Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.     

Kinetics and mechanism of oxidation of some aldoses by vanadium(v) in perchloric acid medium

The kinetics of oxidation of some aldoses by vanadium(V) in perchloric acid media have been investigated. Each reaction is first order with respect to both [Vanadium(V)] and [Aldose]. The reactions are catalysed by acid. The addition of sodium perchlorate accelerates the rate of reaction. Kinetic evidence for the formation of an intermediate compound between vanadium(V) and aldoses is insignificant, and a mechanism is suggested in which vanadium(V) reacts with the aldoses by a fast step to form a transition state, followed by the decomposition of the latter to give the products of reaction in a slow step. The formation of free-radical intermediates has been demonstrated, and one-electron reduction of vanadium(V) by aldoses seems to be the most plausible mechanism. The oxidation rates follow the order: xyloses arabinose galactose mannose. The activation parameters are reported.     

Molecular docking analysis of beta-catenin with compounds derived from Lycopersicon esculentum

Beta-catenin is linked with colorectal cancer (CRC). Therefore, it is of interest to design and develop novel compounds to combat CRC. Hence, we document compounds (chlorogenic acid, gallic acid, protocatechuic acid, quercetin and vanillic acid) from Lycopersicon esculentum with optimal binding features for further consideration.     

MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.     

Purification of muscle enzymes by pseudoaffinity chromatography

A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.     

Stability and dynamics of domain-swapped bovine-seminal ribonuclease

The proteins of the ribonuclease-A (RNase-A) family are monomeric, with the exception of bovine-seminal ribonuclease (BS-RNase). BS-RNase is formed by swapping the N-terminal helices across the two monomeric units. A molecular-dynamics (MD) study has been performed on the protein for a simulation time of 5.5 ns to understand the factors responsible for the stability of the dimer. Essential dynamics analysis and motional correlation of the protein atoms yielded the picture of a stabilising, yet flexible, interface. We have investigated the role of intermolecular H-bonding, protein/water interaction, and protein/water networks in stabilising the dimer. The networks of interchain H-bonds involving side-chain/side-chain or side-chain/main-chain (ScHB) interactions between the two chains have also been studied. The ability of protein atoms in retaining particular H2O molecules was investigated as a function of the accessible surface area (ASA), depth, and hydration parameters, as well as their participation in protein/water networks.     

Chitinase-mediated inhibitory activity of Brassica transgenic on growth of Alternaria brassicae

Chitinase, capable of degrading the cell walls of invading phytopathogenic fungi, plays an important role in plant defense response, particularly when this enzyme is overexpressed through genetic engineering. In the present study, Brassica plant (Brassica juncea L.) was transformed with chitinase gene tagged with an overexpressing promoter 35 S CaMV. The putative transgenics were assayed for their inhibitory activity against Alternaria brassicae, the inducer of Alternaria leaf spot of Brassica both in vitro and under polyhouse conditions. In in vitro fungal growth inhibition assays, chitinase inhibited the fungal colony size by 12-56% over the non-trangenic control. The bioassay under artificial epiphytotic conditions revealed the delay in the onset of disease as well as reduced lesion number and size in 35S-chitinase Brassica as compared to the untransformed control plants.     

Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors

A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.