Functional regulation of PVBV Nuclear Inclusion protein-a protease activity upon interaction with Viral Protein genome-linked and phosphorylation

Regulation of NIa-Pro is crucial for polyprotein processing and hence, for successful infection of potyviruses. We have examined two novel mechanisms that could regulate NIa-Pro activity. Firstly, the influence of VPg domain on the proteolytic activity of NIa-Pro was investigated. It was shown that the turnover number of the protease increases when these two domains interact (as: two-fold; trans: seven-fold) with each other. Secondly, the protease activity of NIa-Pro could also be modulated by phosphorylation at Ser129. A mutation of this residue either to aspartate (phosphorylation-mimic) or alanine (phosphorylation-deficient) drastically reduces the protease activity. Based on these observations and molecular modeling studies, we propose that interaction with VPg as well as phosphorylation of Ser129 could relay a signal through Trp143 present at the protein surface to the active site pocket by subtle conformational changes, thus modulating protease activity of NIa-Pro. (C) 2011 Elsevier Inc. All rights reserved.

Studies on non-specific interference due to serum in the avidin-biotin microELISA for monkey chorionic gonadotropin and a method for its elimination

A study has been carried out on the non-specific interference due to serum in the avidin biotin micro-ELISA for monkey chorionic gonadotropin. Results suggest that it is not due to any proteolytic activity in the serum, but immunoglobulin or associated factors interfering at the level of antigen-antibody interaction. This interference was eliminated by heating samples at 60°C for 30 min.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Trypanosoma evansi is a causative agent of `surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16

We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsinf-old and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.

Varied Immune Response to FVIII: Presence of Proteolytic Antibodies Directed to Factor VIII in Different Human Pathologies

The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation of the complement and its activation, and activation of effector cells. In addition to this plethora of functions, antibodies are capable of expressing enzymatic activity. Antibodies with catalytic function are a result of the productive interplay between the highly evolved machinery of the immune system and the chemical framework used to induce them (antigens). Catalytic antibodies are immunoglobulins with an ability to catalyze the reactions involving the antigen for which they are specific. Catalytic immunoglobulins of the IgM and IgG isotypes have been detected in the serum of healthy donors. In addition, catalytic immunoglobulins of the IgA isotype have been detected in the milk of healthy mothers. Conversely, antigen-specific hydrolytic antibodies have been reported in a number of inflammatory, autoimmune, and neoplastic disorders. The pathophysiological occurrence and relevance of catalytic antibodies remains a debated issue. Through the description of the hydrolysis of coagulation factor VIII as model target antigen, we propose that catalytic antibodies directed to the coagulation factor VIII may play a beneficial or a deleterious role depending on the immuno-inflammatory condition under which they occur.

Isolation and characterization of proteolytic enzymes from the latex of Synadenium grantii Hook, 'f'

Two fractions showing proteolytic enzymes have been obtained from the latex of Synadenium grantii Hook, 'f', using gel-filtration and anion-exchange chromatographic techniques. Both these proteases have the same molecular mass of 76+/-2 kDa each. They exhibit maximal activity at pH 7.0 and at a temperature of 60 degreesC. They display stability over a pH range from 5-10 and are also highly thermostable. Irreversible inhibition by PMSF indicates that they are serine proteases. In addition, histidine residues also appear to play an important role in catalysis as evidenced by inhibition with DEPC. They also exhibit similarity with respect to pH and temperature optima, kinetic properties and thermal stability. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015

Conformation-activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating 1-aminocycloalkane-1-carboxylic acid (Ac(n)c) and alpha,alpha-dialkylglycines (Deg, diethylglycine; Dpg, n,n-dipropylglycine and Dbg, n,n-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx = Ac(7)c, I; Ac(8)c, II; Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded beta-turn conformations in CDCl3 and (CD3)(2)SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C-5) conformations in solution. Peptides I-V exhibit high activity in inducing beta-glucosaminidase release from rabbit neutrophils, with ED(50) values ranging from 1.4-8.0 x 10(-11)M. In human neutrophils the Dxg peptides III-V have ED(50) values ranging from 2.3 x 10(-8) to 5.9 x 10(-10) M, with the activity order being V > IV > III. While peptides I-IV are less active than the parent. For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors.

Spontaneous generation of optical activity in urea inclusion compounds vis-a-vis current theories

A novel test of recent theories of the origin of optical activity has been designed based on the inclusion of certain alkyl 2-methylhexanoates into urea channels.

Synthesis, crystal structure and DNA cleavage activity of (aqua)bis(dipyridophenazine)copper(II) complex

A copper(II) complex of dipyridophenazine, viz., [Cu(dppz)(2)(H2O)](ClO4)(2) (I), has been prepared and structurally characterized by X-ray crystallography. The crystal structure of the complex shows a five-coordinate structure in which two N,N-donor dipyridophenazine (dppz) and one aqua ligand bind to the copper(II) center giving Cu-O and Cu-N bond distances in the range of 1.981(6) to 2.043(6) angstrom. The ESI-MS spectrum of 1 in MeCN shows a peak at m/z value of 313 (100%) indicating the dissociation of the aqua ligand in the solution phase. The complex is one-electron paramagnetic (mu(eff), 1.86 mu(B)). It displays a quasi-reversible Cu(II)/Cu(I) redox process at 0.096 V. The complex is an avid binder to CT DNA giving a binding constant value of 3.5 x 10(5) M-1. It shows significant hydrolytic cleavage of supercoiled pUC19 DNA in dark ill the absence of any external agents. The complex exhibits chemical nuclease activity oil treatment with 3-mercaptopropionic acid as a reducing agent forming hydroxyl radicals. Complex 1 is a model synthetic nuclease and hydrolase showing both modes of DNA cleavage under different reaction conditions. The DNA cleavage activity of 1 is significantly better than its phen analogue but similar to that of the bis-dpq complex.

Identification of amino groups in the carbohydrate binding activity of winged bean acidic agglutinin

Chemical modification studies reveal that the modification of amino groups in WBA II leads to a complete loss in the hemagglutinating and saccharide binding activities. Since WBA II is a dimeric molecule and contains two binding sites, one amino group in each of the binding sites is inferred to be essential for its activity. The presence of amino group which has a potential to form hydrogen bonded interactions with the ligand, substantiates our observation regarding the forces involved in WBA II-receptor and WBA II-simple sugar interactions.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Conjugated Ternary Copper(II) Complexes

Ferrocene-conjugated ternary copper(II) complexes [Cu(L)(B)](ClO4)(2), where L is FcCH(2)N(CH2Py)(2) (Fc = (eta(5)-C5H4)Fe-II(eta(5)-C5H5)) and B is a phenanthroline base, viz., 2,2'-bipyridine (bpy, 1), 1, 10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4), have been synthesized and characterized by various spectroscopic and analytical techniques. The bpy complex 1, as its hexafluorophosphate salt, has been structurally characterized by X-ray crystallography. The molecular structure shows the copper(II) center having an essentially square-pyramidal coordination geometry in which L with a pendant ferrocenyl (Fc) moiety and bpy show respective tridentate and bidentate modes of binding to the metal center. The complexes are redox active, showing a reversible cyclic voltammetric response of the Fc(+)-Fc couple near 0.5 V vs SCE and a quasi-reversible Cu(II)-Cu(I) couple near 0.0 V. Complexes 2-4 show binding affinity to calf thymus (CT) DNA, giving binding constant (K-b) values in the range of 4.2 x 10(4) to 2.5 x 10(5) M-1. Thermal denaturation and viscometric titration data suggest groove binding and/or a partial intercalative mode of binding of the complexes to CT DNA. The complexes show good binding propensity to the bovine serum albumin (BSA) protein, giving K-BSA values of similar to 10(4) M-1 for the bpy and phen complexes and similar to 10(5) M-1 for the dpq and dppz complexes. Complexes 2-4 exhibit efficient chemical nuclease activity in the presence of 3-mercapto-propionic acid (MPA) as a reducing agent or hydrogen peroxide (H2O2) as an oxidizing agent. Mechanistic studies reveal formation of hydroxyl radicals as the reactive species. The dpq and dppz complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to visible light of different wavelengths including red light using an argon-krypton mixed gas ion laser. Mechanistic investigations using various inhibitors reveal the fort-nation of hydroxyl radicals in the DNA photocleavage reactions. The dppz complex 4, which shows efficient photoioduced BSA cleavage activity, is a potent multifunctional model nuclease and protease in the chemistry of photodynamic therapy (PDT) of cancer.

Synthesis, crystal structures, DNA binding and cleavage activity of L-glutamine copper(II) complexes of heterocyclic bases

Ternary L-glutamine (L-gln) copper(II) complexes [Cu(L-gln)(B)(H2O)](X) (B = 2,2'-bipyridine (bpy), X = 0.5SO(4)(2-), 1; B = 1,10-phenanthroline (phen), X = ClO4-, 2) and [Cu(L-gln)(dpq)(ClO4)] (3) (dpq, dipyridoquinoxaline) are prepared and characterized by physicochemical methods. The DNA binding and cleavage activity of the complexes have been studied. Complexes 1-3 are structurally characterized by X-ray crystallography. The complexes show distorted square pyramidal (4+1) CuN3O2 coordination geometry in which the N,O-donor amino acid and the N, N-donor heterocyclic base bind at the basal plane with a H2O or perchlorate as the axial ligand. The crystal structures of the complexes exhibit chemically significant hydrogen bonding interactions besides showing coordination polymer formation. The complexes display a d-d electronic band in the range of 610-630 nm in aqueous-dimethylformamide (DMF) solution (9:1 v/v). The quasireversible cyclic voltammetric response observed near -0.1 V versus SCE in DMF-TBAP is assignable to the Cu(II)/Cu(I) couple. The binding affinity of the complexes to calf thymus (CT) DNA follows the order: 3 (dpq) > 2 (phen) >> 1 (bpy). Complexes 2 and 3 show DNA cleavage activity in dark in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent via a mechanistic pathway forming hydroxyl radical as the reactive species. The dpq complex 3 shows efficient photoinduced DNA cleavage activity on irradiation with a monochromatic UV light of 365 nm in absence of any external reagent. The cleavage efficiency of the DNA minor groove binding complexes follows the order:3 > 2 >> 1. The dpq complex exhibits photocleavage of DNA on irradiation with visible light of 647.1 nm. Mechanistic data on the photo-induced DNA cleavage reactions reveal the involvement of singlet oxygen (O-1(2)) as the reactive species in a type-II pathway. (C) 2008 Elsevier B.V. All rights reserved.

Guanylyl Cyclase C Receptor: Regulation of Catalytic Activity by ATP

Guanylyl cyclase C (GCC), a member of the family of membrane bound guanylyl cyclases is the receptor for the heat-stable enterotoxin (ST) peptides and the guanylin family of endogenous peptides. GCC is activated upon ligand binding to increase intracellular cGMP levels, which in turn activates other downstream signalling events in the cell. GCC is also activated in vitro by nonionic detergents. We have used the T84 cell line as a model system to investigate the regulation of GCC activity by ATP. Ligand-stimulated GCC activity is potentiated in the presence of ATP, whereas detergent-stimulated activity is inhibited. The potentiation of GCC activity by ATP is dependent on the presence of Mg2+ ions, and is probably brought about by a direct binding of Mg-ATP to GCC. The protein kinase-like domain of GCC, which has earlier been shown to play a critical role in the regulation of GCC activity, may be a possible site for the binding of Mg-ATP to GCC.