBD were evaluated by gel shift assay and by monitoring the fluorescence of thioflavin T and 8,1-ANS, respectively. The shape of the aggregates was observed by fluorescence microscopy and the structural transition was monitored by tryptophan fluorescence detection. We found that intracellular osmolytes inhibit amorphous aggregation and denaturation of p53. Furthermore, these osmolytes suppressed the loss of DNA-binding ability associated with aggregation. We have revealed not only the effects of intracellular osmolytes but also the differences in the properties of the two types of aggregates. Interestingly, amyloid aggregates of p53-DBD were hardly formed under conditions of loss of function and were promoted under conditions where the function is retained. Our results overturn the established hypothesis that amyloid is the main cause of loss of function of p53.

Mechanism of protein stabilization in crowded solution and preservation in desiccated sugar glass.

The highly crowded cellular milieu includes countless different macromolecules and osmolytes aimed at regulating osmotic stress while protecting proteins and other macromolecules. Of special interest is the formation of cellular glassy matrices composed of sugars, which strongly correlate with the ability of some yeast, nematodes, and tardigrades to survive desiccation and extreme temperature changes. Remarkably, these carbohydrates stabilize proteins in crowded solutions and preserve them in the dry glassy state. Yet, although considerable efforts have been directed to discern how crowding stabilizes proteins, the molecular-level stabilization mechanism has remained elusive. We have followed the effect of various osmolytes on the native state stability of model miniproteins in crowded solutions using circular dichroism (CD) spectroscopy and our newly developed mean-field theory for crowding. We show that protein native state stability and folding thermodynamic signature can be explained using three distinct types of interactions: excluded volume, solvent-osmolyte non-ideal mixing, and osmolyte-protein soft-interactions. Furthermore, using our combination of synchrotron radiation CD spectroscopy together with sampling enhanced molecular dynamics (MD) simulations we have been able to shed light on the stability and new emerging nanostructures of proteins when they are embedded in sugar glass.

Synergistic effects of osmolytes on solvent exclusion and resulting protein stabilization: Studies with sucrose, taurine and sorbitol individually and in combination

Osmolytes are small molecules which occur naturally and protect biomacromolecules against stress. Mixtures of small organic osmolytes occur in cells of many organisms suggesting that synergy has an important role. In this work, high sensitivity calorimetric as well as spectroscopic approach has been used to reveal individual and combined effects of three osmolytes; sucrose, taurine, and sorbitol on conformational and thermal stability of the protein hen egg-white lysozyme. Sucrose and sorbitol individually show greater extent of thermal stability on lysozyme than taurine. Intrinsic fluorescence and circular dichroism spectroscopic observations suggest absence of appreciable interaction between protein and these osmolytes. Individually taurine does not exert stabilization effect on lysozyme but in combination with sorbitol it provides higher thermal stability to the protein. The results indicate that effect of taurine and sorbitol in combination on the protein is additive at higher molality and at lower pH. In other cases, the extent of protein stability is lesser with individual osmolyte than their combined effect. The results further endorse that osmolytes modify structural properties of water leading to stabilization of the protein. The results obtained in this work provide experimental support to preferential exclusion mechanism of osmolytes in protein stabilization.

PolyQ-Expanded Mutant Huntingtin Forms Inclusion Body Following Transient Cold Shock in a Two-Step Aggregation Mechanism.

Age-dependent formation of insoluble protein aggregates is a hallmark of many neurodegenerative diseases. We are interested in the cell chemistry that drives the aggregation of polyQ-expanded mutant Huntingtin (mHtt) protein into insoluble inclusion bodies (IBs). Using an inducible cell model of Huntington's disease, we show that a transient cold shock (CS) at 4 °C followed by recovery incubation at temperatures of 25-37 °C strongly and rapidly induces the compaction of diffuse polyQ-expanded HuntingtinExon1-enhanced green fluorescent protein chimera protein (mHtt) into round, micron size, cytosolic IBs. This transient CS-induced mHtt IB formation is independent of microtubule integrity or de novo protein synthesis. The addition of millimolar concentrations of sodium chloride accelerates, whereas urea suppresses this transient CS-induced mHtt IB formation. These results suggest that the low temperature of CS constrains the conformation dynamics of the intrinsically disordered mHtt into labile intermediate structures to facilitate de-solvation and hydrophobic interaction for IB formation at the higher recovery temperature. This work, along with our previous observation of the effects of heat shock protein chaperones and osmolytes in driving mHtt IB formation, underscores the primacy of mHtt structuring and rigidification for H-bond-mediated cross-linking in a two-step mechanism of mHtt IB formation in living cells.