Exploring protein-protein interactions using the site-identification by ligand competitive saturation methodology

Protein docking methods are powerful computational tools to study protein-protein interactions (PPI). While a significant number of docking algorithms have been developed, they are usually based on rigid protein models or with limited considerations of protein flexibility and the desolvation effect is rarely considered in docking energy functions, which may lower the accuracy of the predictions. To address these issues, we introduce a PPI energy function based on the site-identification by ligand competitive saturation (SILCS) framework and utilize the fast Fourier transform (FFT) correlation approach. The free energy content of the SILCS FragMaps represent an alternative to traditional energy grids and they can be efficiently utilized to guide FFT-based protein docking. Application of the approach to eight diverse test cases, including seven from Protein Docking Benchmark 5.0, showed the PPI prediction using SILCS approach (SILCS-PPI) to be competitive with several commonly used protein docking methods indicating that the method has the ability to both qualitatively and quantitatively inform the prediction of PPI. Results show the utility of the SILCS-PPI docking approach for determination of probability distributions of PPI interactions over the surface of both partner proteins, allowing for identification of alternate binding poses. Such binding poses are confirmed by experimental crystal contacts in our test cases. While more computationally demanding than available PPI docking technologies, we anticipate that the SILCS-PPI docking approach will offer an alternative methodology for improved evaluation of PPIs that could be used in a variety of fields from systems biology to excipient design for biologics-based drugs.     

Role of chemokines in Leishmania infection

Chemokines are a growing group of chemoattractant cytokines that play important roles in physiological as well as pathological processes. Their roles in various aspects of pathogenesis and inflammation have come to light in the past decade or so. It is becoming increasingly clear that chemokines play a major, perhaps decisive role in Leishmania infections. In this review, we recapitulate important works linking the chemokine system with relation to visceral and cutaneous leishmaniasis over the past decade and attemptto put it all together to propose a single yet unfinished model to account for all the findings.     

GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.     

Endothelial barrier strengthening by activation of focal adhesion kinase

Endothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening.     

A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical

The mechanism of the antiulcer effect of omeprazole was studied placing emphasis on its role to block oxidative damage and apoptosis during ulceration. Dose-response studies on gastroprotection in stress and indomethacin-induced ulcer and inhibition of pylorus ligation-induced acid secretion indicate that omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent mechanism for its antiulcer effect. Time course studies on gastroprotection and acid reduction also indicate that omeprazole almost completely blocks lesions at 1 h when acid inhibition is partial. The severity of lesions correlates well with the increased level of endogenous hydroxyl radical (*OH), which when scavenged by dimethyl sulfoxide causes around 90% reduction of the lesions, indicating that *OH plays a major role in gastric damage. Omeprazole blocks stress-induced increased generation of *OH and associated lipid peroxidation and protein oxidation, indicating that its antioxidant role plays a major part in preventing oxidative damage. Omeprazole also prevents stress-induced DNA fragmentation, suggesting its antiapoptotic role to block cell death during ulceration. The oxidative damage of DNA by *OH generated in vitro is also protected by omeprazole or its analogue, lansoprazole. Lansoprazole when incubated in a *OH-generating system scavenges *OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak at m/z 385, which is 16 mass units higher than that of lansoprazole (m/z 369). The product shows no additional aromatic proton signal for aromatic hydroxylation in (1)H NMR. The product absorbing at 278 nm shows no alkaline shift for phenols, thereby excluding the formation of hydroxylansoprazole. The product is assigned to lansoprazole sulfone formed by the addition of one oxygen atom at the sulfur center following attack by the *OH. Thus, omeprazole plays a significant role in gastroprotection by acting as a potent antioxidant and antiapoptotic molecule.     

Development of genetic markers in cyanobacteria and stability of genetically marked strains in soil

Reporter marker GUS (-glucuronidase gene from Escherichia coli) and luc operon from the American firefly were introduced into cyanobacteria and the stability of these markers in soil was examined. To transfer the integrational vector into cyanobacteria, the genomic DNA library of Synechocystis sp. or Anabaena cylindrica maintained in pBR 322, pCY 100 and pCY 101 were transformed with HB 101 containing pRL 528 and selected for Cm^r and Amp^r. These clones of HB 101 containing pRL 528 and the vectors carrying different cyanobacterial chromosomal DNA fragments were used for triparental mating with HB 101 [pRK 2013/pRK 2073] and cyanobacteria. The frequency of transconjugants for integrational vectors was between 2.1 × 10^–5 and 4.0 × 10^–4. The transfer frequency of RSF 1010 based vectors (pDSK 519 and pCY 106) was 1.0–4.5 × 10^–4 in Synechocystis sp. whereas A. cylindrica failed to maintain these vectors. Low frequency transfer (2.0–2.3 × 10^–6) of RK 2 based vectors pVK 100 and pCY 104 was observed in A. cylindrica but these were unable to replicate in Synechocystis sp. The vectors in general were stable at least by 74.9% for 60 days of incubation in BG-11 medium. The markers were less stable in A. cylindrica (74.9–84.2%) compared to Synechocystis sp. (80.1–88.8%) at 60 days of incubation. Integrational vectors were almost 85% stable in both the strains. The RK 2 derivative of pCY 104 was less stable in A. cylindrica (74.9–77.3%) than the RSF 1010-based vector pCY 106 in Synechocystis sp. (80.1–81.0%). A maximum of 64.7% of the markers were lost in soil. The chromosomal markers through integrational vectors were found to be highly stable and 68.2–72.7% of these markers were retained in cyanobacteria at 60 days of incubation. Plasmid markers were less stable, with a loss of 64.7–48.7% at the end of the experiment. In A. cylindrica 58–65% of the RK 2 vector was lost whereas in Synechocystis sp. 49–61% of RSF 1010 was lost at 60 days of incubation.     

Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo

AIMS: To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. METHODS AND RESULTS: The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4.0-9.0. CONCLUSIONS: The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Lichenin could be a potential candidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.