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Abstract: Osmolytes are naturally occurring small molecules accumulated intracellularly to protect organisms from various denaturing stresses. Similar to the two faces of a coin, several of these osmolytes are stabilizing and destabilizing proteins depending on the concentrations and/or solvent conditions. For example, the well known stabilizing osmolyte, trehalose destabilizes some proteins at high concentration and/or high pH. In spite of the fact that destabilizing aspects of osmolytes can modulate many cellular processes including regulation of protein homeostasis (proteostasis), protein–protein interaction, and protein–DNA interaction, researchers have mostly focused on the stabilizing aspects of osmolytes. Thus, it is important to look into both aspects of osmolytes to determine their precise role under physiological conditions. In this article, we have discussed both stabilizing and destabilizing/denaturant aspects of osmolytes to uncover both sides of the coin. [ABSTRACT FROM AUTHOR]

Missense mutant proteins, such as those produced in individuals with genetic diseases, are often misfolded and subject to processing by intracellular quality control systems. Previously, we have shown using a yeast system that enzymatic function could be restored to I278T cystathionine β-synthase (CBS), a cause of homocystinuria, by treatments that affect the intracellular chaperone environment. Here, we extend these studies and show that it is possible to restore significant levels of enzyme activity to 17 of 18 (94%) disease causing missense mutations in human cystathionine b-synthase (CBS) expressed in Saccharomyces cerevisiae by exposure to ethanol, proteasome inhibitors, or deletion of the Hsp26 small heat shock protein. All three of these treatments induce Hsp70, which is necessary but not sufficient for rescue. In addition to CBS, these same treatments can rescue disease-causing mutations in human p53 and the methylene tetrahydrofolate reductase gene. These findings do not appear restricted to S. cerevisiae, as proteasome inhibitors can restore significant CBS enzymatic activity to CBS alleles expressed in fibroblasts derived from homocystinuric patients and in a mouse model for homocystinuria that expresses human I278T CBS. These findings suggest that proteasome inhibitors and other Hsp70 inducing agents may be useful in the treatment of a variety of genetic diseases caused by missense mutations. [ABSTRACT FROM AUTHOR]

Abstract: The compatible osmolyte glycine betaine (GB) is the most efficient osmoprotectant and best excluder from the protein surface. It can reverse protein aggregation and correct mutant protein defects and counter the harmful effects of urea and salts in vivo and in vitro. In this study we have investigated the pH dependence of the stabilizing effect of GB on three different proteins, namely, α-lactalbumin (α-LA), lysozyme and ribonuclease-A (RNase-A). We show here that (a) GB stabilizes RNase-A at all pH values, and (b) GB has opposite effects on two proteins at high pH and low pH values, namely, α-LA and lysozyme. This conclusion was reached by determining T m (midpoint of denaturation), ΔH m (denaturational enthalpy change at T m), ΔC p (constant-pressure heat capacity change) and ΔG D o (denaturational Gibbs energy change at 25 °C) of proteins in the presence of different GB concentrations. Another conclusion of this study is that ΔH m and ΔC p are not significantly changed in the presence of GB. This study suggests that other methylated glycine osmolytes may also behave in the same manner. [Copyright &y& Elsevier]

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments. These osmolytes protect intracellular macromolecules against denaturing environmental stress. In contrast to the usually benign effects of most organic osmolytes, the waste product urea is a well-known perturbant of macromolecules. Although urea is a perturbing solute which inhibits enzyme activity and stability, it is employed by some species as a major osmolyte. The answer to this paradox was believed to be the discovery of protective osmolytes (methylamines). We review the current state of knowledge on the various ways of counteracting the harmful effects of urea in nature and the mechanisms for this. This review ends with the mechanistic idea that cellular salt (KCl/NaCl) plays a crucial role in counteracting the effects of urea, either by inducing required chaperones or methylamines, or by thermodynamic interactions with urea-destabilised proteins. We also propose future opportunities and challenges in the field. [ABSTRACT FROM AUTHOR]

R4970 --> 650.14 --> Trimethylamine N‐oxide (TMAO), in humans, is obtained from gut microbiota and is known to accumulate in human serum as well as cerebrospinal fluid at high levels. There are several reports for the involvement of serum TMAO in various human diseases including, atherosclerosis, neurodegeneration, cancer etc. One important aspect in neurodegeneration is it's strong association with memory loss. However, the exact molecular mechanism is not yet explored. Keeping in mind that impairment in memory or onset of Alzheimer's is due to decline in cholinergic system of the brain, we thought TMAO could have potentially targeted the cholinergic system. Acetylcholinesterase (AChE) is an important enzyme responsible for the breakdown of neurotransmitter, acetylcholine and therefore plays a crucial role in neuronal plasticity. Recently, our laboratory discovered that TMAO inhibits folding of proteins specifically that are rich in proline content. Since AChE is also a protein characterized by the presence of large number of proline residues, we thought it is worthwhile to investigate if TMAO inhibits it's activity. In the present article we investigated the effect of TMAO on the structure and functional activity of AChE. We explored that TMAO is a specific inhibitor of AChE. The results indicate that TMAO might induce decline in the cholinergic system by impairing the function of AChE. [ABSTRACT FROM AUTHOR]

R1970 --> 792.2 --> Homocysteine (Hcy) is a sulfur containing toxic amino acid synthesized from sulfur metabolism pathway. Elevated level of Hcy is associated with various cardiovascular complications including atherosclerosis. One of the likely mechanisms underlying harmful effects of Hcy is the chemical modification of proteins by homocysteine thiolactone (HTL), a highly reactive cyclic thioester of Hcy. HTL preferentially forms amide bonds with ɛ‐ amino group of protein lysine, arginine or histidine residues in a non‐enzymatic mechanism; a process referred to as "protein N‐homocysteinylation resulting in the impairment of protein functions. Therefore, novel approaches that could inhibit or delay protein N‐homocysteinylation would be therapeutically important to curb hyperhomocysteinemia. In the present communication, we screened for different cellular molecules produced under stressful insults for their ability to suppress covalent modification of proteins by HTL. We found that out of several molecules, proline has the potential to inhibit protein N‐homocysteinylation in a non‐enzymatic fashion. We also discovered that proline treatment increases Hcy levels in HeLa cells and induces cell cycle arrest. Our study highlights the importance of proline treatment in reducing toxicity load in hyperhomocysteinemic/homocystinuric patients. [ABSTRACT FROM AUTHOR]

Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine β-synthase protein, 12781, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to 1278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of 1278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper 12781 folding. In contrast, deletion of HSP26 results in increased 1278T protein and activity, whereas overexpression of Hsp26 results in reduced 1278T protein. The Hsp26-1278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS), a key enzyme in the metabolism of homocysteine, has previously been shown to require a heme co-factor for maximal activity. However, the biochemical function of the CBS heme is not well defined. Here, we show that expression of human CBS in heme-deficient strains of Saccharomyces cerevisiae and Escherichia coli results in production of an enzyme that is misfolded and degraded. Addition of exogenous heme, porphyrins with non-iron metal, or porphyrin lacking metal entirely produced stable and active CBS enzyme. Purification of recombinant CBS enzyme expressed in the presence of various metalloporphyrins confirmed that Mn(III) and Co(III) had 30- 60% of the specific activity of Fe(III)-CBS, and still responded to allosteric activation by S-adenosyl-L-methio- nine. Treatment of S. cerevisiae with the chemical chaperone trimethylamine-N-oxide resulted in near complete restoration of function to human CBS produced in a heme-deficient strain. Taken together, these results suggest that porphyrin moiety of the heme plays a critical role in proper CBS folding and assembly, but that the metal ion is not essential for this function or for allosteric regulation by S-adenosyl-L-methionine. [ABSTRACT FROM AUTHOR]

Abstract: Missense mutations in the cystathionine beta-synthase (CBS) gene, such as I278T, are responsible for CBS deficiency, the most common inherited disorder in sulfur metabolism. Expression of human mutant CBS proteins in Saccharomyces cerevisiae reveals that most disease causing mutations severely inhibit enzyme activity and cannot support growth of yeast on cysteine-free media. Here, we show that the osmolyte chemical chaperones glycerol, trimethylamine-N-oxide, dimethylsulfoxide, proline or sorbitol, when added to yeast media, allows growth on cysteine-free media and causes increased enzyme activity from I278T and three other mutant CBS proteins. Rescuable mutants are ones that are predicted to cause a decrease in solvent accessible surface area. The increase in enzyme activity is associated with stabilization of the tetramer form of the enzyme. This effect is not specific to yeast, as addition of the chaperone glycerol resulted in increased I278T activity when the enzyme is produced either in Escherichia coli or in a coupled in vitro transcription/translation reaction. However, no stimulation of specific activity was observed when chaperones were added directly to purified I278T indicating that the presence of chemical chaperones is required during translation. We also found that by mixing different chaperones we could achieve rescue at significantly lower chaperone concentrations. Taken together, our data show that chemical chaperones present during the initial folding process can facilitate proper folding of several mutant CBS proteins and suggest it may be possible to treat some inborn errors of metabolism with agents that enhance proper protein folding. [Copyright &y& Elsevier]

Foxtail millet (Setaria italica), an annual grass plant, produces seeds that possess health‐promoting properties owing to its unique protein composition containing a high content of essential amino acids. The mature foxtail seeds mainly consist of proline‐rich, alcohol‐soluble proteins (prolamin) called setarins, comprising about 60% of the total protein, with less content of disulfide cross‐linked proteins than with other cereal and millets. Protein fractionation schemes are an important tool and provide preliminary information on the nature of foxtail proteins for their applications in the field of agriculture, food pharma, and bio‐based materials. Variation in the methods of preparation can influence the composition, structure, and nutritional quality of the protein concentrate. Moreover, foxtail protein or its hydrolysate has shown several bioactive effects that can be explored further for the management of chronic diseases in humans. Additionally, owing to its low cost and excellent functional properties of flour and protein concentrate, foxtail millet can be considered as good candidate for replacing animal protein foods. Furthermore, there is huge potential for successfully developing low‐cost, protein‐rich functional food products helpful in the prevention and management of lifestyle‐related chronic diseases. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The present study was designed to explore the hydrophobicity and concentration dependence of imidazolium based surface active ionic liquids (SAILs) effects on the structural-functional integrity of proteins. Specifically, we investigated the impact of SAILs viz. 1-octyl-3-methylimidazolium dodecylbenzenesulfonate ([OMIM][DBS]) and 1-dodecyl-3-methylimidazolium dodecylbenzenesulfonate ([DDMIM][DBS]) on activity, structure and stability of lysozyme. Activity measurements revealed that, in contrast to [DDMIM][DBS] that renders lysozyme either feebly active or inactive, [OMIM][DBS] significantly enhances the lysozyme activity in the concentration range of critical aggregation concentrations (CAC) to C s (SAIL saturation concentration of protein backbone) i.e., 0.5 mM–1.35 mM. Tensiometric results in agreement with turbidity measurements inferred significant composition and concentration dependence of the lysozyme-SAIL interactions. Spectroscopic investigations revealed that compared to destabilizing behaviour of [DDMIM][DBS], [OMIM][DBS] significantly enhances both conformational as well as thermal stability of lysozyme in the CAC to C s concentration regime. Altogether, results obtained do indicate that [OMIM][DBS], in the concentration regime of CAC to C s , serves as an efficient stabiliser with an ability to appreciably enhance the activity, thermal stability and overall conformational stability of lysozyme. We firmly believe that [OMIM][DBS], at least in the CAC to C s concentration ranges, can be exploited as a promising stabiliser and activity enhancer for numerous industrially important enzymes. [ABSTRACT FROM AUTHOR]

Urea is a strong denaturant and inhibits many enzymes but is accumulated intracellularly at very high concentrations (up to 3–4 M) in mammalian kidney and in many marine fishes. It is known that the harmful effects of urea on the macromolecular structure and function is offset by the accumulation of an osmolytic agent called methylamine. Intracellular concentration of urea to methylamines falls in the ratio of 2:1 to 3:2 (molar ratio). At this ratio, the thermodynamic effects of urea and methylamines on protein stability and function are believed to be algebraically additive. The mechanism of urea-methylamine counteraction has been widely investigated on various approaches including, thermodynamic, structural and functional aspects. Recent advances have also revealed atomic level insights of counteraction and various molecular dynamic simulation studies have yielded significant molecular level informations on the interaction between urea and methylamines with proteins. It is worthwhile that urea-methylamine system not only plays pivotal role for the survival and functioning of the renal medullary cells but also is a key osmoregulatory component of the marine elasmobranchs, holocephalans and coelacanths. Therefore, it is important to combine all discoveries and discuss the developments in context to physiology of the mammalian kidney and adaptation of the marine organisms. In this article we have for the first time reviewed all major developments on urea-counteraction systems to date. We have also discussed about other additional urea-counteraction systems discovered so far including urea-NaCl, urea-myoinsoitol and urea-molecular chaperone systems. Insights for the possible future research have also been highlighted. [ABSTRACT FROM AUTHOR]

Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea’s harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea’s effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction. [ABSTRACT FROM AUTHOR]

Human kidney cells are under constant urea stress due to its urine concentrating mechanism. It is believed that the deleterious effect of urea is counteracted by methylamine osmolytes (glycine betaine and glycerophosphocholine) present in kidney cells. A question arises: Do the stabilizing osmolytes, non-methylamines (myo-inositol, sorbitol and taurine) present in the kidney cells also counteract the deleterious effects of urea? To answer this question, we have measured structure, thermodynamic stability (ΔGDo) and functional activity parameters (Km and kcat) of different model proteins in the presence of various concentrations of urea and each non-methylamine osmolyte alone and in combination. We observed that (i) for each protein myo-inositol provides perfect counteraction at 1∶2 ([myo-inositol]:[urea]) ratio, (ii) any concentration of sorbitol fails to refold urea denatured proteins if it is six times less than that of urea, and (iii) taurine regulates perfect counteraction in a protein specific manner; 1.5∶2.0, 1.2∶2.0 and 1.0∶2.0 ([taurine]:[urea]) ratios for RNase-A, lysozyme and α-lactalbumin, respectively. [ABSTRACT FROM AUTHOR]

We report the effects of stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) on the stability and function of RNase-A under physiological conditions (pH 6.0 and 25 °C). Measurements of Gibbs free energy change at 25 °C (Δ GD°) and kinetic parameters, Michaelis constant ( Km) and catalytic constant ( kcat) of the enzyme mediated hydrolysis of cytidine monophosphate, enabled us to classify stabilizing osmolytes into three different classes based on their effects on kinetic parameters and protein stability. (a) Polyhydric alcohols and amino acids and their derivatives do not have significant effects on Δ GD° and functional activity ( Km and kcat). (b) Methylamines increase Δ GD° and kcat, but decrease Km. (c) Sugars increase Δ GD°, but decrease both Km and kcat. These findings suggest that, among the stabilizing osmolytes, (a) polyols, amino acids and amino acid derivatives are compatible solutes in terms of both stability and function, (b) methylamines are the best refolders (stabilizers), and (c) sugar osmolytes stabilize the protein, but they apparently do not yield functionally active folded molecules. [ABSTRACT FROM AUTHOR]

In this study, the effect of ZnO buffer layer on the electrical properties of PbZrTiO3/BiFeO3 (PZT/BFO) multilayers has been reported. For this, PZT/BFO multilayers were spin-coated with and without ZnO buffer layer on platinized silicon wafers. X-ray diffraction results of both the films showed polycrystalline phase pure perovskite structure. Both the films show a dense and homogeneous grain structure. The electric properties of the films were measured. The ZnO buffered multilayer thin film showed ∼3 times improvement in remnant polarization compared to the multilayer thin film with no buffer. The buffered samples were found to have higher dielectric constant (1000 at 100 Hz) compared to that of sample (580 at 100 Hz)) with no buffer. Dielectric constants of both the films were found to be ∼30% tunable at 5 V. The buffered film also showed low leakage current density and higher dielectric breakdown compared to the multilayer thin film without buffer. [ABSTRACT FROM AUTHOR]

Withania somnifera exhibits different pharmacological activities which mainly stem from its broad range of bioactive molecules. Majority of these bioactive molecules, fall into the groupings of alkaloids, steroidal lactones, phenolic compounds and glycoproteins. In this study, we evaluated a novel protein fraction, named here as WSPF, isolated from Withania somnifera roots for its cytotoxic properties against various human cancer cell lines. WSPF exhibited apoptotic activity for each cancer cell line tested, demonstrating significant activity against MDA-MB-231 human breast cancer cells with an IC 50 value of 92 μg/mL. WSPF induced mitochondrial-mediated apoptosis of MDA-MB-231 cells via extensive reactive oxygen species generation, dysregulation of Bax/Bcl-2, loss of mitochondrial membrane potential and caspase-3 activation. Additionally, we observed G2/M-phase cell cycle arrest, cleavage of nuclear lamin A/C proteins, and nuclear morphological changes. The present results highlight the anti-cancer properties of WSPF, indicating that the proteins in this fraction can be potential therapeutic agents for triple negative breast cancer treatment. [ABSTRACT FROM AUTHOR]

We have synthesized a furan-based acetylating agent, 2,5-bisacetoxymethylfuran (BAMF) from carbohydrate derived 5-hydroxymethylfurfural (HMF) and studied its acetylation activity with amines and cytochrome c . The results show that BAMF can modify proteins in biological conditions without affecting their structure and function. The modification of cytochrome c with BAMF occurred through the reduction of heme center, but there was no change in the coordination property of iron and the tertiary structure of cytochrome c . Further analysis using MALDI-TOF-MS spectrometer suggests that BAMF selectively targeted lysine amino acid of cytochrome c under our experimental conditions. Kinetics study revealed that the modification of cytochrome c with BAMF took place at faster rates than aspirin. [ABSTRACT FROM AUTHOR]

An abstract of the conference paper "Emergence of unique recombinant forms (URFs) in Indian HIV-1 epidemic: Data from nationwide clinical cohort between 2007 and 2011," by S. Gupta and colleagues is presented.