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 (K_i = 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.     

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

Abstract

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     

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.     

Paclobutrazol affects growth of almond fruits and germination of almond seeds

Paclobutrazol (PBZ) applied to almond fruits 25 days after full bloom delayed the growth of fruits and seeds. The period of the delay and the amount of retardation depended on the paclobutrazol concentration applied.Seeds from the treated fruits germinated well, except those treated twice with 4000 mg L^–1 which showed only a low percentage of germination. Seeds treated just before sowing failed to germinate.Abbreviations PBZ Paclobutrazol     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparative phylogenetic analysis of cystatin gene families from arabidopsis,rice and barley

The plant cystatins or phytocystatins comprise a family of specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes and in defence against pests and pathogens. Extensive searches in the complete rice and Arabidopsis genomes and in barley EST collections have allowed us to predict the presence of twelve different cystatin genes in rice, seven in Arabidopsis, and at least seven in barley. Structural comparisons based on alignments of all the protein sequences using the CLUSTALW program and searches for conserved motifs using the MEME program have revealed broad conservation of the main motifs characteristic of the plant cystatins. Phylogenetic analyses based on their deduced amino acid sequences have allowed us to identify groups of orthologous cystatins, and to establish homologies and define examples of gene duplications mainly among the rice and barley cystatin genes. Moreover, the absence of a counterpart between the two monocots, as well as strong variations in the motifs that interact with the cysteine proteinases, may be related to a species-specific evolutionary process. This cystatin classification should facilitate the assignment of proteinase specificities and functions to other cystatins as new information is obtained.Electronic Supplementary Material Supplementary material is available for this article at     

Transgalactosylation activity of sweet almond α-galactosidase: Synthesis of saccharides

Sweet almond α-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) catalyses hydrolytic, synthetic (de novo) and transfer reactions. Transfer products were formed using p-nitrophenyl α-d-galactoside as the galactosyl donor and glucose, galactose, sucrose, maltose and lactose as acceptors; several of the products were identified. The enzyme also caused elongation of the oligosaccharide chain of two substrates (melibiose and raffinose). In addition, the enzyme catalysed condensation of free galactose, yielding oligosaccharides. The products were identified in all cases by chromatography.     

巴旦杏授粉试验及花粉管生长的荧光显微观察

对巴旦杏4个主栽品种的授粉试验结果表明：自花授粉试验中“鹰嘴”、“双果”坐果率分别为19．74％和4．29％,“纸皮”和“麻壳”坐果率为零;自然授粉状态下4个主栽品种坐果率在9．37％－20．13％之间;人工异花授粉试验中4个品种9个授粉组合坐果率绝大多数都在40％以上,其中混合花粉授粉试验结果最好;人工养蜂条件下的坐果率明显高于无蜂自然授粉的坐果率,昆虫传粉可达到人工混合花粉授粉的效果。授粉试验还表现出巴旦杏雌蕊柱头的有效授粉时间为开花后花期的前4d,授粉越早,越有利于坐果。荧光显微观察表明,巴旦杏花粉管最早约于授粉第7天通过花柱基部到达子房室,第8天前后到达胚珠。     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.     

Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.     

Development and transportability across Prunus species of 42 polymorphic almond microsatellites

We report 47 new simple sequence repeats (SSRs) obtained from a CT/AG enriched genomic library of almond cv. Texas (syn. Mission). Forty‐two of them were polymorphic in a sample of eight almond cultivars and 31 of these were single‐locus. The average values of the number of alleles per locus (6.6), and mean observed (65%) and expected (76%) heterozygosities for these 31 SSRs indicated a high level of variability. All cultivars studied could be individually identified using any one of the five SSRs. Transportability to other Prunus species (peach, sweet cherry, Japanese plum and apricot) was also high (83–100%).     

Plastocyanin: Structural and functional analysis

Plastocyanin is one of the best characterized of the photosynthetic electron transfer proteins. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 Å resolution. Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between theChlamydomonas and poplar proteins), the three-dimensional structures are remarkably conserved (e.g., 0.76 Å rms deviation in the C positions between theChlamydomonas and poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel -barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochromef and Photosystem I, and validated the model of two functionally significant electron transfer paths in plastocyanin. One putative electron transfer path is relatively short (4 Å) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (12–15 Å) and involves the nearly conserved residue Tyr-83 in the negative patch.     

Effect of curcumin on the nitric oxide induced structural and functional modifications of high molecular mass goat brain cystatin

Cystatins are thiol proteinase inhibitors ubiquitously present in the mammalian body. In brain, they prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Under physiological conditions nitric oxide can be found in almost all the tissues, but under pathological conditions NO has damaging effects. Its increased concentration, under various neural diseases leads to cell damage through formation of highly reactive peroxynitrite. Our present study was designed to investigate the protective effect of curcumin against NO induced damage of HM-GBC. NO caused intensive structural and functional damage of HM-GBC, resulting in 89% loss of its antiproteolytic activity after 2 h of incubation. Structural damage occurs in the form of protein degradation. Curcumin significantly protected HM-GBC against this damage. This suggests that curcumin has a significant potential in the treatment of diseases caused by nitrogen free radicals and this potential must be further explored for the development of novel drugs. This text was submitted by the authors in English.     

Structural and functional diversity within the cystatin gene family of Hordeum vulgare

Phytocystatins are inhibitors of cysteine proteinases from plants putatively involved in defence and as endogenous regulators of protein turnover. Seven genes encoding cystatins (HvCPI-1 to HvCPI-7), identified from EST collections and from an endosperm cDNA library, have been characterized. The intron-exon structure of their corresponding ORFs has been determined and the predicted three-dimensional models for the seven barley cystatins have been established, based on the known crystal structure of oryzacystatin I from rice. Only one out of the seven deduced proteins, HvCPI-7, had sequence variations affecting the three conserved motifs implicated in the enzyme-inhibitor interaction. In three cases, HvCPI-5, HvCPI-6, and HvCPI-7, amino acid differences lead to the prediction of important structural changes in their three-dimensional structures. Northern blot analysis indicated that the seven genes have different expression patterns in barley tissues. The recombinant proteins expressed in Escherichia coli showed distinct inhibitory properties in vitro, with different K(i) values, against the three cysteine proteinases tested: papain, cathepsin B, and cathepsin H. Moreover, these recombinant proteins presented differential fungicidal characteristics inhibiting the growth of phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum in vitro. The resulting implications for the structural and functional diversity of the seven barley cystatins studied are discussed.     

Interaction of terbium and calcium with chicken cystatin

The emission intensity of the fluorescent lanthanide, terbium, is shown to be enhanced upon binding to chicken cystatin. Fluorescence titrations indicate the presence of a single high affinity binding site per molecule. Binding of the terbium results in a 29% quenching of the fluorescence of the single tryptophan residue in the molecule. Calcium displaces the terbium from cystatin as judged by the decrease of terbium fluorescence in competition titrations. Similar titrations with magnesium or strontium demonstrate that the metal binding site of cystatin exhibits specificity for calcium or terbium. Analysis of the N-terminal sequence of chicken cystatin suggests the presence of a putative consensus sequence for a metal binding site between residues 13 and 24. Calcium causes a 17% decrease in the tryptophan fluorescence of cystatin, indicating that an induced conformational change accompanies metal binding. The increased quenching observed with terbium appears to be the result of resonance energy transfer from tryptophan to terbium. From the critical distance for energy transfer from tryptophan to terbium, it is estimated that the terbium binding site lies approximately 12 A from the single tryptophan residue in the molecule.     

Paclobutrazol affects the extension growth and the levels of endogenous IAA of almond seedlings

The effect of paclobutrazol on plant height and the concentration of free indole-3-acetic acid (IAA) in almond seedlings grown in 0.5 L pots with moist perlite and subjected to 15°C and photoperiod of 16 hours was studied.In the first two experiments the paclobutrazol was applied as a drench at 0, 0.5, 1.5 and 5 mg/pot. Seven and sixteen days respectively after application a gradual decrease in the growth rate and concentration of free IAA in the tops of the growing shoots of almond seedlings on which paclobutrazol was applied, was observed. Furthermore, a gradual increase in the concentration of free IAA in the four younger leaves at the time of application was also detected.In the third experiment the effect of paclobutrazol (5 mg/pot) on plant height and the concentration of free IAA measured over 10 day period was studied. From the 4th day following application differences in the height of seedlings and in the concentration of free IAA in the growing tops between the control and treated seedlings were observed.Abbreviations IAA Indoleacetic acid - GA₃ Gibberellic acid - CCC 2 Chloroethanephosphonic acid - B-995 N-dimethyl aminosuccinamic acid     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

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 × 10⁶ 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.Abbreviations: LC, liver cystatin; ADR, adriamycin; CD, circular dichroism; Ka, binding constant; HBV, human hepatitis B virus     

Risk management decisions for pesticides and threatened and endangered species: The role of uncertainty analysis

Despite data gaps and information shortfalls, government agencies in the United States are expected to produce timely and defensible decisions to regulate pesticide use under the Federal Insecticide, Fungicide, and Rodenticide Act and in compliance with the Endangered Species Act. The decision to register a pesticide is predicated on a conclusion that no unreasonable effects will accrue to the environment, including threatened and endangered species. We recognize that the definition of acceptable risk is a policy judgment stemming from legislative language and judicial interpretation. However, a common risk assessment approach with similar technical underpinnings and a high degree of transparency used by all the agencies would be cost effective and more likely to achieve consensus among interested parties. Quantitative probabilistic risk assessment (PRA) methods can be used to develop risk estimates and to describe the level of confidence in these estimates. PRA methods can also differentiate among the contributions of natural stochasticity, measurement variability, and lack of knowledge. Because this approach enhances transparency and increases understanding of the implications of limited data sets and associated assumptions, we encourage the appropriate agencies to implement PRA methods as a means of reaching common ground when assessing risks of pesticides to listed species.