Laishram Rajendrakumar Singh

Forty Years of Research on Osmolyte-Induced Protein Folding and Stability

Most organisms that have adapted to environmental stresses have done so by production and accumulation of certain small organic molecules, known as osmolytes that arose by natural selection and have the ability to stabilize intracellular proteins against the environmental stress. It is well known that osmolytes stabilize proteins and induce folding of aberrant proteins and therefore, it is of therapeutic use for a large number of protein misfolding diseases. Thus, it is very important that the present knowledge of the ability and mechanism of osmolyte-induced protein folding and structural stabilization should reach to researchers working in different avenues. In around 40 years of research, we have gained great advances in various aspects of protein folding and structural stabilization induced by osmolytes. To summarize and discuss the original findings, many short review articles and few long reviews have also been available but almost all have focuses on specific aspects. To get a clear picture of the effect of osmolytes on protein folding and structural stabilization, it is necessary for the benefits of the general readers, to combine and discuss all findings made during its 40 years of life. This review article is therefore, designed to give a collective knowledge on almost all facets of the progresses made on osmolyte-protein interaction to-date.

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.

Denatured State Structural Property Determines Protein Stabilization by Macromolecular Crowding: A Thermodynamic and Structural Approach

Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total macromolecular concentration never surpasses 10 g l−1. However, biological macromolecules are known to evolve and function under crowded intracellular environments that comprises of proteins, nucleic acids, ribosomes and carbohydrates etc. Crowded environment is known to result in altered biological properties including thermodynamic, structural and functional aspect of macromolecules as compared to the macromolecules present in our commonly used experimental dilute buffers (for example, Tris HCl or phosphate buffer). In this study, we have investigated the thermodynamic and structural consequences of synthetic crowding agent (Ficoll 70) on three different proteins (Ribonuclease-A, lysozyme and holo α-lactalbumin) at different pH values. We report here that the effect of crowding is protein dependent in terms of protein thermal stability and structure. We also observed that the structural characteristics of the denatured state determines if crowding will have an effect or not on the protein stability.

Molecular Linkages Between Diabetes and Alzheimer's Disease: Current Scenario and Future Prospects

After the revolutionary Rotterdam study that suggested there was an increased risk of developing Alzheimers disease (AD) in patients with type-2 diabetes mellitus (T2DM), a number of studies have provided direct evidence for the linkage between AD and T2DM. In recent years, AD is considered as a neuroendocrine disorder, also referred as type-3 diabetes. There is a growing list of evidence to suggest that, in addition to impaired insulin signaling, there are a number of additional factors that may act as mechanistic links between AD and T2DM. These factors mainly include hypercholesterolemia, dyslipidemia, hypercystinemia, inflammation, impaired insulin signaling and impaired central nervous response to the adipose tissue-derived hormone leptin. Increased cholesterol plays a crucial role in the abnormal metabolism of the amyloid precursor protein, leading to the accumulation of β-amyloid. In addition to impaired insulin signaling, diabetes has been found to accelerate the appearance of cerebrovascular inflammation and β-amyloid peptide (Aβ) deposition. Increased oxidative stress and production of advanced glycation end products are other probable marker linkages. However, the details of many of these molecular links still require extensive investigation. It is possible that a number of common molecular linkages exist between T2DM and AD. Understanding and analyzing the various molecular linkages between AD and T2DM may shed light on new tools that can be used for the early diagnosis and treatment of AD and also accelerate the identification of T2DM patients who are at high risk of AD.

Homocystinuria: Therapeutic approach.

Homocystinuria is a disorder of sulfur metabolism pathway caused by deficiency of cystathionine β-synthase (CBS). It is characterized by increased accumulation of homocysteine (Hcy) in the cells and plasma. Increased homocysteine results in various vascular and neurological complications. Present strategies to lower cellular and plasma homocysteine levels include vitamin B6 intake, dietary methionine restriction, betaine supplementation, folate and vitamin B12 administration. However, these strategies are inefficient for treatment of homocystinuria. In recent years, advances have been made towards developing new strategies to treat homocystinuria. These mainly include functional restoration to mutant CBS, enhanced clearance of Hcy from the body, prevention of N-homocysteinylation-induced toxicity and inhibition of homocysteine-induced oxidative stress. In this review, we have exclusively discussed the recent advances that have been achieved towards the treatment of homocystinuria. The review is an attempt to help clinicians in developing effective therapeutic strategies and designing novel drugs against homocystinuria.

Macromolecular crowding decelerates aggregation of a β-rich protein, bovine carbonic anhydrase: a case study

The majority of in vitro investigations concerning protein aggregation have been performed in dilute systems, which poorly reflect the crowded in vivo scenario. Cell interior is highly crowded with soluble and insoluble macromolecules that alter macromolecular properties. Macromolecular crowding is known to enhance the rate and/or extent of protein aggregation. However, most of the understandings were derived from studies with α-rich or predominantly α-proteins. Indeed, α-proteins fold faster than β-proteins and conversion of α-helices to cross β-sheets are responsible for aggregate/amyloid formation. Therefore, it is important to investigate how macromolecular crowding affects the aggregation propensity of β-rich proteins. In this study, we investigated the effect of synthetic macromolecular crowders on bovine carbonic anhydrase (BCA, a β-rich protein) aggregation. In contrast to the effect of macromolecular crowding on α-rich proteins, BCA aggregation was observed to be reduced due to decrease in the population of aggregation-prone intermediates as a consequence of increased native state stability. In addition, the extent of aggregation was found to depend on the nature of the crowder under consideration. Combining the published data on α-proteins and this study, we conclude that macromolecular crowding can have opposite consequences on protein aggregation process depending on the fold type of the protein.

Unique Medicinal Properties of Withania somnifera: Phytochemical Constituents and Protein Component

Withania somnifera is an important medicinal herb that has been widely used for the treatment of different clinical conditions. The overall medicinal properties of Withania somnifera make it a viable therapeutic agent for addressing anxiety, cancer, microbial infection, immunomodulation, and neurodegenerative disorders. Biochemical constituents of Withania somnifera like withanolideA, withanolide D, withaferin A and withaniamides play an important role in its pharmacological properties. Proteins like Withania somnifera glycoprotein and withania lectin like-protein possess potent therapeutic properties like antimicrobial, anti-snake venom poison and antimicrobial. In this review, we have tried to present different pharmacological properties associated with different extract preparations, phytochemical constituents and protein component of Withania somnifera. Future insights in this direction have also been highlighted.

N-homocysteinylation induces different structural and functional consequences on acidic and basic proteins.

One of the proposed mechanisms of homocysteine toxicity in human is the modification of proteins by the metabolite of Hcy, homocysteine thilolactone (HTL). Incubation of proteins with HTL has earlier been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation). It has been believed that protein N-homocysteinylation is the pathological hallmark of cardiovascular and neurodegenerative disorders as homocysteinylation induces structural and functional alterations in proteins. In the present study, reactivity of HTL towards proteins with different physico-chemical properties and hence their structural and functional alterations were studied using different spectroscopic approaches. We found that N-homocysteinylation has opposite consequences on acidic and basic proteins suggesting that pI of the protein determines the extent of homocysteinylation, and the structural and functional consequences due to homocysteinylation. Mechanistically, pI of protein determines the extent of N-homocysteinylation and the associated structural and functional alterations. The study suggests the role of HTL primarily targeting acidic proteins in eliciting its toxicity that could yield mechanistic insights for the associated neurodegeneration.

Existence of molten globule state in homocysteine-induced protein covalent modifications.

Homocysteine thiolactone is a toxic metabolite produced from homocysteine by amino-acyl t-RNA synthetase in error editing reaction. The basic cause of toxicity of homocysteine thiolactone is believed to be due to the adduct formation with lysine residues (known as protein N-homocysteinylation) leading to protein aggregation and loss of enzyme function. There was no data available until now that showed the effect of homocysteine thiolactone on the native state structural changes that led to aggregate formation. In the present study we have investigated the time dependent structural changes due to homocysteine thiolactone induced modifications on three different proteins having different physico-chemical properties (cytochrome-c, lysozyme and alpha lactalbumin). We discovered that N-homocysteinylation leads to the formation of molten globule state—an important protein folding intermediate in the protein folding pathway. We also found that the formation of the molten globule state might be responsible for the appearance of aggregate formation. The study indicates the importance of protein folding intermediate state in eliciting the homocysteine thiolactone toxicity.

Testing the Ability of Non-Methylamine Osmolytes Present in Kidney Cells to Counteract the Deleterious Effects of Urea on Structure, Stability and Function of Proteins

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 (ΔG D o) and functional activity parameters (K m and k cat) 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.