Urea induced unfolding of rai seed cystatin: Influence of glycerol as a chemical chaperone.

Cystatin superfamily members, by virtue of their thiol protease regulatory properties, show involvement in myriad physiological processes important for survival and well-being. The current study involves urea-induced denaturation of a novel variant of the cystatin superfamily, rai seed cystatin (RSC), employing a variety of biophysical assays in order to characterize different folding intermediates generated on unfolding. Urea as a denaturant presented the passage of RSC through a series of events resulting in the loss of RSC functional capability, accompanied by changes in the archetype at secondary and tertiary structural levels, as evident from protease inhibitory, UV absorption, and intrinsic fluorescence assays, respectively. ANS fluorescence also revealed routing of RSC through discrete multiple sub-states thus presenting the generation of intermediate states somewhat close to the pre-molten globule and/or molten globule forms of RSC. Furthermore, far-UV circular dichroism analysis revealed a concentration-dependent gradual loss in typical -helical RSC peaks, indicating a nearly 50 % loss in secondary structural elements around 5 M urea treatment. The study also reports the possible role of glycerol in the refolding and/or reactivation of the urea unfolded RSC form. Glycerol presented itself as a potent structural stabilizer as it assisted in the refolding and reactivation of the unfolded RSC in a dosage-dependent manner, concomitantly paving the way for unravelling the mechanistic approach involved in the phenomenon, which can facilitate future studies.

Protein aggregation as a consequence of non-enzymatic glycation: Therapeutic intervention using aspartic acid and arginine.

Non-enzymatic glycation tempted AGEs of proteins are currently at the heart of a number of pathological conditions. Production of chemically stable AGEs can permanently alter the protein structure and function, concomitantly leading to dilapidated situations. Keeping in perspective, present study aims to report the glycation induced structural and functional modification of a cystatin type isolated from rai mustard seeds, using RSC-glucose and RSC-ribose as model system. Among the sugars studied, ribose was found to be most potent glycating agent as evident from different biophysical assays. During the course of incubation, RSC was observed to pass through a series of structural intermediates as revealed by circular dichroism, altered intrinsic fluorescence and high ANS binding. RSC incubation with ribose post day 36 revealed the possible buildup of ß structures as observed in CD spectral analysis, hinting towards the generation of aggregated structures in RSC. High thioflavin T fluorescence and increased Congo red absorbance together with enhanced turbidity of the modified form confirmed the aggregation of RSC. The study further revealed anti-glycation and anti-aggregation potential of amino acids; aspartic acid and arginine as they prevented and/or slowed down the process of AGEs and ß structure buildup in a concentration dependent manner with arginine proving to be the most effective one.

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.

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.

Binding of λ-carrageenan (a food additive) to almond cystatin: An insight involving spectroscopic and thermodynamic approach.

Carrageenan is a high molecular weight linear sulphated polysaccharide, primarily used in food industry as gelling, thickening, and stabilizing agent. Almond milk prepared from almonds is low in fat, but high in antioxidants, energy, proteins, lipids and fibre. Purified almond cystatin was incubated with increasing concentrations of carrageenan at 25°C for different time interval and significant loss in inhibitory activity was observed. Interaction between carrageenan and cystatin resulted in complex formation as depicted by the decrease in fluorescence intensity with increase in the concentration of carrageenan. Stern-volmer analysis of fluorescence quenching data showed binding constant to be 1.84±0.20×104M-1 and number of binding sites close to unity. These results were further confirmed by supporting results obtained in UV-vis spectroscopy. FTIR analysis shows significant shift in the peak intensity and this change clearly depict change in the structure of cystatin from that of α helix to ß-sheet. CD spectra further confirmed the structural transition of the cystatin from α helix to ß-sheet structure on interaction with increased concentrations of carrageenan. The contributing thermodynamic parameters were determined by ITC. The negative ΔH° and positive TΔS° values suggest involvement of electrostatic forces and hydrophobic interaction in the formation of the λ-carrageenan-cystatin complex.

Interaction of almond cystatin with pesticides: Structural and functional analysis.

Pesticides are chemical substances that eliminate or control a variety of agricultural pests that damage crops and livestock. They not only affect the targeted pests but also affect the nontargeted systems, raising more concerns for their effect on both plant and animal systems. Cystatins (cysteine protease inhibitor) are ubiquitously present in all living cells and show a variety of important physiological functions. The present study shows the effect of different pesticides (pendimethalin, methoxyfenozide, and CuII hydroxide) on purified almond cystatin. Almond cystatin showed concentration-dependent loss in papain inhibitory activity on interaction with the pesticides, showing maximum loss in the presence of Cu(II) hydroxide and minimum in the case of methoxyfenozide. Native polyacrylamide gel electrophoresis showed maximum degradation of purified cystatin in the presence of Cu(II) hydroxide with insignificant effect in the presence of methoxyfenozide. Structural alterations were significant in the case of Cu(II) hydroxide and less in the case of methoxyfenozide as revealed by UV and fluorescence spectral studies. Secondary structural alterations were further conformed by circular dichroism and Fourier transform infrared spectroscopy. The α-helix content of almond cystatin decreases from 35.64% (native) to 34.83%, 30.79%, and 29.62% for methoxyfenozide-, pendimethalin-, and Cu(II) hydroxide-treated cystatin, respectively. A Fourier transform infrared study shows an amide I band shift for almond cystatin from 1649.15 ± 0.5 to 1646.48 ± 0.6, 1640.44 ± 0.6, and 1635.11 ± 0.3 cm-1 for methoxyfenozide, pendimethalin, and Cu(II) hydroxide, respectively. Values obtained for different thermodynamic parameters (ΔH0 , ΔG0 , N, and ΔS0 ) by isothermal titration calorimetric experiments reveal maximum binding of almond cystatin with Cu(II) hydroxide followed by pendimethalin and little interaction with methoxyfenozide.

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.

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

Thiol 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 ß-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit increased Thioflavin T fluorescence.