A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Thermal inactivation of alpha-galactosidase from Penicillium canescens

The kinetics and mechanism of thermal inactivation of Penicillium canescens alpha-galactosidase in the temperature range of 55-65 degrees C have been studied. The kinetic scheme of alpha-galactosidase thermal inactivation was proposed which included the reversible dissociation of active hexamers into associating monomers and irreversible denaturation of monomers. The kinetic constants of thermal inactivation have been determined. The effect of enzyme concentration and purification efficiency has been investigated. A possibility of defence of protein molecule from thermal inactivation in the presence of BSA, glycerol, melibiose, raffinose and stachyose is shown.     

A protein aggregation based test for screening of the agents affecting thermostability of proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K _agg(T−T ₀)², where K _agg is the parameter characterizing the initial rate of aggregation and T ₀ is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T ₀ corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets.     

Influence of metal ions and specific chemical reagents on activity of alpha-L-rhamnosidase of Eupenicillium erubescens

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide methiodide, EDTA, o-phenanthroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the activity of alpha-L-rhamnosidase of Eupenicillium erubescens has been investigated. The essential role of Ag+ and Hg2+ which inhibit the alpha-L-rhamnosidase activity by 47-73% has been shown. Whereas L-cysteine exhibits the protective effect, rhamnose in concentration of 1-5 mM does not protect the enzyme from negative effect of Ag+ and Hg2+. Basing on the inhibitory and kinetic analysis it was supposed that the carboxyl group of C-terminal aminoacid and imidazole group of histidine take part in the catalytic action of alpha-L-rhamnosidase. It was assumed that sulphydryl groups took part in catalysis, carried out by alpha-L-rhamnosidase of E. erubescens, since the activity of alpha-L-rhamnosidase inhibited by p-ChMB and thiol reagents such as dithiothreitol, L-cysteine, beta-mercaptoethanol did not remove its inhibitory action.     

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (R_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (R_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.     

Aspergillus niger alpha-N-acetylgalactosaminidase: isolation, purification and properties

alpha-N-acetylgalactosaminidase has been isolated from liquid culture of micromycete Aspergillus niger and purified 600 times by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels with specific activity 10.5 U/mg of protein. The preparation was homogenic: its molecular mass by the data of gel-filtration on Sepharose 6B was 430 kDa, on PAAGE in the system of DDSNa--70 kDa. That gives every reason to suppose oligomeric structure of the enzyme molecule. The carbohydrate component, including mannose, galactose, glucosamine and two nonidentified hexosamines was observed in alpha-N-acetylgalactosaminidase. Thermo- and pH- optima were 60 degrees C and pH 3.5, respectively. The enzyme was thermo- and pH-stable, resistant in storage. The enzyme was found to exhibit strict specificity in respect ofglycon. It was shown that enzyme was competitively inhibited by substrate and reaction product. Km and Vmax with respect to nitrophenyl substrate were 1.25 mM and 10.5 mkmole/min/mg of protein. The activity of glycosidase tested was independent of the presence of metal ions. The presence of carboxylic group of C-terminal aminoacid and imidazol group of hystidine in active centre of molecule was established. A number of natural and synthetic substrates were able to activate (50-200%) production of A. niger alpha-N-acetylgalactosaminidase.     

Coordinative compounds of zinc with N-substituted thiocarbamoyl-N'-pentamethylensulfenamides--activity modifiers of enzymes of proteolytic and glycolytic action

The influence of a number of coordinative compounds of zinc with N-substituted thiocarbamoil-N'-pentamethylensulfenamides on activity of elastase, alpha-L-rhamnosidase and alpha-galactosidases evidence for a possibility of their usage as stimulators or inhibitors of enzymes tested have been studied. It was shown that all the compounds in concentration of 0.1 and 0.01% inhibited by 90-100% Bacillus thuringiensis 27-88Els+ elastase activity. [Zn(L2)Br2], [Zn(L1)(NCS)2] and [Zn(L3)(NCS)2] at 20 h exposition activated Cryptococcus albidus 1001 alpha-L-rhamnosidase activity. The rest of compounds influenced it on the control level or inhibited it by 7-23%. The obtained results testify that essential role is not played by separate fragments (L-ligand and anions), but by molecules of zinc complexes as a whole. All the studied complexes, exept for [Zn(L3)(NCS)2], induced alpha-L-rhamnosidase activity of Eupenicillium erubescens 248 (7 to 60%). All zinc compounds (concentration 0.01%, exposition time - 60 min) influenced at the control level Aspergillus niger and Cladosporium cladosporioides alpha-galactosidases activity, however inhibited (up to 20%) activity of Penicillium canescens alpha-galactosidase. The increasing of exposition time of the compounds tested with enzymes up to 20 h testify to selective action of separate compounds on enzymes tested. The data obtained prove, that the character of interaction of zinc complexes is changed depending on the enzyme tested and its strain-producer.     

AmylPepPred: Amyloidogenic Peptide Prediction tool

We present an efficient computational architecture designed using supervised machine learning model to predict amyloid fibril forming protein segments, named AmylPepPred. The proposed prediction model is based on bio-physio-chemical properties of primary sequences and auto-correlation function of their amino acid indices. AmylPepPred provides a user friendly web interface for the researchers to easily observe the fibril forming and non-fibril forming hexmers in a given protein sequence. We expect that this stratagem will be highly encouraging in discovering fibril forming regions in proteins thereby benefit in finding therapeutic agents that specifically aim these sequences for the inhibition and cure of amyloid illnesses.

Availability

AmylPepPred is available freely for academic use at www.zoommicro.in/amylpeppred