Mammalian cystatin and protagonists in brain diseases.

Cystatins are the thiol Proteinase inhibitors, present ubiquitously in mammalian body. They prevent unwanted proteolysis and play important role in several diseases. Regulation of cysteine Proteinase and their inhibitors is of utmost importance in neurodegenerative diseases like Alzheimer, amyloid angiopathy and in many other diseases. The action of these cysteine proteases is biologically controlled by proteinase inhibitors namely cystatins(cys) they constitute a superfamily of homologous proteins. The major role of cystatins is to protect the organism against endogenous proteases released from lysosomes, invading microorganisms and parasites that use cysteine proteases to enter the body. An enormous progress has been made in understanding of protein degradation process under normal and pathological conditions; in fact proteases are now clearly viewed as important drug targets. Some studies have suggested that cystatin C is a target for intervention in neurological disorders because its expression increases in response to human neurological disorders and in animal models of neurodegenerative states. Although, these studies did not clarify whether CysC up-regulation is a pathogenic factor in neurodegenerative disorders or whether it represents a neuroprotective compensatory response of the organisms aimed to prevent progression of the disease. However, for other diseases in some cases cystatins other than cys C are up regulated and in some it is down regulated.Cystatins have been implicated in the processes of neuronal degeneration and repair of the nervous system. Both CysC and CysB are potent, reversible inhibitors of most of the currently known cathepsins. The extent of proteolytic activity at any given time and location is the result of a balance between active proteases and physiological inhibitors. Uncontrolled proteolysis as a result of imbalance between active proteases and their endogenous inhibitors has been associated with neuronal cell death in different neuronal diseases, including brain tumors, stroke, some forms of epilepsy, Alzheimer's disease, and neurological autoimmune diseases.An antidepressant is a psychiatric medication used to alleviate mood disorders, major depression and other brain diseases. Drugs including the monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), and serotonin-norepinephrine reuptake inhibitors (SNRIs) are most commonly used antidepressant. They are also used to treat other conditions, such as anxiety disorders, obsessive compulsive disorder, eating disorders, and chronic pain. Although the mechanisms of the action of these antidepressants are not precisely understood, their principal target of action is at the monoamine transporter proteins located at nerve endings. Monoamine neurotransmitter transporters act to terminate synaptic neurotransmission. Selective serotonin reuptake inhibitors or SSRIs are also most widely used class of antidepressants. They work by increasing the level of serotonin in the brain. SSRIs have fewer and milder side effects, fewer drug interactions, and are much less likely to be associated with suicide than TCAs.These antidepressants shows binding when incubated with cystatin, presenting the involvement of these antidepressant in cascade of disease, as leaving no cystatin to inhibit the cathepsin showing the myriad side effect after the administration of antidepressant. This might be one of the reason in the mechanism of action of antidepressant.So this review expound about the role of cystatins in neurological diseases which is considered to be highly significant as it pave the way for commanding tool in the drug design.Communicated by Ramaswamy H. Sarma.

An Update on the Association of Protein Kinases with Cardiovascular Diseases.

BACKGROUND: Protein kinases are the enzymes involved in phosphorylation of different proteins which leads to functional changes in those proteins. They belong to serine-threonine kinases family and are classified into the AGC (Protein kinase A/ Protein kinase G/ Protein kinase C) families of protein and Rho-associated kinase protein (ROCK). The AGC family of kinases are involved in G-protein stimuli, muscle contraction, platelet biology and lipid signaling. On the other hand, ROCK regulates actin cytoskeleton which is involved in the development of stress fibres. Inflammation is the main signal in all ROCK-mediated disease. It triggers the cascade of a reaction involving various proinflammatory cytokine molecules. METHODS: Two ROCK isoforms are found in mammals and invertebrates. The first isoforms are present mainly in the kidney, lung, spleen, liver, and testis. The second one is mainly distributed in the brain and heart. RESULTS: ROCK proteins are ubiquitously present in all tissues and are involved in many ailments that include hypertension, stroke, atherosclerosis, pulmonary hypertension, vasospasm, ischemia-reperfusion injury and heart failure. Several ROCK inhibitors have shown positive results in the treatment of various disease including cardiovascular diseases. CONCLUSION: ROCK inhibitors, fasudil and Y27632, have been reported for significant efficiency in dropping vascular smooth muscle cell hyper-contraction, vascular inflammatory cell recruitment, cardiac remodelling and endothelial dysfunction which highlight ROCK role in cardiovascular diseases.

Role of phytocystatin in combating metal ion induced conformational alterations in glutathione reductase.

Glutathione reductase (GR) is a flavoprotein that catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione (2GSH) in the presence of coenzyme NADPH. The importance of glutathione stems from the fact that it serves an important role in various metabolic processes. Plants growing in highly polluted areas are exposed to higher concentration of metal ions; thereby feeling abiotic stress and affecting various regulatory enzyme activities. In this study, effect of metal ions has been studied on GR. Phytocystatins show an increased expression in abiotic stress conditions. Here in, the effect of cystatin isolated from yellow mustard seeds (YMP) on heavy metals induced conformational changes in GR was investigated making use of GR activity assay, UV-absorption spectroscopy, fluorescence spectroscopy, FTIR, CD, ITC and SEM analysis. The results obtained clearly reveals that metal ions like Cu2+ and Zn+2 induces concentration dependent conformational changes in GR; YMP restores these alterations in way decreasing the effective concentration of metal ions.

Spectroscopic studies on free radical coalescing antioxidants and brain protein cystatin.

Cystatins are the inhibitors of thiol proteinases and are ubiquitously present in mammalian system. In brain, they put off unwanted proteolysis and are also involved in several neurodegenerative diseases. In the present study, it was demonstrated that photo-activated HOCl-induced modifications in brain cystatin leading to its inactivation and degradation due to hydroxyl radicals. It has been shown that oxidation of cystatin by ROS in vivo leads to oxidative modification which may direct the damage of this significant protein, as it is so well pronounced in vitro. The interplay between free radicals, antioxidants and co-factors is important in maintaining health, aging and age-related diseases. Body's endogenous antioxidant systems stabilize free radical-induced oxidative stress by the ingestion of exogenous antioxidants. If the generation of free radicals goes beyond the protective effect of antioxidants, this can cause oxidative damage which accumulates during the life cycle and has been implicated in aging and age-related diseases such as cardiovascular disease, cancer, neurodegenerative disorders and other chronic conditions. Activation of neutrophils in certain diseases (e.g., inflammatory conditions and atherosclerosis) results in the production of highly reactive species, such as OH• and the release of the enzyme myeloperoxidase. Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. Hypochlorous acid (HOCl) is a potent oxidant formed by myeloperoxidase that causes aggregation of many proteins and damage of proteins by reaction with amino-acid side-chains or backbone cleavage. Communicated by Ramaswamy H. Sarma.

Purification and characterization of a cystatin like thiol protease inhibitor from Brassica nigra.

Phytocystatins or plant cystatins belong to a group of thiol protease inhibitors present ubiquitously in living system. They play a crucial role in cellular protein turnover thereby showing involvement in a wide array of physiological processes in plants. With wide importance and tremendous potential applications in the fields of genetic engineering, medicine, agriculture, and food technology, it is imperative to identify and isolate such protease inhibitors from different cheap and easily available plant sources. Present study focuses on the isolation, purification and characterization of a cystatin like thiol protease inhibitor from the seeds of Brassica nigra (rai mustard) following a simple two-step method using ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with 51.85% yield and 151.50 fold purification. Rai seed cystatin (RSC) gave a molecular mass of ~19.50 kDa as determined by SDS PAGE and gel filtration behaviour. Stokes radius and diffusion coefficient of RSC were 19.80 Šand 11.21 × 10-7 cm2 s-1 respectively. Kinetic analysis revealed a reversible and non-competitive mode of inhibition with RSC showing highest inhibition towards papain (Ki = 1.62 × 10-7 M) followed by ficin and bromelain. Purified RSC possessed an α helical content of 35.29% as observed by far-UV CD spectroscopy. UV, fluorescence, CD and FTIR spectral studies revealed a significant conformational alteration in one or both the proteins upon RSC-papain complex formation. Isothermal Titration Calorimetry (ITC) analysis further revealed the values for different thermodynamic parameters involved in complex formation, indicating the process to be enthalpically as well as entropically driven with forces involved in binding the proteins to be electrostatic in nature. Additionally binding stoichiometry (N) of 0.95 ±â€¯0.08 sites indicates that each molecule of RSC is surrounded by nearly one papain molecule.

Oxidation of cystatin imparted by riboflavin generated free radicals: Spectral analysis.

Thiol Protease inhibitors (cystatins) are endogenous natural inhibitors of cysteine proteases. They are present in all mammalians cells and body fluids. Cystatin are allocated into three major families. Family -I stefins, family -II cystatins and family -III kininogens, according to their amino acid sequence, molecular weight, carbohydrate content and disulphide bonds. It has been investigated that thiol proteases (cathepsin) and their endogenous inhibitor, cystatins have been closely associated with diseases like Alzheimer's, Prions, neurodegenerative diseases, cancer and diabetes. Photodynamic effect of various sensitizers' have long been applied to delineate structural and functional properties of biologically active proteins. Flavins are well known to photo oxidize amino acids which effects conformation of proteins. Riboflavin (Vit B2) with a recommended daily requirement of approximately 2-3 mg is a yellow pigment, It is widely distributed in human tissues and blood, in both free and conjugated forms. In the present Study it has been shown that cystatin purified from buffalo brain (BC) is susceptible to reactive oxygen species generated by photo activation of riboflavin. It was observed that Photo activated riboflavin leads to inactivation of BC. Major Loss of tryptophan intensity was observed in the presence of purified thiol protease inhibitor upon incubation with 50 µM of riboflavin. In order to inspect the type of reactive oxygen species involved in inactivation of the inhibitor, different scavenger's were used namely glucose, potassium Iodide, sodium azide, manitol, thiourea, sodium benzoate, curcumin, quercetin, ascorbic acid and uric acid. It was found that Glucose, Potassium Iodide and sodium azide, have preventive effect on photo inactivation of the purified cystatin whilst other scavengers illustrated diminutive defensive effect.

Damage of cystatin due to ROS-generation and radical-scavenging activity of antioxidants and associated compounds.

Cystatins are thiol proteinase inhibitors present ubiquitously in mammalian system. They prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Hydrogen peroxide, hydroxyl radical and superoxide radical were produced during the autoxidation of various cytotoxic agents namely 6-hydroxydopamine, 6-aminodopamine, 6,7-dihydroxytryptamine, and dialuric acid. Reactive oxygen species (ROS) such as singlet oxygen [O2-] and hydroxyl radicals result in nonspecific reactivity, thus involved in several pathological conditions such as inflammation and reperfusion injury leading to damage of different biological molecules due to the production of enzyme myeloperoxidase because of activation of neutrophils in these diseases. As Myeloperoxidase [MPO] is released extracellularly by activated monocytes and is highly basic protein that damages the localized sites. The onset and progression of several diseases is a complex process in which oxidation is believed to play significant role. Oxidized lipids and proteins that are detected in early, intermediate and advanced stage of human neurodegenerative diseases (plaques) have revealed considerably elevated levels of reactive oxygen species showing their involvement in brain diseases. In the present study, it has been demonstrated that H2O2 induced modifications in brain cystatin leads to its inactivation and degradation. It was also found that the damage from the oxidants occurred mainly because of the (ROS) hydroxyl radicals [OH] produced by H2O2 which in turn changes the conformation of the Cystatin, a major factor for the damage of this significant protein. It is one of the cause of protease-antiprotease imbalance in mammals leading to progression of several diseases.

Glycation induced conformational alterations in caprine brain cystatin (CBC) leads to aggregation via passage through a partially folded state.

Glycation induced advanced glycation end products (AGEs) of proteins formed as a result of Maillard reaction is currently at the heart of a number of pathological conditions. The formation of chemically stable AGEs can permanently alter protein structure and function; hence can serve as an implication in long term complications. Cystatins with high amyloidogenic inclination are implicated in various diseases including cancer and neurodegenerative conditions. The aggregates of cystatin purified from caprine brain have been studied on addition of glucose and ribose using UV absorption, fluorescence emission, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). In the present study AGEs have been monitored and characterized. CBC was incubated for varying time intervals up to 41days in the presence of 17 and 100mM each of glucose and ribose. Ribose at both the concentrations was found to be more potent glycating agent as compared to glucose at these concentrations which is evident from UV and fluorescence spectroscopic studies. Altered intrinsic and high ANS fluorescence for 100mM and 17mM sugar concentrations respectively, suggested the existence of molten globule state of CBC. Glycated CBC as AGEs and aggregates were observed on day 27 and 41 respectively. Formation of AGEs was confirmed by employing AGEs specific fluorescence studies. CBC aggregates confirmed the presence of ß-sheet structure as shown by far-UV CD, dye binding assay and transmission electron microscopy (TEM). Current study is of immense importance as cystatin is a potential candidate of amyloidogenic tendency and a potent endogenous regulator of thiol proteases; hence serves to be an attractive model to study amyloidogenesis of brain cysteine protease inhibitor.

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus.

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10-7 cm2 s-1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (-5.78 kcal mol-1) and positive ΔS (11.01 cal mol-1 deg-1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (-9.19 kcal mol-1) value implies a spontaneous CBC-papain interaction.

Differential effects of anti-cancer and anti-hepatitis drugs on liver cystatin.

The drug-protein interaction has been the subject of increasing interest over the decades. In the present communication, the interaction of liver cystatin with anti-cancer (adriamycin) and anti-hepatitis (adevofir dipivoxil) drugs was studied by thiol-protease inhibitory assay, UV absorption, fluorescence spectroscopy and circular dichroism (CD). A static type of quenching was observed between the protein and the drug molecules. Binding constant (Ka) of adriamycin to liver cystatin (LC) was found to be 1.08 × 10(6) M(-1). Moreover, binding site number was found to be 2. Importantly, cystatin loses its activity in the presence of adriamycin. However, intrinsic fluorescence studies in the presence of adevofir dipivoxil showed enhancement in the fluorescence intensity suggesting that binding of adevofir to LC caused unfolding of the protein. The unfolding of the test protein was also accompanied by significant loss of inhibitory activity. CD spectroscopy result showed, both adriamycin and adevofir dipivoxil caused perturbation in the secondary structure of liver cystatin. The possible implications of these results will help in combating drug induced off target effects.

Antidepressant Fluoxetine Modulates the In Vitro Inhibitory Activity of Buffalo Brain Cystatin: A Thermodynamic Study Using UV and Fluorescence Techniques.

Cystatins constitute a superfamily of homologous proteins. The major role of cystatins is to regulate the unwanted proteolysis and to protect the organism against endogenous proteases released from lysosomes, invading microorganisms and parasites that use cysteine proteases to enter the body. Imbalance in regulation of proteolytic activity may lead to a wide range of human diseases. An enormous progress has been made in understanding of protein degradation process under normal and pathological conditions; infact proteases are now clearly viewed as important drug targets. Fluoxetine a selective serotonin reuptake inhibitor (SSRI) is an antidepressant. It is used to treat major depressive disorders. In the present study binding of fluoxetine to cystatin was studied by UV and fluorescence quenching technique. Intrinsic fluorescence of fluoxetine complexed with purified buffalo brain cystatin (BC) was measured by selectively exciting the tryptophan residues. Gradual quenching was observed on complex formation. When cystatin was added to fluoxetine solutions at a molar ratio of 1 : 0.5, it not only quenched more than half of its fluorescence but also reduced the activity of cystatin. Stern-Volmer plots obtained from experiments carried out at 25(°)C showed the quenching of fluorescence to be a collisional phenomenon. Our results suggest the prime binding site for fluoxetine on BC to be at or near tryptophan residues. Fluoxetine quenched the fluorescence by a static process, which specifically indicates the formation of a complex.

Studies on interaction of buffalo brain cystatin with donepezil: an Alzheimer's drug.

When drugs bind to a protein, the intramolecular structures can be altered, resulting in conformational change of the protein. Donepezil, an Acetyl Cholinesterase inhibitor (AChE), is commonly prescribed to patients with Alzheimer's disease (AD) to enhance cholinergic neurotransmission. It is the "first-line" agents in the treatment of Alzheimer's disease used to improve cognitive function in the disease. In the present study, a cysteine protease inhibitor (cystatin) has been isolated from buffalo brain using alkaline treatment, 40 to 60% ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with % yield of 64.13 and fold purification of 384.7. The purified inhibitor (Buffalo Brain Cystatin, (BBC)) was eluted as a single papain inhibitory peak which migrated as single band on native PAGE; however, on SDS-PAGE with and without beta mercaptoethanol ( ß ME) BBC gave two bands of M W 31.6 and 12.4 KDa, respectively. The molecular weight determined by gel filtration came out to be 43.6 KDa. The UV spectra of cystatin on interaction with donepezil suggested a conformational change in the protein. The fluorescence spectra of BC-donepezil composite show structural changes indicating 40 nm red shift with significant increase in fluorescence intensity of cystatin in the presence of donepezil representing an unfolding of cystatin on interaction, which is an indication of side effect of donepezil during the use of this drug.

Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver.

Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.

Studies on the chemical modification of goat liver cystatin and the effect on its anti-papain inhibitory activity.

Goat liver cystatin was subjected to various chemical modifications in order to ascertain the amino acid residues responsible for its structural and functional integrity. Modification of tryptophan by HNBB led to the complete inactivation of the protein. The inactivation was also accompanied by the complete loss of tryptophan fluorescence at 340 nm. The reaction of liver cystatin with HNBB yielded a characteristic decrease in absorbance at 280 nm. Acetylation of the amino groups of liver cystatin was carried out in the presence of acetic anhydride. The acetylated cystatin showed a decrease in fluorescence intensity at 335 nm which could be attributed to the modification of tyrosine residue due to side reaction.

Purification and characterization of buffalo brain cystatin.

UNLABELLED: Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. AIMS: To purify and characterize Thiol protease inhibitor from buffalo brain and to compare its properties with respect to tissue and organ difference from other mammalian cystatins. MAIN METHODS: Inhibitor has been isolated and purified using alkaline treatment; ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with a % yield of 64.13 and fold purification of 384.72.The inhibitor was studied by U.V and fluorescence spectroscopy. Papain inhibitory activity was measured using casein as substrate. KEY FINDING: The molecular weight of the buffalo brain cystatin (BC), determined by gel filtration and SDS PAGE came out to be 43.6 KDa and 44.20 KDa respectively. BC was found to be stable in broad pH and temperature range. The inhibitor was devoid of any sulphydryl group and carbohydrate content. These properties led to conclusion that BC is variant of type-I cystatin. The stokes radius and diffusion coefficient of the inhibitor were found to be 27 A° and 8.1 x 10⁻7 cm²/sec respectively, the f/f0 ratio was 1.12 signifying that purified cystatin is nearly globular in shape. Kinetic data revealed binding stoichiometry of BC with papain as 1:1. The Ki value with papain ficin and bromelain were found to be 1, 1.85 and 2.25 nM respectively suggesting that cystatin has higher affinity with papain as compared to ficin and bromelain. The fluorescence and UV spectra of BC- papain complex showed significant conformational changes indicative of perturbation in the micro environment of aromatic amino acid residues on the formation of complex. SIGNIFICANCE: This work proliferates our knowledge about cystatins of the mammalian brain on the basis of their physiochemical properties.