A multi-source molecular network representation model for protein-protein interactions prediction.

The prediction of potential protein-protein interactions (PPIs) is a critical step in decoding diseases and understanding cellular mechanisms. Traditional biological experiments have identified plenty of potential PPIs in recent years, but this problem is still far from being solved. Hence, there is urgent to develop computational models with good performance and high efficiency to predict potential PPIs. In this study, we propose a multi-source molecular network representation learning model (called MultiPPIs) to predict potential protein-protein interactions. Specifically, we first extract the protein sequence features according to the physicochemical properties of amino acids by utilizing the auto covariance method. Second, a multi-source association network is constructed by integrating the known associations among miRNAs, proteins, lncRNAs, drugs, and diseases. The graph representation learning method, DeepWalk, is adopted to extract the multisource association information of proteins with other biomolecules. In this way, the known protein-protein interaction pairs can be represented as a concatenation of the protein sequence and the multi-source association features of proteins. Finally, the Random Forest classifier and corresponding optimal parameters are used for training and prediction. In the results, MultiPPIs obtains an average 86.03% prediction accuracy with 82.69% sensitivity at the AUC of 93.03% under five-fold cross-validation. The experimental results indicate that MultiPPIs has a good prediction performance and provides valuable insights into the field of potential protein-protein interactions prediction. MultiPPIs is free available at https://github.com/jiboyalab/multiPPIs .

Cell surface expression of nucleolin mediates the antiangiogenic and antitumor activities of kallistatin.

Kallistatin is a unique serine proteinase inhibitor and heparin-binding protein. A previous study conducted by our group indicated that kallistatin has antiangiogenic and antitumoral activities. In the present study, we report that kallistatin specifically binds to membrane surface-expressed nucleolin with high affinity. Antibody-mediated neutralization or siRNA-induced nucleolin knockdown results in loss of kallistatin suppression of endothelial cell proliferation and migration in vitro and tumor angiogenesis and growth in vivo. In addition, we show that kallistatin is internalized and transported into cell nuclei of endothelial cells via nucleolin. Within the nucleus, kallistatin inhibits the phosphorylation of nucleolin, which is a critical step required for cell proliferation. Thus, we demonstrate that nucleolin is a novel functional receptor of kallistatin that mediates its antiangiogenic and antitumor activities. These findings provide mechanistic insights into the inhibitory effects of kallistatin on endothelial cell growth, tumor cell proliferation, and tumor-related angiogenesis.

The GH18 family of chitinases: their domain architectures, functions and evolutions.

The glycoside hydrolase 18 (GH18) family of chitinases is a multigene family that plays various roles, such as ecdysis, embryonic development, allergic inflammation and so on. Efforts are still needed to reveal their functional diversification in an evolutionary and systematic manner. We collected 85 GH18 genes from eukaryotic representatives. The domain architectures of GH18 proteins were analyzed and several conserved patterns were identified. It was observed that some (11 proteins) GH18 members in Ecdysozoa or fungi possess repeats of catalytic domains and/or chitin-binding domains (ChtBs). The domain repeats are likely to meet requirements for higher efficiency of chitin degradation in chitin-containing species. On the contrary, all vertebrate GH18 proteins contain no more than one catalytic domain or ChtB. The results from homologous analysis, domain architectures, exon arrangements and synteny loci supported two evolutionary paths for the GH18 family. One path experienced gene expansion and contraction several times during evolution, covering most of GH18 members except CHID1 (stabilin-1 interacting partner) and its homologs. Proteins in this path underwent frequent domain gain and loss, as well as domain recombination, that could achieve versatility in function. The other path is comparatively conserved. The CHID1 gene evolved without gene duplication except in Danio rerio. Domain architectures of CHID1 orthologs are all identical. The diverse phylogeny of the GH18 family in arthropod is also presented.

Inhibitory kinetics of citric acid on beta-N-acetyl-d-glucosaminidase from prawn (Litopenaeus vannamei).

NAGase (EC.3.2.1.52) from crustaceans has the important roles in immunity, molting and digestion of chitinous foods. In this paper, the effects of citric acid on the activity of NAGase from Litopenaeus vannamei for the hydrolysis of pNP-NAG have been studied. The results showed that appropriate concentrations of citric acid could lead to reversible inhibition on NAGase and IC(50) was estimated to be 5.00 +/- 0.35 mM. Using the plots of Lineweaver-Burk, the inhibition of NAGase by citric acid belongs to competitive type, the inhibitory equilibrium constant for citric acid binding with free NAGase, K(I), is 3.26 +/- 0.25 mM. The inhibitory kinetics of citric acid on NAGase in the appropriate concentrations of citric acid has been studied using the kinetic method of substrate reaction. The time course of NAGase for the hydrolysis of pNP-NAG in the presence of different concentrations of citric acid showed that at each citric acid concentration, the rate decreased with increasing time until a straight line was approached. The results show that the inhibition of NAGase by citric acid is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction on citric acid with NAGase.

Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei.

GH18 chitinase is a multi-gene family. The family plays important physiological roles in Crustacea, e.g. ecdysis and defense against pathogen. However, data about GH18 family are rather limited in Crustacea. In the study, different cloning strategies were adopted to clone chitinase genes of Litopenaeus vannamei, which is the most widely cultured shrimp. Seven chitinase family members were identified. Analysis of domain architectures showed the repeated CBM18 modules and catalytic domain of enzymatically inactive chitolectin in Crustacea for the first time. Comparing to the three known groups of crustacean chitinase, four of the seven members are located on new evolutionary clades thus enriched the chitinase family of Crustacea. Tissue expression profiles were investigated in eight tissues. Expression of CHT5 and CHID1 were both detected in the hemocyte by which the innate immunity activity was carried out. The domain architectures, evolutionary relationships and tissue expression patterns all provide reasonable explanation for the existence of multiple genes in crustacean chitinase family.

Inhibitory kinetics of betaine on beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei).

The effects of betaine on prawn beta-N-acetyl-D-glucosaminidase (NAGase) activity for the hydrolysis of p-nitrophenyl-N-acetyl- beta-D-glucosaminide (pNP-NAG) have been studied. The results showed that appropriate concentrations of betaine could lead to reversible inhibition against NAGase, and the IC(50) value was estimated to be 15.00 +/- 0.30 mM. The inhibitory kinetics assay showed that betaine was a mixed type inhibitor with a K(I) value of 9.17 +/- 0.85 mM and a K(IS) value of 45.58 +/- 2.52 mM. The inhibitory model was set, and the microscopic rate constants were determined using the kinetic method of the substrate reaction. The time course of the hydrolysis of pNP-NAG catalyzed by NAGase in the presence of different betaine concentrations showed that at each betaine concentration, the rate decreased with an increase in time until a straight line was approached, indicating that the inhibition of NAGase by betaine is a slow, reversible reaction with fractional residual activity. The fact that k(+0) is much larger than k(+0)(') indicated that the free enzyme molecule is more fragile than the enzyme-substrate complex against betaine. It is suggested that the presence of the substrate offers marked protection of NAGase against inhibition by betaine.

Purification and characterization of trypsin from the intestine of hybrid tilapia (Oreochromis niloticusxO.aureus).

Trypsin from the intestine of hybrid tilapia (Oreochromis niloticus x O.aureus) was purified by the following techniques: acetone precipitation, ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and DEAE-sephacel ion exchange chromatography. The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight was estimated as 22,000 Da. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 9.0 and 60 degrees C, respectively. The enzyme was stable over a broad pH range from 7.0 to 12.0 at 30 degrees C, and the enzyme was inactive at temperatures above 50 degrees C. The behavior of the enzyme for the hydrolysis of casein followed Michaelis-Menten kinetics with Km of 0.46 mg/mL. The purified enzyme was inhibited by the general serine protease inhibitor phenyl methyl sulphonyl fluoride (PMSF) and also by the specific trypsin inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK) using Nalpha-CBZ-L-lysine p-nitrophenyl ester hydrochloride (CBZ-Lys.pNP) as a substrate. The protease was inhibited by the following ions in decreasing order: Zn2+>Fe3+>Cu2+>Al3+>Co2+=Pb2+>Cd2+>Mn2+. The ions Li+, Na+, K+, Mg2+, and Ba2+ had little effect on the enzyme, and Ca2+ can partially promote its activity at low concentration.

Modification and modificatory kinetics of the active center of prawn beta-N-acetyl-D-glucosaminidase.

Beta-N-acetyl-D-glucosaminidase (NAGase, EC3.2.1.52) plays important role in molting, digestion of chitinous foods, and defense systems against parasites in prawn (Litopenaeus vannamei). However, study on functional groups and catalytic mechanism of NAGase are yet limited. The modification of the active center of NAGase from prawn has been first studied. The results demonstrate that the disulfide bonds and the carbamidine groups of arginine residues are not essential to the enzyme's activity. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 276 nm, indicating that tryptophan is essential residue to the enzyme. The modificatory kinetics of NAGase in the appropriate concentrations of NBS solution has been studied and the numbers of essential tryptophan residues have been determined using the kinetic method of the substrate reaction. The result shows that only one tryptophan residue is essential for enzyme activity. And the modifications of histidine, lysine residue, and the carboxyl groups also inactivate the enzyme completely or incompletely. The results showed that the carboxyl groups of acidic amino acid, imidazole groups of histidine residue, amino groups of lysine residue, and indole group of tryptophan were essential for the activity of enzyme.

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion.

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.

Inhibitory kinetics of bromacetic acid on beta-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei).

beta-N-Acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52), a composition of chitinases, cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine (NAG). NAGase from prawn (Penaeus vannamei) is involved in digestion and molting processes. The investigation of enzymatic properties, functional groups and catalytic mechanism is an essential mission to its commercial application. Bromacetic acid (BrAc) is a specific modifier for the histidine residue in specific condition. In this paper, the effect of BrAc on prawn NAGase activity for the hydrolysis of pNP-NAG has been investigated. The results showed that BrAc can reversibly and non-competitively inhibit the enzyme activity at appropriate concentrations and the value of IC(50) was estimated to be 17.05+/-0.65 mM. The inhibition kinetics of the enzyme by BrAc has been studied using the kinetic method of the substrate reaction. And the inhibition model was set up and the microscopic rate constants for the reaction of the inhibitor with free enzyme and the enzyme-substrate complexes were determined for inactivation and reactivation. The rate constant of the forward inactivation (k(+0)), which is 1.25 x 10(-3)s(-1), is about eight times as much as that of the reverse reactivation (k(-0)), which is 1.64 x 10(-4)s(-1). Therefore, when the BrAc concentration is sufficiently large, the enzyme is completely inactivated.

Inhibition kinetics of hydrogen peroxide on beta-n-acetyl-D-glucosaminidase from prawn (Penaeus vannamei).

The effects of hydrogen peroxide (H2O2) on prawn NAGase activity for the hydrolysis of pNP-beta-D-GlcNAc have been studied. The results show that H2O2 can reversible inhibit the enzyme (IC50 = 0.85 M) and the inhibition is of a mixed type. The kinetics show that k+o is much larger than k+0, indicating the free enzyme is more susceptible than the enzyme-substrate complex in the H2O2 solution. It is suggested that the presence of the substrate offers marked protection against inhibition by H202. Changes of activity and conformation of the enzyme in different concentrations of H202 have been compared by measuring the fluorescence spectra and residual activity and show that the change of conformation is more rapidly than that of the residual activity, which implies that the whole conformation of the enzyme changes more rapidly than the conformation of the active centre of the enzyme in the H2O2 solution.

Inactivation kinetics of guanidinium chloride on Penaeus vannamei beta-N-acetyl-D-glucosaminidase and the relationship of enzyme activity and its conformation.

The effects of guanidinium chloride (GuHCl) on the activity of Penaeus vannamei beta-N-acetyl-D-glucosaminidase (NAGase) have been studied. The results show that GuHCl, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 0.6 M. Changes of activity and conformation of the enzyme in different concentrations of GuHCl have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreases distinctly with increasing GuHCl concentrations, and the emission peaks appear red-shifted (from 339.4 to 360 nm). Changes in the conformation and catalytic activity of the enzyme are compared. The extent of inactivation is greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. The kinetics of inactivation has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k(+0) is larger than that of k'(+0) which suggests that the enzyme is protected by substrate to a certain extent during guanidine denaturation.

Effects of mercuric ion on the conformation and activity of Penaeus Vannamei beta-N-acetyl-d-glucosaminidase.

beta-N-acetyl-d-glucosaminidase (NAGase, EC.3.2.1.52), a composition of the chitinases, catalyzes the cleavage of N-acetylglucosamine polymers into N-acetylglucosamine. In this paper, the effects of mercuric ion on the activity of NAGase from Penaeus vannamei for the hydrolysis of pNP-NAG have been studied. The results show that HgCl2 can lead to irreversible inactivation to this enzyme. The inactivation process follows a first-order reaction and the inactivation rate constants have been determined. The relationship between the inactivation rate constants and HgCl2 concentration has been studied and the result shows that only one molecule of HgCl2 binds to the enzyme molecule to lead the enzyme lose its activity. Moreover, the conformational changes of the enzyme inactivated by HgCl2 were studied by following changes in the intrinsic fluorescence emission and ultraviolet absorption spectra.

Inactivation kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei) in dioxane solution.

beta-N-Acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) catalyzes the cleavage of N-acetylglucosamine polymers. It is in the composition of the chitinases and cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine. In this work, the effects of dioxane on the enzyme activity for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide from the prawn (Penaeus vannamei) have been studied. The results show that appropriate concentrations of dioxane can lead to reversible inactivation of the enzyme, and the IC(50) is estimated to be 1.1 M. The kinetics of inactivation of NAGase in the appropriate concentrations of dioxane solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show that the free enzyme molecule is more fragile than the enzyme-substrate complex in the dioxane solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by dioxane.