Implications of trimethylamine N-oxide (TMAO) and Betaine in Human Health: Beyond Being Osmoprotective Compounds.

Osmolytes are naturally occurring small molecular weight organic molecules, which are accumulated in large amounts in all life forms to maintain the stability of cellular proteins and hence preserve their functions during adverse environmental conditions. Trimethylamine N-oxide (TMAO) and N,N,N-trimethylglycine (betaine) are methylamine osmolytes that have been extensively studied for their diverse roles in humans and have demonstrated opposing relations with human health. These osmolytes are obtained from food and synthesized endogenously using dietary constituents like choline and carnitine. Especially, gut microbiota plays a vital role in TMAO synthesis and contributes significantly to plasma TMAO levels. The elevated plasma TMAO has been reported to be correlated with the pathogenesis of numerous human diseases, including cardiovascular disease, heart failure, kidney diseases, metabolic syndrome, etc.; Hence, TMAO has been recognized as a novel biomarker for the detection/prediction of several human diseases. In contrast, betaine acts as a methyl donor in one-carbon metabolism, maintains cellular S-adenosylmethionine levels, and protects the cells from the harmful effects of increased plasma homocysteine. Betaine also demonstrates antioxidant and anti-inflammatory activities and has a promising therapeutic value in several human diseases, including homocystinuria and fatty liver disease. The present review examines the multifarious functions of TMAO and betaine with possible molecular mechanisms towards a better understanding of their emerging and diverging functions with probable implications in the prevention, diagnosis, and treatment of human diseases.

C10Pred: A First Machine Learning Based Tool to Predict C10 Family Cysteine Peptidases Using Sequence-Derived Features.

Streptococcus pyogenes, or group A Streptococcus (GAS), a gram-positive bacterium, is implicated in a wide range of clinical manifestations and life-threatening diseases. One of the key virulence factors of GAS is streptopain, a C10 family cysteine peptidase. Since its discovery, various homologs of streptopain have been reported from other bacterial species. With the increased affordability of sequencing, a significant increase in the number of potential C10 family-like sequences in the public databases is anticipated, posing a challenge in classifying such sequences. Sequence-similarity-based tools are the methods of choice to identify such streptopain-like sequences. However, these methods depend on some level of sequence similarity between the existing C10 family and the target sequences. Therefore, in this work, we propose a novel predictor, C10Pred, for the prediction of C10 peptidases using sequence-derived optimal features. C10Pred is a support vector machine (SVM) based model which is efficient in predicting C10 enzymes with an overall accuracy of 92.7% and Matthews' correlation coefficient (MCC) value of 0.855 when tested on an independent dataset. We anticipate that C10Pred will serve as a handy tool to classify novel streptopain-like proteins belonging to the C10 family and offer essential information.

Trimethylamine N-oxide alters structure-function integrity of ß-casein: Structural disorder co-regulates the aggregation propensity and chaperone activity.

Intrinsically disordered proteins (IDPs), involved in the regulation and function of various cellular processes like transcription, translation, cell cycle etc., exist as ensembles of rapidly interconverting structures with functional plasticity. Among numerous cellular regulatory mechanisms involved in structural and functional regulation of IDPs, osmolytes are emerging as promising regulatory agents due to their ability to affect the structure-function integrity of IDPs. The present study investigated the effect of methylamine osmolytes on ß-casein, an IDP essential for maintaining the overall stability of casein complex in milk. It was observed that trimethylamine N-oxide induces a compact structural state in ß-casein with slightly decreased chaperone activity and insignificant aggregation propensity. However, the other two osmolytes from this group, i.e., sarcosine and betaine, had no significant effect on the overall structure and chaperone activity of the IDP. The present study hints towards the possible evolutionary selection of higher structural disorder in ß-casein, compared to α-casein, for stability of the casein complex and prevention of amyloidosis in the mammary gland.

Protein kinases as regulators of osmolyte accumulation under stress conditions: An overview.

Accumulation of osmolytes, during cell volume perturbations, as cell volume regulators is ensured through their de novo synthesis, decreased degradation and transport from their site of synthesis to the site of utility through various transport systems. Among these, transport system mediated accumulation has been observed to be quite significant during long term cell volume perturbation. Under stress conditions, these osmolyte transporters are regulated at transcriptional as well as translational level. At translational level, protein kinases carry out phosphorylation of osmolyte transporters and have been shown to play a crucial role in cell volume homeostasis. In fact phosphorylation of osmolyte transporters on their conserved residues regulates the uptake and efflux of osmolytes by cells. Additionally, accumulated osmolytes in turn have been shown to modulate the structure and functioning of protein kinases. The present review has tried to provide an overview about the role of protein kinases in regulation of osmolyte accumulation under stress conditions. Due to their ability of regulating osmolyte accumulation, potential of protein kinases as therapeutic targets for diseases like cancer has also been highlighted.

Trimethylamine N-oxide abolishes the chaperone activity of α-casein: an intrinsically disordered protein.

Osmolytes (small molecules that help in circumventing stresses) are known to promote protein folding and prevent aggregation in the case of globular proteins. However, the effect of such osmolytes on the structure and function of intrinsically disordered proteins (IDPs) has not been clearly understood. Here we have investigated the effect of methylamine osmolytes on α-casein (an IDP present in mammalian milk) and discovered that TMAO (Trimethylamine-N-oxide) but not other methylamines renders α-casein functionless. We observed that the loss of chaperone activity of α-casein in presence of TMAO was due to the induction of an unstable aggregation-prone intermediate. The results indicate that different osmolytes may have different structural and functional consequences on IDPs, and therefore might have clinical implications for a large number of human diseases (e.g., amyloidosis, cancer, diabetes, and neurodegeneration) where IDPs are involved.

Antioxidant and hepatoprotective effects of Crataegus songarica methanol extract.

The protective activity of the methanolic extract of the Crataegus songarica leaves was investigated against CCl4- and paracetamol-induced liver damage. On folklore levels, this plant is popularly used to treat various toxicological diseases. We evaluated both in vitro and ex vivo antioxidant activity of C. songarica. At higher concentration of plant extract (700 µg/ml), 88.106% inhibition on DPPH radical scavenging activity was observed and reducing power of extract was increased in a concentration-dependent manner. We also observed its inhibition on Fe2+/ascorbic acid-induced lipid peroxidation on rat liver microsomes in vitro. In addition, C. songarica extract exhibited antioxidant effects on calf thymus DNA damage induced by Fenton reaction. Hepatotoxicity was induced by challenging the animals with CCl4 (1 ml/kg body weight, i.p.) and paracetamol (500 mg/kg body weight) and the extract was administered at three concentrations (100, 200, and 300 mg/kg body weight). Hepatoprotection was evaluated by determining the activities of liver function marker enzymes and antioxidant status of liver. Administration of CCl4 elevated the levels of liver function enzymes, SGOT, SGPT, and LDH. We also observed a dramatic increase in ALT, AST, bilirubin, and alkaline phosphatase levels in rats administered 500 mg/kg body weight of paracetamol. Decreased antioxidant defense system as glutathione (GSH), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione-S-transferase (GST), and superoxide dismutase (SOD) were observed in rats treated with CCl4 and paracetamol. Pretreatment with the extract decreased the elevated serum GOT, GPT, LDH, bilirubin, and alkaline phosphatase activities and increased the antioxidant enzymes in a dose-dependent manner. Therefore, C. songarica methanol extract may be an effective hepatic protective agent and viable candidate for treating hepatic disorders and other oxidative stress-related diseases.

In vitro antioxidant and cytotoxic activities of Arnebia benthamii (Wall ex. G. Don): a critically endangered medicinal plant of Kashmir Valley.

Arnebia benthamii is a major ingredient of the commercial drug available under the name Gaozaban, which has antibacterial, antifungal, anti-inflammatory, and wound-healing properties. In the present study, in vitro antioxidant and anticancer activity of different extracts of Arnebia benthamii were investigated. Antioxidant potential of plant extracts was evaluated by means of total phenolics, DPPH, reducing power, microsomal lipid peroxidation, and hydroxyl radical scavenging activity. The highest phenolic content (TPC) of 780 mg GAE/g was observed in ethyl acetate, while the lowest TPC of 462 mg GAE/g was achieved in aqueous extract. At concentration of 700 µg/mL, DPPH radical scavenging activity was found to be highest in ethyl acetate extract (87.99%) and lowest in aqueous extract (73%). The reducing power of extracts increased in a concentration dependent manner. We also observed its inhibition on Fe(2+)/ascorbic acid-induced lipid peroxidation (LPO) on rat liver microsomes in vitro. In addition, Arnebia benthamii extracts exhibited antioxidant effects on Calf thymus DNA damage induced by Fenton reaction. Cytotoxicity of the extracts (10-100 µg/mL) was tested on five human cancer cell lines (lung, prostate, leukemia, colon, and pancreatic cell lines) using the Sulphorhodamine B assay.

Glycine betaine may have opposite effects on protein stability at high and low pH values.

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, alpha-lactalbumin (alpha-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, alpha-LA and lysozyme. This conclusion was reached by determining T(m) (midpoint of denaturation), DeltaH(m) (denaturational enthalpy change at T(m)), DeltaC(p) (constant-pressure heat capacity change) and DeltaG(D)(o) (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different GB concentrations. Another conclusion of this study is that DeltaH(m) and DeltaC(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.