Bilqees Bano

Cystatins in Health and Diseases

The Cystatins constitute a large group of evolutionary related proteins with diverse biological activities. They have been recently realized as instrumental in myriad of pathophysiological conditions. They have been implicated in various pathological conditions. The degree of malignancy of various types of cancer cells has been found to be inversely associated with the expression of cystatins. Cystatins have been found to have various antimicrobial, antiviral and immunomodulatory properties. Keeping in view as their being prospective drug targets and anti-disease options this review explores the role of cytoplasmic and cell secreted cystatins in various human diseases.

Photosynthetic and growth responses of two mustard cultivars differing in phytocystatin activity under cadmium stress

Cadmium inhibits photosynthetic capacity of plants by disturbing protein conformations, whereas phytocystatins prevent degradation of target proteins and are involved in abiotic stress tolerance. Two mustard (Brassica juncea L.) cultivars, Ro Agro 4001 and Amruta, were grown with Cd (50 µM) in order to study physiological and biochemical basis of differences in Cd tolerance. Amruta accumulated higher Cd and H2O2 concentrations in leaves than that of Ro Agro 4001. Cd significantly decreased photosynthesis and growth of plants in both cultivars by reducing a chlorophyll content, gas exchange parameters, and activity of Rubisco; the effects were more prominent in Amruta than those in Ro Agro 4001. The greater photosynthesis and growth of Ro Agro 4001 under Cd stress might be attributed to its higher phytocystatin activity together with greater ascorbate peroxidase activity, photosynthetic nitrogen-use efficiency, sulphur assimilation (ATP-sulphurylase activity and S content), and contents of cysteine and reduced glutathione compared to Amruta. In contrast, the activity of superoxide dismutase (SOD) was higher in Amruta than that of Ro Agro 4001 under control conditions, whereas the Cd treatment increased significantly the SOD activity in both cultivars with the greater increase in Ro Agro 4001. The fluorescence spectra of phytocystatin showed a lesser change in Ro Agro 4001 under Cd stress than that in Amruta suggesting higher resistance of Ro Agro 4001 to Cd. The higher phytocystatin activity under Cd stress in Ro Agro 4001 compared to Amruta enabled the plants to protect their proteins more efficiently. This resulted in a greater increase of photosynthetic capacity in Ro Agro 4001 than that of Amruta. Thus, the phytocystatin activity may be considered as a physiological parameter for augmenting photosynthesis and growth of mustard under Cd stress.

Employing in vitro analysis to test the potency of methylglyoxal in inducing the formation of amyloid-like aggregates of caprine brain cystatin

AbstractThiol protease inhibitors (cystatins) are implicated in various disease states from cancer to neurodegenerative conditions and immune responses. Cystatins have high amyloidogenic propensity and they are prone to form fibrillar aggregates leading to amyloidosis. Particularly challenging examples of such disorders occur in type 2 diabetes, Alzheimer’s and Parkinson’s diseases. The aim of the present study is to find an interaction between the compound methylglyoxal (MG) which is particularly elevated in type 2 diabetes with caprine brain cystatin (CBC). Results have shown that elevated concentration of MG forms amyloid aggregates of CBC. This was achieved by allowing slow growth in a solution containing moderate to high concentrations of MG. When analysed with microscopy, the protein aggregate present in the sample after incubation consisted of extended filaments with ordered structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit increased Thioflavin T fluorescence.

Purification and biochemical characterization of phytocystatin from Brassica alba

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two‐step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S‐100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180‐fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10−7 cm2s−1 respectively. The isolated phytocystatin was found to be stable in the pH range of 6–8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non‐competitive type of inhibition and inhibited papain more efficiently (Ki = 3 × 10−7 M) than ficin (Ki = 6.6 × 10−7 M) and bromelain (Ki = 7.7 × 10−7 M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content. Copyright

Conformational behaviour and aggregation of chickpea cystatin in trifluoroethanol: effects of epicatechin and tannic acid.

Conformational alterations and aggregates of chickpea cystatin (CPC) were investigated upon sequential addition of trifluoroethanol (TFE) over a range of 0-70% v/v. CPC on 30% and 40% v/v TFE addition exhibited non-native β-sheet, altered intrinsic fluorescence, increased thioflavin T fluorescence, prominent red shifted shoulder peak in Congo red absorbance, and enhanced turbidity as well as Rayleigh scattering, suggesting the aggregate formation. TEM results confirmed the formation of fibrillar aggregates at 30% and 40% v/v TFE. On increasing concentration of TFE to 70% v/v, CPC showed retention of native-like secondary structure, increased intrinsic and ANS fluorescence. Thus our results show that favourable condition for fibrillation of CPC is in the range of 30-40% TFE. Moreover, anti-aggregational effects of polyphenols, epicatechin (EC) and tannic acid (TA) were analysed using ThT binding assay and other biophysical assays. EC and TA produced a concentration dependent decline in ThT fluorescence suggesting inhibition of the fibril formation. Furthermore, TA in comparison to EC, served as a more effective inhibitor against amyloid fibril formation of CPC. This work supports the universality of the amyloid-like aggregation not restricted to some special categories of protein and the fact that this aggregation can be prevented.

Purification and Characterizaion 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.

Comparative effect of olive oil and fish oil supplementation in combating gentamicin induced nephrotoxicity in rats.

The present study is related to the comparative effects of fish oil and olive oil supplementation on gentamicin induced nephrotoxicity in rats. Three treatment groups (Pretrement, Co-treatment and post treatment) were chosen for the study. Nephrotoxicity in rats was induced by intraperitonial administration of gentamicin (80 mg/kg/d) for 3,5,7,10,& 12 consecutive days. The animals were sacrificed 12 hrs after last treatment in each group. The maximum nephrotoxicity was developed on 10 days treatment of gentamicin. For each group a control group was taken without any oil or gentamicin treatment. Beneficial effects of oils were evidenced by reduced serum urea and creatinine concentrations in the group receiving oils compared to the non oil treatment animals receiving gentamicin only. Further, the changed values of alkaline phosphatase and acid phosphatase activity retumed to normal in kidney and liver tissue homogenates after fish and olive oil treatment. In this study, it was found that co-treatment of fish and olive oil is more effective antagonist of gentamicin induced nephrotoxicity. However fish oil was found to be more effective. Hypercholesteromia associated with gentamicin induced nephrotoxicity is also lowered by oil supplementations. The beneficial effects of these oils are due to counteracting effect of the biochemical alterations induced by the drug.

Purification, characterization and kinetics of thiol protease inhibitor from goat ( Capra hircus ) lung

In the present study, two molecular forms of goat lung cystatin (GLC), I and II, were purified to homogeneity by a two-step procedure including ammonium sulfate precipitation (40–60%) and ion exchange chromatography. The inhibitor forms migrated as single bands under native and SDS-PAGE with and without reducing agent giving molecular mass of 66.4 and 76.4 kDa, respectively. GLC-I possesses 0.07% and GLC-II 2.3% carbohydrate content and no -SH groups. GLC-I showed greater affinity for papain than for ficin and bromelain. Immunological studies showed that the inhibitor was pure and there was cross reactivity between anti-GLC-I serum and goat brain cystatin. Both inhibitor forms were stable in the pH range of 3–10 and up to 75°C. GLC-I was found to possess 49% α-helical structure by CD spectroscopy. The inhibitor-papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies.

Conformational transitions induced by in vitro macromolecular crowding lead to the amyloidogenesis of buffalo heart cystatin

The biological cells and extracellular matrix exhibit a highly crowded environment, called as macromolecular crowding. Crowding significantly influences protein structure and may lead to its aggregation. In the present study, buffalo heart cystatin (BHC), after purification from buffalo heart tissue, has been used as a model protein for studying effect of macromolecular crowding in the presence of high concentrations of bovine serum albumin (BSA), poly‐ethylene glycol‐1000 (PEG‐1000), and poly‐ethylene glycol‐4000 (PEG‐4000). Cystatins are thiol protease inhibitors and found to be involved in various important physiological processes. Functional inactivation of BHC was observed upon crowding, which varied as a function of concentration and molecular weight of crowding agents as well as incubation time. Structural changes of BHC at tertiary and secondary level were detected with the help of fluorescence and CD spectroscopy. CD analysis showed changes of α‐helix to β‐sheet, which could be due to aggregation. The ANS‐fluorescence study suggested the unfolding and presence of some partially folded intermediates. Increase in ThT‐fluorescence and absorption of Congo red spectra with red shift, confirmed the amyloid type aggregation of BHC in the presence of various crowding agents. Finally, electron microscopy provided the physical evidence about the formation of amyloid fibrils. Results suggested that among the various crowding agents used, amyloidogenesis of BHC was maximal in case of BSA followed by PEG‐4000 and least for PEG‐1000. The present work makes an important contribution in crowding mediated protein aggregation, which can have implications of potential interest. Copyright

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 104 M−1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.