Thiol Protease Inhibitor Research Articles

The Roles of Cystatin B in the Brain and Pathophysiological Mechanisms of Progressive Myoclonic Epilepsy Type 1.

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder, also known as Unverricht-Lundborg disease (ULD). EPM1 patients suffer from photo-sensitive seizures, stimulus-sensitive myoclonus, nocturnal myoclonic seizures, ataxia and dysarthria. In addition, cerebral ataxia and impaired GABAergic inhibition are typically present. EPM1 is caused by mutations in the Cystatin B gene (CSTB). The CSTB protein functions as an intracellular thiol protease inhibitor and inhibits Cathepsin function. It also plays a crucial role in brain development and regulates various functions in neurons beyond maintaining cellular proteostasis. These include controlling cell proliferation and differentiation, synaptic functions and protection against oxidative stress, likely through regulation of mitochondrial function. Depending on the differentiation stage and status of neurons, the protein localizes either to the cytoplasm, nucleus, lysosomes or mitochondria. Further, CSTB can also be secreted to the extracellular matrix for interneuron rearrangement and migration. In this review, we will review the various functions of CSTB in the brain and discuss the putative pathophysiological mechanism underlying EPM1.

Antiglycation potential of metal ions and polyphenolic extract of chickpea on thiol-protease inhibitor: A management for diabetic complications

Glycation is the non-enzymatic adduct formation between reducing sugars or dicarbonyls with proteins and is a crucial molecular event under hyperglycaemic conditions of diabetes. The accumulation of advanced glycation end products (AGEs) due to glycation of proteins has been implicated in several diseases associated with ageing and diabetes. Thus, investigating the antiglycation potential of some trace metal ions (Manganese; Mn2+, and Zinc; Zn2+) and polyphenolic extract of chickpea seeds (PEC) on the methylglyoxal (MGO) induced glycation of a phytocystatin isolated from chickpea was taken up to find an inexpensive and non-toxic therapeutic means of medicating protein glycation and associated diabetic complications. The current study focused on the comparative analyses of these micronutrients and herbal extracts in inhibiting protein glycation and AGEs formation in a quest to develop nutraceuticals for managing diabetes. The effect of metals (Mn2+, Zn2+) and PEC on protein glycation was assessed by different techniques, i.e., glycation-specific AGE fluorescence and absorbance, thiol protease inhibitory activity assay, and conformational alterations by spectroscopic assays. This study revealed the significant anti-glycation potencies of Mn2+, Zn2+, and PEC against the MGO-induced glycation of CPC, which might pave the way for resolving pathological complications of diabetes by combining higher levels of efficacy, selectivity, and safety in humans. Moreover, characterization and identification of different AGEs formed during the glycation process in diabetics was done to apply the same for determining the onset of glycation at the early stage so that appropriate steps be taken to address the menace of diabetic complications.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Recognize and Hydrolyze Oligopeptides Corresponding to the S-Protein of SARS-CoV-2.

The S-protein is the major antigen of the SARS-CoV-2 virus, against which protective antibodies are generated. The S-protein gene was used in adenoviral vectors and mRNA vaccines against COVID-19. While the primary function of antibodies is to bind to antigens, catalytic antibodies can hydrolyze various substrates, including nucleic acids, proteins, oligopeptides, polysaccharides, and some other molecules. In this study, antibody fractions with affinity for RBD and S-protein (RBD-IgG and S-IgG) were isolated from the blood of COVID-19 patients vaccinated with Sputnik V. The fractions were analyzed for their potential to hydrolyze 18-mer oligopeptides corresponding to linear fragments of the SARS-CoV-2 S-protein. Here, we show that the IgG antibodies hydrolyze six out of nine oligopeptides efficiently, with the antibodies of COVID-19-exposed donors demonstrating the most significant activity. The IgGs of control donors not exposed to SARS-CoV-2 were found to be inactive in oligopeptide hydrolysis. The antibodies of convalescents and vaccinated patients were found to hydrolyze oligopeptides in a wide pH range, with the optimal pH range between 6.5 and 7.5. The hydrolysis of most oligopeptides by RBD-IgG antibodies is inhibited by thiol protease inhibitors, whereas S-IgG active centers generally combine several types of proteolytic activities. Ca2+ ions increase the catalytic activity of IgG preparations containing metalloprotease-like active centers. Thus, the proteolytic activity of natural antibodies against the SARS-CoV-2 protein is believed to be due to the similarity of catalytic antibodies' active centers to canonical proteases. This work raises the question of the possible physiological role of proteolytic natural RBD-IgG and S-IgG resulting from vaccination and exposure to COVID-19.

Effects of Nutrient Elements on Growth and Expression of Insect-Defense Response Genes in Zanthoxylum bungeanum Maxim

In China, Zanthoxylum bungeanum Maxim, known as “Huajiao,” has a pleasant, fragrant flavor and several therapeutic properties. The nutritional content of plants is necessary for their defense response to insects. In this study, we analyzed the effects of soil fertilization treatments such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and special compound fertilizer for pepper (HZ) on the different growth parameters and expression of insect-defense-response genes in Z. bungeanum. The results show that the height and weight of prickly ash significantly differed after the application of fertilizers with different concentrations. Additionally, seedlings that were treated with low concentrations of nutrient fertilizers (N1, P1, K1, Ca1, and HZ1) were significantly higher, and they were selected for transcriptome analysis. According to transcriptomic analysis, a total of 65,566 unigenes were discovered, among which 61,379 corresponded to annotated protein-coding genes and 4187 to transcripts of novel protein-coding genes. A total of 294 unigenes were detected as candidate genes for regulating the defense response to insects, including 204 protease inhibitors, 29 plant lectins, and 61 other defense response genes. Additionally, trypsin inhibitors, cystatin, phytepsin, metalloproteinase, MMP, caffeic acid, resveratrol, and thiol proteinase inhibitors, ACA, TDC, and 28 BES1 were enriched in Z. bungeanum. Specifically, the leaves of Z. bungeanum that were treated with Ca and HZ fertilizations were dominated by the protease inhibitors. In addition, the type of fertilizer significantly affects gene expression in plants. The functional annotations were predicted by the number of differentially expressed genes and classified by GO and KEGG ontology enrichment analysis. Moreover, according to the GO database, biological processes were the largest group and contained a high frequency of differentially expressed genes. According to KEGG pathway results, significantly enriched genes belonged to the biosynthesis of secondary metabolisms, amino acid metabolism, and folding, sorting, and degradation. Overall, it was found that the type of fertilizer with low concentrations had an effect on Z. bungeanum’s primary and secondary metabolism, and these findings provided grounds for further research in forest protection science.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Transcriptome profiles of pre-pubertal and adult in vitro matured ovine oocytes obtained from FSH-stimulated animals.

Pre-pubertal females produce more oocytes than adult individuals, but the ability of oocytes to undergo embryonic development and produce viable offspring is less in pre-pubertal animals than in adult animals. Oocyte quality is associated with abnormal gene expression. To date, the transcriptome profiles of 1-month-old lamb oocytes after in vitro maturation (IVM) are poorly reported. This study aimed to identify differentially expressed genes (DEGs) in lamb oocytes with varying competencies, affecting oocyte competence. Using RNA sequencing (RNA developmental -seq) technology, the transcriptome profiles of 1-month-old lamb oocytes after IVM were assessed, and oocytes from adult sheep large follicles were used as controls. We found 11 up-regulated and 39 down-regulated DEGs in lamb oocytes. Gene Ontology analysis of DEGs showed that molecular functions were clustered in catalytic and binding activities, while biological processes were clustered in metabolic process, cellular process, single-organism process and biological regulation. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs were involved in oxidative phosphorylation, transforming growth factor-beta signalling pathway, or DNA replication. In lamb MII oocytes, down-regulation of oxidative phosphorylation genes (ATP5E, NDUFA7 and COX6C), thiol protease inhibitor (CSTB) and 26S proteasome component (SHFM1) and up-regulation of CUL1, MARCH7 and TRIM17 might cause low competence of lamb embryos. In conclusion, this study provided detailed information on mRNA transcriptomes in lamb oocytes after IVM, which offers insights into the reduced developmental potential of lamb oocytes.

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K a) of methotrexate to muscle cystatin was found to be 1 × 10−7 M−1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile. 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 Å and 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 ± 0.08 sites indicates that each molecule of RSC is surrounded by nearly one papain molecule.

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

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.

Deciphering the toxic effects of iprodione, a fungicide and malathion, an insecticide on thiol protease inhibitor isolated from yellow Indian mustard seeds

Pesticides are being used globally to improve agricultural production. They are applied specifically to combat with pathogens that are a major threat for reduced optimum yield of crops. This study was carried out to see the effect of commercially used pesticides on a specific plant protein viz. phytocystatin isolated from yellow mustard seeds (YMP). Phytocystatin is a thiol proteinase inhibitor, which regulates endogenous and exogenous cysteine proteinases and plays a vital physiological role in plants. Different classes of pesticides like fungicide (iprodione) of dicarboximide class and an insecticide (malathion) of class organophosphate are retorted for our study. In the presence of these pesticides, biophysical and biochemical changes were observed in phytocystatin. These changes were evaluated making use of caseinolytic activity assay, UV–vis spectroscopy, fluorescence spectroscopy, FTIR, and circular dichroism. Isothermal titration calorimetry was employed to see interaction pattern of these pesticides with phytocystatin. The results obtained clearly depict that the pesticides bind with the phytocystatin thereby changing its native conformation and reducing its intrinsic property of inhibition on cysteine proteinase as evident by reduced anti-papain inhibition in the presence of pesticides. Furthermore, CD and FTIR spectroscopy results clearly show a decrease in α-helical content upon interaction with malathion and iprodione. Among the two pesticides, iprodione has far more pronounced effect on YMP evident from striking changes in UV, Fluorescence, CD and FTIR spectroscopy. 2,4-dinitrophenylhydrazine spectrophotometric assay was also carried out to check production of ROS, generation of ROS was observed in the presence of these pesticides thus implying that ROS might be responsible for changes in native structure of phytocystatin induced by pesticides.

Oxadiargyl induced conformational transition of cystatin isolated from yellow mustard seeds: Biophysical and biochemical approach

Phytocystatins are thiol proteinase inhibitors crucial due to their inhibitory activity in plants. These play important roles in improving crop yield, protection against insects and pathogens and modulation of apoptosis. In this chemical era, various pesticides are being used globally to increase the crop biomass. These pesticides accumulate in plant body and produce harmful effects on plants itself by interacting with essential proteins. In this present study, we have monitored the interaction of a herbicide; oxadiargyl, with phytocystatin isolated from yellow mustard seeds (YMP) by employing spectroscopic techniques viz. UV, fluorescence, FTIR and CD spectroscopy and Isothermal titration calorimetry (ITC). UV and fluorescence spectroscopy shows YMP transformation from native to non-native form apparent by decreased absorbance and decreased fluorescence. FTIR and CD spectroscopy further confirmed secondary structural disruption of YMP. Anti-papain activity assay was also carried out; a reduction in activity was observed in presence of oxadiargyl. Thermodynamic parameters obtained from ITC shows affinity of oxadiargyl towards phytocystatin. Oxadiargyl was also found to induce ROS generation in YMP as evident by DNPH assay. Thus oxadiargyl binds to phytocystatin causing structural alterations reducing its physiological benefits and altering its functionality and ultimately leading to reduced crop yield.

Lysosomal Cell Death and Apoptosis Crosstalk - Synergistic Role in Bcl-2 Inhibitor (ABT-263) Mediated Cell Death in B-Cell Precursor Acute Lymphoblastic Leukemia

Although improved treatment of pediatric Acute Lymphoblastic Leukemia (ALL) has become increasingly successful with cure rates of up to 90%, leukemia reoccurrence is clearly associated with inferior prognosis. Treatment failure and resistance in leukemias are considered to involve defects in apoptosis signaling, which forms the basis for apoptogenic treatment approaches such as the use of Bcl-2 inhibitors. Identification of alternative cell death programs like lysosomal membrane permeabilization-induced cell death may provide a novel strategy for the treatment of ALL. Previous studies showed that lysosomal activity, as well as their traffic properties are greatly altered during tumorigenesis. Intralysosomal hydrolases such as cathepsin B, L and D have been implicated in cancer progression and high expression levels are generally associated with poor prognosis. Lysosomotropic agents modulating lysosomal integrity may overcome cell death resistance and might therefore also improve the outcome of high risk or ALL relapse patients.In this study, we investigated the efficacy of the lysosomotropic compound B10, a derivative of Betulinic acid, as well as its combination with the Bcl-2 inhibitor ABT-263 in B-cell precursor- (BCP-) ALL.In BCP-ALL cell lines, B10 showed induction of cell death combined with increased DNA fragmentation. Interestingly, additional treatment with the pan-caspase inhibitor zVAD.fmk only partially rescued B10 triggered loss of cell viability, indicating that B10 is not exclusively inducing caspase-dependent apoptosis, but also induces an additional alternative cell death program. B10 permeabilized lysosomes as indicated by the significant decrease of LysotrackerRED positive populations detected by flow cytometry, and treatment with E-64d, a potent inhibitor of thiol proteases and cathepsins, reduced B10-induced cell death, thus emphasizing the cathepsin-dependent effect of B10. To exclude that B10-induced lysosomal permeabilization might represent a secondary effect, a panel of different substances was tested for their activity on lysosomal membrane integrity. While treatment with dexamethasone, chloroquine, vincristine and asparaginase also showed lysosomal permeabilization, co-treatment with E-64d did not reduce cell death indicating a secondary effect of these compounds on lysosomes. In addition to cell lines, we tested the effect of B10 on 15 primary leukemia samples isolated from ALL bearing mice of established patient-derived NOD/SCID/hu BCP-ALL xenografts. In a majority of these individual patient-derived leukemias, B10 induced cell death that could be inhibited by E-64d, thus demonstrating a lysosomal protease-dependent death program also in primary ALL.In addition to its classical role in triggering the intrinsic mitochondria dependent apoptosis pathway, the pro-apoptotic Bcl-2 family member Bax was previously described to be involved in the regulation of lysosomal membrane integrity, pointing to a possible synergistic effect of B10 and BH3-mimetics. Intriguingly, in cell fractionation assays we observed increased Bax recruitment to lysosomal membranes when B10 is present. Interestingly, co-treatment with the Bcl-2 inhibitor ABT-263, which displaces Bcl-2 from its inhibitory binding to Bax, led to increased lysosomal permeabilization, loss of mitochondrial membrane potential, and caspase activation indicating involvement of the intrinsic apoptosis pathway. The importance of lysosomal Bax recruitment for the activity of ABT-263 and its concomitant effect on lysosomal membrane permeabilization was further supported by Bax knockdown experiments, since induction of lysosomal disruption, release of cathepsins and their subsequent effect on cell death activation by B10 was reduced.Taken together, our findings suggest an important role of lysosomal membrane permeabilization-induced cell death for the activity of Bcl-2 inhibitors such as ABT-263. The combination of BH3 mimetics with lysosomotropic compounds may provide the basis for novel molecular directed treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Structural transition of kidney cystatin induced by silicon dioxide nanoparticles: An implication for renal diseases

Nanotechnology is one of the fastest growing fields of science owing to use of nanomaterials in industries and medicine across the globe. Currently silicon dioxide nanoparticles (SiO2 NPs) are one of the most popular nanomaterials owing to their inert toxicity profile and hence exposure to SiO2 nanoparticles is on the increase. Cystatins are thiol proteinase inhibitors (TPIs) ubiquitously distributed in plants and animals and they are now at the heed of a number of normal and pathological conditions and shouldn’t be regarded solely as TPIs. Up till now many studies have targeted the potential toxicity of NPs on pulmonary target; although little focus is given to kidney which is a secondary target organ. The objective of this work is to study the structural changes in buffalo kidney cystatin (BKC) induced by SiO2 NPs. UV and Fluorescence spectroscopy shows BKC transformation from native to non-native form evident by decreased absorbance and increased fluorescence. FTIR and CD spectroscopy further confirmed secondary structure disruption of BKC. Isothermal titration calorimetry (ITC) and microscopy were resorted to visualize interaction between SiO2 NPs and BKC. Comet assay and MTT assay were utilized to perceive the toxicity of SiO2 NPs incubated BKC; decreased cell viability clearly suggesting toxicity of SiO2 NPs incubated BKC. Our work suggests that SiO2 NPs have a deteriorating effect on BKC thereby causing a decrease in its ability to inhibit papain and hence less functionality. This study also shows that BKC transforms to a toxic non-native form in presence of SiO2 NPs.

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.

Physico-chemical and in-silico analysis of a phytocystatin purified from Brassica juncea cultivar RoAgro 5444.

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% β-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

Structural transition of kidney cystatin in dimethylnitrosamine-induced renal cancer in rats: identification as a novel biomarker for kidney cancer and prognosis

In our study, renal cancer is induced in rats making use of dimethylnitrosamine (DMN). G1 – Group 1 were control rats and G2 – Group 2 rats were given a single intra-peritoneal injection of DMN of 50 mg/kg body weight resulting in 100% incidences of renal tumors after 12 months. SEM and histopathology confirmed the presence of renal cancer in the DMN-treated rats. Making use of ammonium sulfate precipitation and gel filtration chromatography on Sephacryl S-100HR column, a thiol protease inhibitor was isolated from kidney of control rats known as Rat kidney Cystatin (RKC) as well as from kidney of cancerous rat called as Cancerous Rat Kidney Cystatin (CRKC). Both these inhibitors were characterized, and interestingly, it was found that CRKC showed greater anti-papain activity and also it was stable in a broad pH and temperature range thus implying that CRKC is more stable as compared to RKC. UV and fluorescence spectroscopy point out in structural difference between RKC and CRKC which was further confirmed by Circular dichroism (CD) and FTIR spectroscopy. Our study clearly showed that kidney cystatin is structurally modified in the case of renal cancer and performs its role in a more efficacious manner.

Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders

In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10−4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and −20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.

Purification and Biochemical Characterization of a Cystatin-Like Thiol Proteinase Inhibitor from Cicer arietinum (Chickpea)

Cystatins are thiol proteinase inhibitors ubiquitously present in many plants as well as animal tissues. In the present study, a cystatin like a thiol proteinase inhibitor was isolated from chickpea using ammonium sulfate fractionation (40-60%) and gel filtration chromatography on a Sephacryl S-100 HR column with 21.09% yield and fold purification of 78.89. It gave a molecular mass of about 25.3 kDa as determined by SDS-PAGE, Dynamic Light Scattering (DLS), and from its gel filtration behavior. The inhibitor was found to have very low carbohydrate content (0.34%) and meager amount (1.45%) of sulfhydryl content. The Stokes radius, frictional, diffusion and sedimentation coefficients of CPC were 4.299 × 10-8 g/s, 22.82 A, 12.68 × 10-7 cm2 s-1 and 2.64 S, respectively. Kinetic analysis of CPC with thiol proteinases (papain, ficin, bromelain) revealed the reversible and competitive mode of inhibition with CPC showing the highest inhibitory efficiency against papain (Ki=0.82 nM) followed by ficin (Ki=17.6 nM) and then bromelain (Ki=41.3 nM). CPC possessed about 25.34% α-helical content as assessed by CD spectroscopy. UV-Visible and fluorescence spectra indicated that upon formation of papain-CPC complex there is a significant conformational change in one or both the proteins of the complex.