The gut metabolite, trimethylamine N-oxide inhibits protein folding by affecting cis-trans isomerization and induces cell cycle arrest.

Trimethylamine N-Oxide (TMAO) is an important metabolite, which is derived from choline, betaine, and carnitine in various organisms. In humans, it is synthesized through gut microbiota and is abundantly found in serum and cerebrospinal fluid (CSF). Although TMAO is a stress protectant especially in urea-rich organisms, it is an atherogenic agent in humans and is associated with various diseases. Studies have also unveiled its exceptional role in protein folding and restoration of mutant protein functions. However, most of these data were obtained from studies carried on fast-folding proteins. In the present study, we have investigated the effect of TMAO on the folding behavior of a well-characterized protein with slow folding kinetics, carbonic anhydrase (CA). We discovered that TMAO inhibits the folding of this protein via its effect on proline cis-trans isomerization. Furthermore, TMAO is capable of inducing cell cycle arrest. This study highlights the potential role of TMAO in developing proteopathies and associated diseases.

Conformational status of cytochrome c upon N-homocysteinylation: Implications to cytochrome c release.

One of the proposed mechanisms of homocysteine (Hcy) toxicity is the post-translational modification of proteins by its metabolite, homocysteine thiolactone (HTL). Incubation of proteins with HTL has been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation) which ultimately results in structural and functional alterations of the modified proteins. In the present study, the effects of HTL on the conformational and heme status of cytochrome c (cyt c) were investigated. Spectroscopic analyses revealed that HTL-modified cyt c undergoes certain conformational alterations leading to disturbed heme-Trp distance and packing of the apolar groups. These alterations were accompanied with the reduction of the heme moiety and activation of peroxidase-like function of cyt c, which is known to be a crucial event for initiation of the intrinsic apoptotic pathway. Further structural characterization revealed that disruption of the heme-Met80 interaction, thereby converting the hexa-coordinate cyt c to a penta-coordinate species (with a free heme ligand), was responsible for the activation of the peroxidase activity. The study provides insights for the possible role of cyt c N-homocysteinylation in eliciting its toxicity and cell death.

Macromolecular crowding: Macromolecules friend or foe.

BACKGROUND: Cellular interior is known to be densely crowded due to the presence of soluble and insoluble macromolecules, which altogether occupy ~40% of the total cellular volume. This results in altered biological properties of macromolecules. SCOPE OF REVIEW: Macromolecular crowding is observed to have both positive and negative effects on protein folding, structure, stability and function. Significant data has been accumulated so far on both the aspects. However, most of the review articles so far have focused on the positive aspect of macromolecular crowding and not much attention has been paid on the deleterious aspect of crowding on macromolecules. In order to have a complete knowledge of the effect of macromolecular crowding on proteins and enzymes, it is important to look into both the aspects of crowding to determine its precise role under physiological conditions. To fill the gap in the understanding of the effect of macromolecular crowding on proteins and enzymes, this review article focuses on the deleterious influence of crowding on macromolecules. MAJOR CONCLUSIONS: Macromolecular crowding is not always good but also has several deleterious effects on various macromolecular properties. Taken together, the properties of biological macromolecules in vivo appears to be finely regulated by the nature and level of the intracellular crowdedness in order to perform their biological functions appropriately. GENERAL SIGNIFICANCE: The information provided here gives an understanding of the role played by the nature and level of cellular crowdedness in intensifying and/or alleviating the burden of various proteopathies.

Protein N-homocysteinylation: From cellular toxicity to neurodegeneration.

BACKGROUND: Homocysteine (Hcy) is a sulfur containing non-protein amino acid that occupies a central role in metabolism of thiol compounds. The past decade had noticed an explosion in interests of Hcy and this very interest came primarily from the fact that increased Hcy level is related to various neurodegenerative and vascular complications. SCOPE OF REVIEW: Several factors responsible for the Hcy-associated neurotoxicity have been proposed and well documented in literature, including oxidative stress and apoptosis. In addition, protein covalent modification by the metabolite of Hcy, Hcy thiolactone (HTL), has now been shown to be another cause of cellular Hcy toxicity. This mechanism, termed as "protein N-homocysteinylation", is known to result in protein denaturation, enzyme inactivation and even amyloid formation. The role of protein N-homocysteinylation and the resulting consequences with regard to neurodegeneration have not yet been extensively discussed. The present review describes major advances in understanding protein N-homocysteinylation and their role in neurodegeneration. MAJOR CONCLUSIONS: Formation of protein aggregates/amyloids are crucial events in various human pathologies including neurodegenerative diseases. Since elevated Hcy has been closely linked to neurodegeneration, N-homocysteinylation-induced protein modification and aggregates/amyloids formation could be one possible mechanism for the neurodegenerative conditions. GENERAL SIGNIFICANCE: The information highlighted here provides us an understanding of the role protein modification by N-homocysteinylation in neurodegenerative diseases.

Osmolyte mixtures have different effects than individual osmolytes on protein folding and functional activity.

Osmolytes are small organic molecules accumulated by organisms under stress conditions to protect macromolecular structure and function. In the present study, we have investigated the effect of several binary osmolyte mixtures on the protein folding/stability and function of RNase-A. For this, we have measured ΔGD(o) (Gibbs free energy change at 25°C) and specific activity of RNase-A mediated hydrolysis of cytidine 2'-3' cyclic monophosphate in the presence and absence of individual and osmolyte mixtures. It was found that the osmolyte mixtures have different effect on protein stability and function than that of individual osmolytes. Refolding studies of RNase-A in the presence of osmolyte mixtures and individual osmolytes also revealed that osmolyte mixtures have a poor refolding efficiency relative to the individual osmolytes.

pH might play a role in regulating the function of paired amphipathic helices domains of human Sin3B by altering structure and thermodynamic stability.

Human Sin3B (hSin3B), a transcription regulator, is a scaffold protein that binds to different transcription factors and regulates transcription. It consists of six conserved domains that include four paired amphipathic helices (PAH 1-4), histone deacetylase interaction domain (HID), and highly conserved region (HCR). Interestingly, the PAH domains of hSin3B are significantly homologous to each other, yet each one interacts with a specific set of unique transcription factors. Though various partners interacting with hSin3B PAH domains have been characterized, there is no structural information available on the individual PAH domains of hSin3B. Here we characterize the structure and stability of different PAH domains of hSin3B at both nuclear and physiological pH values by using different optical probes. We found that the native state structure and stability of different PAH domains are different at nuclear pH where hSin3B performs its biological function. We also found that PAH2 and PAH3 behave differently at both nuclear and physiological pH in terms of native state structure and thermodynamic stability, while the structural identity of PAH1 remains unaltered at both pH values. The study indicates that the structural heterogeneity of different PAH domains might be responsible for having a unique set of interacting transcription factors.

Structural and Functional Diversity of the Peroxiredoxin 6 Enzyme Family.

Significance: Peroxiredoxins (Prdxs) with a single peroxidative cysteine (CP) in a conserved motif PXXX(T/S)XXCP within its thioredoxin fold, have been classified as the peroxiredoxin 6 (Prdx6 ) family. All Prdxs can reduce H2O2 and short chain hydroperoxides while Prdx6 in addition, can reduce phospholipid hydroperoxides (PLOOH) due to its ability to interact with peroxidized phospholipid substrate. The single CP of Prdx6 uses various external electron donors including glutathione thioredoxin, and ascorbic acid for resolution of its peroxidized state and, therefore, its peroxidase activity. Prdx6 proteins also exhibit Ca2+-independent phospholipase A2 (PLA2), lysophosphatidylcholine acyltransferase (LPCAT), and chaperone activities that depend on cellular localization and the oxidation and oligomerisation states of the protein. Thus, Prdx6 is a "moonlighting" enzyme. Recent Advance: Physiologically, Prdx6s have been reported to play an important role in protection against oxidative stress, repair of peroxidized cell membranes, mammalian lung surfactant turnover, activation of some NADPH oxidases, the regulation of seed germination in plants, as an indicator of cellular levels of reactive O2 species through Nrf-Klf9 activation, and possibly in male fertility, regulation of cell death through ferroptosis, cancer metastasis, and oxidative stress-related signalling pathways. Critical Issues: This review outlines Prdx6 enzyme unique structural features and explores its wide range of physiological functions. Yet, existing structural data falls short of fully revealing all of human Prdx6 multifunctional roles. Further endeavour is required to bridge this gap in its understanding. Although there are wide variations in both the structure and function of Prdx6 family members in various organisms, all Prdx6 proteins show the unique a long C-terminal extension that is also seen in Prdx1, but not in other Prdxs. Future Directions: As research data continues to accumulate, the potential for detailed insights into the role of C-terminal of Prdx6 in its oligomerisation and activities. There is a need for thorough exploration of structural characteristics of the various biological functions. Additionally, uncovering the interacting partners of Prdx6 and understanding its involvement in signalling pathways will significantly contribute to a more profound comprehension of its role.

Functionally active cross-linked protein oligomers formed by homocysteine thiolactone.

Deposition of high-order protein oligomers is a common hallmark of a large number of human diseases and therefore, has been of immense medical interest. From the past several decades, efforts are being made to characterize protein oligomers and explore how they are linked with the disease pathologies. In general, oligomers are non-functional, rather cytotoxic in nature while the functional (non-cytotoxic) oligomers are quite rare. In the present study, we identified new protein oligomers of Ribonuclease-A and Lysozyme that contain functionally active fractions. These functional oligomers are disulfide cross-linked, native-like, and obtained as a result of the covalent modification of the proteins by the toxic metabolite, homocysteine thiolactone accumulated under hyperhomocysteinemia (a condition responsible for cardiovascular complications including atherosclerosis). These results have been obtained from the extensive analysis of the nature of oligomers, functional status, and structural integrity of the proteins using orthogonal techniques. The study implicates the existence of such oligomers as protein sinks that may sequester toxic homocysteines in humans.

AGE-RAGE axis culminates into multiple pathogenic processes: a central road to neurodegeneration.

Advanced glycation end-products (AGEs; e.g., glyoxal, methylglyoxal or carboxymethyl-lysine) are heterogenous group of toxic compounds synthesized in the body through both exogenous and endogenous pathways. AGEs are known to covalently modify proteins bringing about loss of functional alteration in the proteins. AGEs also interact with their receptor, receptor for AGE (RAGE) and such interactions influence different biological processes including oxidative stress and apoptosis. Previously, AGE-RAGE axis has long been considered to be the maligning factor for various human diseases including, diabetes, obesity, cardiovascular, aging, etc. Recent developments have revealed the involvement of AGE-RAGE axis in different pathological consequences associated with the onset of neurodegeneration including, disruption of blood brain barrier, neuroinflammation, remodeling of extracellular matrix, dysregulation of polyol pathway and antioxidant enzymes, etc. In the present article, we attempted to describe a new avenue that AGE-RAGE axis culminates to different pathological consequences in brain and therefore, is a central instigating component to several neurodegenerative diseases (NGDs). We also invoke that specific inhibitors of TIR domains of TLR or RAGE receptors are crucial molecules for the therapeutic intervention of NGDs. Clinical perspectives have also been appropriately discussed.

Urea ameliorates trimethylamine N-oxide-Induced aggregation of intrinsically disordered α-casein protein: the other side of the urea-methylamine counteraction.

Trimethylamine N-oxide (TMAO) is generally accumulated by organisms and cells to cope with denaturing effects of urea/hydrodynamic pressure on proteins and can even reverse misfolded or aggregated proteins so as to sustain proteostasis. However, most of the work regarding this urea-TMAO counteraction has been performed on folded proteins. Compelling evidence of aggregation of intrinsically disordered proteins (IDPs) like tau, α-synuclein, amyloid ß etc., by TMAO and its potential to impact various protein processes in absence of stressing agents (such as urea) suggests that the contrary feature of interaction profiles of urea and TMAO maximizes their chances of offsetting the perturbing effects of each other. Recently, our lab observed that TMAO induces aggregation of α-casein, a model IDP. In this context, the present study, for the first time, evaluated urea for its potential to counteract the TMAO-induced aggregation of α-casein. It was observed that, at the biologically relevant ratios of 2:1 or 3:1 (urea:TMAO), urea was able to inhibit TMAO-induced aggregation of α-casein. However, urea did not reverse the effects of TMAO on α-casein. In addition to this, α-casein in presence of 1:1 and 2:1 urea:TMAO working ratios show aggregation-induced cytotoxic effect on HEK-293, Neuro2A and HCT-116 cell lines but not in presence of 3:1 working ratio, as there was no aggregation at all. The study infers that the accumulation of TMAO alone in the cells, in absence of stress (such as urea), might result in loss of conformational flexibility and aggregation of IDPs in TMAO accumulating organisms.Communicated by Ramaswamy H. Sarma.

Organosulfurs, S-allyl cysteine and N-acetyl cysteine sequester di-carbonyls and reduces carbonyl stress in HT22 cells.

Diabetes, characterized by high blood glucose level, is a progressive metabolic disease that leads to serious health complications. One of the major pathological consequences associated with diabetes is the accumulation of highly reactive carbonyl compounds called advanced glycation end products (AGEs). Most of the AGEs are dicarbonyls and have the potential to covalently modify proteins especially at the lysine residues in a non-enzymatic fashion (a process termed as glycation) resulting in the functional impairment and/or toxic gain in function. Therefore, non-toxic small molecules that can inhibit glycation are of interest for the therapeutic intervention of diabetes. In the present communication, we have investigated the effect of organosulfurs (S-allyl cysteine, SAC and N-acetyl cysteine, NAC) that are major principal components of Allium sativa against the glycation of different proteins. We discovered that both SAC and NAC are potent anti-glycating agents. We also found that both SAC and NAC reduce ROS level and inhibit apoptosis caused by protein glycation.

TMAO to the rescue of pathogenic protein variants.

Trimethylamine N-oxide (TMAO) is a chemical chaperone found in various organisms including humans. Various studies unveiled that it is an excellent protein-stabilizing agent, and induces folding of unstructured proteins. It is also well established that it can counteract the deleterious effects of urea, salt, and hydrostatic pressure on macromolecular integrity. There is also existence of large body of data regarding its ability to restore functional deficiency of various mutant proteins or pathogenic variants by correcting misfolding defects and inhibiting the formation of high-order toxic protein oligomers. Since an important class of human disease called "protein conformational disorders" is due to protein misfolding and/or formation of high-order oligomers, TMAO stands as a promising molecule for the therapeutic intervention of such diseases. The present review has been designed to gather a comprehensive knowledge of the TMAO's effect on the functional restoration of various mutants, identify its shortcomings and explore its potentiality as a lead molecule. Future prospects have also been suitably incorporated.

Effect of polyol osmolytes on the structure-function integrity and aggregation propensity of catalase: A comprehensive study based on spectroscopic and molecular dynamic simulation measurements.

Owing to the ability of catalase to function under oxidative stress vis-à-vis its industrial importance, the structure-function integrity of the enzyme is of prime concern. In the present study, polyols (glycerol, sorbitol, sucrose, xylitol), were evaluated for their ability to modulate structure, activity and aggregation of catalase using in vitro and in silico approaches. All polyols were found to increase catalase activity by decreasing Km and increasing Vmax resulting in enhanced catalytic efficiency (kcat/Km) of the enzyme. Glycerol was found to be the most efficient polyol with a kcat/Km increase from 4.38 × 104 mM-1 S-1 (control) to 5.8 × 105 mM-1 S-1. Correlatively with this, enhanced secondary structure with reduced hydrophobic exposure was observed in all polyols. Furthermore, increased stability, with an increase in melting temperature by 15.2 °C, and almost no aggregation was observed in glycerol. Overall, ability to regulate structure-function integrity and aggregation propensity was highest for glycerol and lowest for xylitol. Simulation studies were performed involving structural dynamics measurement, principal component analysis and free energy landscape analysis. Altogether, all polyols were stabilizing in nature and glycerol, in particular, has potential to efficiently prevent not only the aggregation of the antioxidant defense system but might also serve as a stability aid during industrial processing of catalase.

An in silico approach to identify potential medicinal plants for treating Alzheimer disease: a case study with acetylcholinesterase.

Alzheimer's disease (AD) is a progressive neurological disorder affecting an estimated 10 million people worldwide. There is no cure for AD, and only a handful of drugs are known to provide some relief of the symptoms. The prescription drug donepezil has been widely used to treat to slow the progression and onset of the disease; however, the unpleasant side effects have paved the way to find alternative medicines. Many herbs are known to improve brain function, but evidence of medicinal plants that can treat AD is limited due to the lack of concrete rational evidences. Moreover, the traditional method of randomly screening plant extract against AD targets takes time and resources. In this study, a receptor-based in silico method has been implemented which serves to accelerate the process of identification of medicinal plants useful for treatment of AD. A database of natural compounds was compiled to identify hits against acetylcholinesterase (AChE). Receptor-based pharmacophore screening was performed, and selected hits were subjected to docking and molecular dynamics simulations. Molecular Mechanics/Generalized Born surface area (MM/GBSA) calculations were carried out to identify the best scoring hits further. In vitro assays were done for the plant extracts containing the top-scoring hits against AChE. Three plant extracts showed favorable inhibitory activity.Communicated by Ramaswamy H. Sarma.

Brain Metabolite, Myo-inositol, Inhibits Catalase Activity: A Mechanism of the Distortion of the Antioxidant Defense System in Alzheimer's disease.

A strong correlation between brain metabolite accumulation and oxidative stress has been observed in Alzheimer's disease (AD) patients. There are two central hypotheses for this correlation: (i) coaccumulation of toxic amyloid-ß and Myo-inositol (MI), a significant brain metabolite, during presymptomatic stages of AD, and (ii) enhanced expression of MI transporter in brain cells during oxidative stress-induced volume changes in the brain. Identifying specific interactive effects of MI with cellular antioxidant enzymes would represent an essential step in understanding the oxidative stress-induced AD pathogenicity. This study demonstrated that MI inhibits catalase, an essential antioxidant enzyme primarily inefficient in AD, by decreasing its k cat (turnover number) and increasing K m (Michaelis-Menten constant) values. This inhibition of catalase by MI under in vivo studies increased cellular H2O2 levels, leading to decreased cell viability. Furthermore, MI induces distortion of the active heme center with an overall loss of structure and stability of catalase. MI also alters distances of the vital active site and substrate channel residues of catalase. The present study provides evidence for the involvement of MI in the inactivation of the antioxidant defense system during oxidative stress-induced pathogenesis of AD. Regulation of MI levels, during early presymptomatic stages of AD, might serve as a potential early-on therapeutic strategy for this disease.

Functional inhibition of redox regulated heme proteins: A novel mechanism towards oxidative stress induced by homocysteine.

Homocysteine (Hcy) is a sulfur containing non-protein toxic amino acid synthesized from methionine. Elevated level of Hcy is associated with cardiovascular complications and neurodegeneration. Hcy is believed to induce organ damage and apoptosis via oxidative stress. The pro-oxidant nature of Hcy is considered to originate from the metal-induced oxidation of thiol group-containing molecules forming disulfides (Hcy-Hcy, Hcy-cysteine, Hcy-glutathione, etc) or with free cysteine residues of proteins (a process called protein S-homocysteinylation). Formation of such disulfides indeed results in the generation of reactive oxygen species (ROS) which eventually leads to loss of cellular integrity. In the present manuscript, we performed systematic investigation of the effect of Hcy on iron containing proteins. We discover a novel mechanism of Hcy toxicity wherein Hcy oxidation is linked with the functional loss of the protein with iron as cofactors. Our results indicate that redox regulated heme proteins might be primarily involved in the Hcy toxicity and associated oxidative stress.

pH induced conformational alteration in human peroxiredoxin 6 might be responsible for its resistance against lysosomal pH or high temperature.

Peroxiredoxin 6 (Prdx6), the ubiquitously expressed enzyme belonging to the family of peroxidases, namely, peroxiredoxins, exhibits a unique feature of functional compartmentalization within cells. Whereas, the enzyme localized in cytosol shows glutathione peroxidase activity, its lysosomal counterpart performs calcium independent phospholipase A2 (aiPLA2) activity. Like any true moonlighting protein, these two activities of Prdx6 are mutually exclusive of each other as a function of the pH of the cellular compartments. Differential substrate preference at different pH (i.e. peroxidised phospholipids at neutral pH and reduced phospholipids at acidic pH) is considered to be the reason for this behavior. To gain insight into the pH-induced structural-functional interplay we have systematically evaluated conformational variations, thermodynamic stability of the protein and quaternary state of the conformers at both pH 7.0 and 4.0. Our findings suggest that change in pH allows alterations in native states of Prdx6 at pH 7.0 and 4.0 such that the changes make the protein resistant to thermal denaturation at low pH.

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.

Brain Metabolite, N-Acetylaspartate Is a Potent Protein Aggregation Inhibitor.

Deposition of toxic protein inclusions is a common hallmark of many neurodegenerative disorders including Alzheimer's disease, Parkinson disease etc. N-acetylaspartate (NAA) is an important brain metabolite whose levels got altered under various neurodegenerative conditions. Indeed, NAA has been a widely accepted biological marker for various neurological disorders. We have also reported that NAA is a protein stabilizer. In the present communication, we investigated the role of NAA in modulating the aggregation propensity on two model proteins (carbonic anhydrase and catalase). We discovered that NAA suppresses protein aggregation and could solubilize preformed aggregates.