Protein and Peptide Letters - Volume 15, Issue 3, 2008

Increasing antibiotic resistance has led to an urgent need for new therapeutic approaches. Host defense peptides are known to be antimicrobial and have revealed broad immunomodulatory functions for both innate and adaptive immunity. This review will focus on the role of host defense peptides in infection and immune response and discuss its potential and limitations as a future therapeutical agent.

Proteins with the double stranded beta-helix (DSBH, also known as cupin) fold perform a diverse range of functions. In this study, Bacillus subtilis quercetinase was used as a model system to understand the conformational determinants of functional diversity within the cupin fold. Controlled proteolysis experiments revealed that this enzyme is active, thermo-stable and maintains its quaternary arrangement even after substantial (ca 33 %) cleavage of the protein. The results presented in this manuscript thus show that the DSBH scaffold offers a novel balance between protein stability and function by locating the active site and substrate recognition features in the most stable region of the protein.

Cytochrome c oxidases of prion protein (PrP) gene-deficient (Prnp-/-) and Prnp+/+ mice were examined in vivo and in vitro. Non-invasive near-infrared spectra revealed that oxidation of copper and heme a+a3 in cytochrome c oxidase of Prnp-/- mice was similar to that in Prnp+/+ mice. Biochemical analysis of mitochondrial fractions also supported the results. PrP might not be involved in regulation of cytochrome c oxidase.

Intestinal trefoil factor (ITF), a member of the trefoil factor family, plays an important role in protecting the epithelial layer of the intestinal tract from damage and repairing epithelium after injury. In the present study, we expressed recombinant hITF by Picha pastoris expression system in shake flasks to 50mg/L. rhITF was purified up to 95% by ammonium sulfate precipitation, Ni-NTA affinity chromatography and ultrafiltration. In conditions simulating intestinal tract environment, rhITF was shown to exhibit resistance to proteases, heat, acid and alkali. In addition, rhITF was found to be biologically active in an in vitro restitution model. This study lays the foundation for the commercial production of rhITF and provides theoretical evidence for the oral use of rhITF.

The primary specificity residue of a substrate or an inhibitor, called the P1 residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S1 pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine α-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P1 with the sequence Ac-Phe-Ala-Thr-XAnb 5,2-NH2 were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO2), 4-aminophenylalanine - Phe(p- NH2), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonylphenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (kcat, KM and kcat/KM) of the peptides synthesized with bovine α- chymotrypsin were determined. The highest value of the specificity constant kcat/KM, reaching 6.0x105 [M-1xs-1], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO2)-Anb5,2-NH2. The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of kcat more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO2) in position P1 and potent chymotrypsin inhibitors were obtained. The dissociation constant (Ki) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO2)-CHO, amounted to 1.12x10-8 M.

Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a ß-hydroxy fatty acid. A biosurfactant-producing strain, Bacillus Sabtilis HSO121, was isolated from the production water of an oil field. The strain was able to produce eight surfactin isoforms which have been isolated by acid-precipitation followed by extraction into methanol. A novel procedure for the purification of surfactin was achieved. It consists of a solid-phase extraction on C18 gel followed by reversed-phase high performance liquid chromatography using Prep. HiQ sil C18W, column. The surfactin isoforms were eluted by linear acetonitrile gradient from 80-100%%. The peaks were analyzed by TLC on silica gel, and after acid hydrolysis their amino acid compositions were determined by HPLC analysis. Eight isoforms of surfactin had nearly the same amino acid composition and appeared a single spot in TLC. According to the Rf values with the amino acid composition, these peaks belong to the surfactin group of lipopeptides. Infrared analysis of the purified samples also revealed a pattern similar to that of surfactin. This is a very effective method for isolating and fractionating lipopeptides, of the same or different nature.

Erythrina velutina vicilin, EvV, is a dimeric glycoprotein with Mr of 124.6 kDa. EvV was tested for anti-insect activity against bean bruchid larvae. EvV had LD50 of 0.10% and ED50 of 0.14% for Z. subfasciatus and LD50 of 0.26% and ED50 of 0.19% for C. maculatus. EvV was not digested by bean larvae enzymes until 12 h of incubation, and at 24 h EvV was more resistant to Z. subfasciatus enzymes.

In the present study, seven novel dimeric analogues of endomorphin-2 with longer spacers were designed and synthesized. Through dimerization, their affinity for δ-opioid receptor was mostly increased, especially the δ-opioid receptor preferred dimeric analogue, DEM12. The results were confirmed by the in vitro bioassay. The structure-activity relationships were also discussed.

Plant profilins form a well-known panallergen family responsible for cross-sensitization between plant foods and pollens. We sought to map T and B-cell epitopes on the Iranian Crocus sativus profilin by bioinformatics tools. The predicted peptides are useful for further vaccine development.

Protein subcellular localization, which tells where a protein resides in a cell, is an important characteristic of a protein, and relates closely to the function of proteins. The prediction of their subcellular localization plays an important role in the prediction of protein function, genome annotation and drug design. Therefore, it is an important and challenging role to predict subcellular localization using bio-informatics approach. In this paper, a robust predictor, AdaBoost Learner is introduced to predict protein subcellular localization based on its amino acid composition. Jackknife crossvalidation and independent dataset test were used to demonstrate that Adaboost is a robust and efficient model in predicting protein subcellular localization. As a result, the correct prediction rates were 74.98% and 80.12% for the Jackknife test and independent dataset test respectively, which are higher than using other existing predictors. An online server for predicting subcellular localization of proteins based on AdaBoost classifier was available on http://chemdata.shu. edu.cn/sl12.

A computer-aided docking study was carried out to quickly clarify the binding structure of the ligand-receptor complex between bisphenol A (BPA), a well-known endocrine disruptor, and estrogen-related receptor γ (ERRγ). The resulting complex indicated that BPA binds to the ligand-binding pocket of ERRγ without any disruptions of the activation conformation.

Phytases play important roles in agricultural and feed industries. In this study, the stability of a beta-propeller phytase, PhyL, from Bacillus licheniformis was successfully improved by introducing Xaa→Pro and Gly→Ala substitutions at consensus positions. Our results suggest that Gly→Ala substitution is a more promising strategy to improve protein stability.

G pretein coupled-receptors (GPCR) have been shown to form heterodimers or heterooligomers with various biochemical and/or pharmacological activity that are distinct from those of the corresponding monomers or homomers. Recent in vitro and in vivo experimental data have also shown novel functional roles of several orphan GPCRs as modulatory units of heterodimers and potentials for development of heterodimer-selective clinical agents. In this review, we summarize essential and latest knowledge as to GPCR heterodimerizations and the current issues to be solved in the near future.

As a part of our spectroscopic and structural elucidation of small peptides, their salts and metal complexes, the polarized IR-spectroscopic and structural elucidation of the protonated form of glycyl-L-phenylalanyl-glycine as hydrochloride monohydrate (H-Gly-Phe-Gly-OHxHClxH2O) is reported. The obtained structure of the protonated tripeptide is supported by quantum chemical ab initio calculations at UHF level of theory and 6-31++G**, showing only one stable conformer with two intermolecular NH3 +…OH2 and (C=O)OH…OH2 hydrogen bonds. The Nterminus amide O=C-N-H amide fragment is flat trans-configured with a dihedral angle value of 179.0(7)°, while the corresponding group at Cterminus is with transoide-configurated and value of the dihedral angle of 151.1(3)°, respectively. In addition data of 1H- and 13C magnetic resonance spectroscopy (NMR), thermogravimetry (TGA), differential scanning calorimetry (DSC) and high performed liquid chromatography (HPLC) with tandem mass spectrometry (HPLC-MS/MS) as presented.

Several autoantibodies found in RA are directed to epitopes in citrullinated proteins. One of them is anti modified citrullinated vimentin (Anti-MCV). We tested the value a newly developed ELISA for the detection of antibodies againts a genetically modified citrullinated vimentin (anti-MCV) in comparison with an anti-CCP based ELISA system for the diagnosis of RA. Thirty-five patients with RA (mean age; 42.6 ± 10.87 years, mean disease duration; 9.37 ± 3.98 years) were enrolled in this study. Twenty -five ankylosing spondylitis (mean age; 35.88 ± 6.64 years, mean disease duration; 10.25 ± 4.61 years), and 19 healthy subjects (mean age; 40.26 ± 5.11 years) served as controls. Anti-CCP antibodies and Anti-MCV antibodies were measured using ELISA. In all RA patients, mean anti- CCP level was 69.07 ± 90.43 U/ml and anti-MCV level was 665.77 ± 1040.19 U/ml. In patients with AS, the mean anti-CCP level was 10.7 ± 5.22 U/ml and anti-MCV level was 40.54 ± 20.15 U/ml. In healthy controls, the mean anti-CCP level was 11.11 ± 7.65 U/ml, anti-MCV level was 23.12 ± 12.04 U/ml. In patients with active RA, the mean serum anti-CCP level was 100.54 ± 98.07 U/ml and anti-MCV level was 998.74 ± 1154.93 U/ml. In patients with inactive RA, the mean serum anti-CCP level was 8.77 ± 1.55 U/ml and anti-MCV level was 27.59 ± 23.10 U/ml. According to these results; In patients with RA, the mean serum anti-MCV and anti-CCP levels were significantly high compared to patients with AS and healthy controls (p=0.002, p=0.001, p=0.002, p=0.001 respectively). The mean serum anti-MCV and anti- CCP levels were significantly higher in active patients with RA than in inactive patients with RA patients (p=0.001 and p=0.001 respectively). In inactive patients with RA, the mean serum anti-MCV and anti-CCP levels were similar in patients with AS and patients (p=0.484, p=0.308, p=0.09 and p=0.222 respectively). The mean serum anti-MCV levels were correlated with DAS 28 (r=0.531, p=0.001), VAS score (r=0.332, p=0.01), ESR (r=0.458, p=0.001), serum CRP levels (r=0.568, p=0.01), serum RF levels (r=0.529, p=0.001), swollen joints number (r=0.525, p=0.001) and tender joints number (r=0.638, p=0.001). As a result; measurement of serum anti-MCV levels is useful for diagnosis of RA and combined use of anti-MCV and RF may be more useful prognostic factor than either method alone, RF and anti-CCP.

Hemoglobin (Hb) is a tetrameric protein, which contains four heme prosthetic groups, and each one is associated with a polypeptide chain. Herein, we report the rabbit hemoglobin which has intrinsically high oxygen affinity and possess highest sequence identity with human hemoglobin. The purified hemoglobin has been tried to crystallize in different crystallization conditions owing to its formation of various crystal systems. The rabbit Hb crystals were grown using PEG 3350 as the precipitant at 18° C. The crystals of rabbit Hb belongs to triclinic space group P1 with one molecule (α2β2) in the asymmetric unit.