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.

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.

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.

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.

Structural and Functional Characterization of Covalently Modified Proteins Formed By a Glycating Agent, Glyoxal.

Glycation, the main consequence of hyperglycemia, is one of the major perpetrators of diabetes and several other conditions, including coronary and neurodegenerative complications. Such a hyperglycemic condition is represented by a large increase in levels of various glycation end products including glyoxal, methylglyoxal, and carboxymethyl-lysine among others. These glycation end products are known to play a crucial role in diabetic complications due to their ability to covalently modify important proteins and enzymes, specifically at lysine residues (a process termed as glycation), making them non-functional. Previous studies have largely paid attention on characterization and identification of these reactive glycating agents. Structural and functional consequences of proteins affected by glycation have not yet been critically investigated. We have made a systematic investigation on the early conformational changes and functional alterations brought about by a glycating agent, glyoxal, on different proteins. We found that the early event in glycation includes an increase in hydrodynamic diameter, followed by minor structural alterations sufficient to impair enzyme activity. The study indicates the importance of glyoxal-induced early structural alteration of proteins toward the pathophysiology of hyperglycemia/diabetes and associated conditions.

N-Acetylaspartate Is an Important Brain Osmolyte.

Most of the human diseases related to various proteopathies are confined to the brain, which leads to the development of various forms of neurological disorders. The human brain consists of several osmolytic compounds, such as N-Acetylaspartate (NAA), myo-inositol (mI), glutamate (Glu), glutamine (Gln), creatine (Cr), and choline-containing compounds (Cho). Among these osmolytes, the level of NAA drastically decreases under neurological conditions, and, hence, NAA is considered to be one of the most widely accepted neuronal biomarkers in several human brain disorders. To date, no data are available regarding the effect of NAA on protein stability, and, therefore, the possible effect of NAA under proteopathic conditions has not been fully uncovered. To gain an insight into the effect of NAA on protein stability, thermal denaturation and structural measurements were carried out using two model proteins at different pH values. The results indicate that NAA increases the protein stability with an enhancement of structure formation. We also observed that the stabilizing ability of NAA decreases in a pH-dependent manner. Our study indicates that NAA is an efficient protein stabilizer at a physiological pH.

Osmolytes in vaccine production, flocculation and storage: a critical review.

Small molecule osmolytes, responsible for protecting stresses have long been known to rescue proteins and enzymes from loss of function. In addition to protecting macromolecules integrity, many osmolytes also act as potential antioxidant and also help to prevent protein aggregation, amyloid formation or misfolding, and therefore are considered promising molecules for neurodegenerative and many other genetic diseases. Osmolytes are also known to be involved in the regulation of several key immunological processes. In the present review we discuss in detail the effect of these compounds on important aspects of vaccines i.e., increasing the efficiency, production and purification steps. The present review therefore will help researchers to make a better strategy in vaccine production to formulation by incorporating specific and appropriate osmolytes in the processes.