Protein and Peptide Letters - Volume 17, Issue 5, 2010

The recognition of human leukocyte antigen (HLA) molecules by specific receptors is a crucial step in the regulation of natural killer (NK) cell function. Killer cell immunoglobulin-like receptor (KIR) 3DS1 is one of the activating receptors of NK cell and is implicated in slowing disease progression in HIV infection. KIR3DS1 play an important role in the outcome of multiple diseases associated with viral infections. In contrast to the inhibitory receptor, much less is known about the ligands of KIR3DS1. In order to achieve a better understanding of the biology of KIR3DS1 and its ligand systems, it is necessary to identify the ligands of KIR3DS1. In this work, we utilized recombinant HLA-B2705 molecules and DsbA-KIR3DS1 fusion protein to monitor the interaction between HLA-B2705 complexes and DsbAKIR3DS1 using BIAcore 3000 SPR sensor and found that the specific binding between KIR3DS1 and HLA-B2705 existed and the affinity was 6.95×10-6 mol//L. So we concluded that HLA-B2705 is a possible ligand of KIR3DS1.

Histone deacetylase 6 (HDAC6) is a cytosolic enzyme that catalyzes deacetylation of several proteins. Acetylated tubulin has been recently identified as a physiological substrate of HDAC6. However in previous reports, all in vitro binding and enzymatic assays were accomplished with only partially purified protein samples. Therefore, it still remained unclear whether HDAC6 alone could interact with tubulin and catalyze deacetylation. In this study, both binding and enzymatic assays were conducted using recombinant-derived HDAC6 and purified α/β tubulin to eliminate possible contamination. The results clearly demonstrated that interaction between HDAC6 and tubulin is independent of other proteins. In addition, HDAC6 can independently catalyze deacetylation of both tubulin dimer and microtubule polymer.

We use approximate entropy and hydrophobicity patterns to predict G-protein-coupled receptors. Adaboost classifier is adopted as the prediction engine. A low homology dataset is used to validate the proposed method. Compared with the results reported, the successful rate is encouraging. The source code is written by Matlab.

MMP-7 is the smallest metalloproteinase. Its unregulated activities and existence in serum are recently known to be tightly related with life-threatening disease such as cardiac disease and several cancers. The protein production is thought to be useful for its characterization and antibody generation. Although many attempts at bacterial expressions have been conducted, they were recovered as insoluble and inactive protein. In this study, after soluble expression, singlestep purification and conversion to active protease, it was applied for the screening secretory metalloproteinase inhibitors in conditioned media of human cancer cells.

Cockroach infestations have been linked with allergic diseases such as asthma in humans. Bla g 5, sigma class glutathione S-transferase (GST), is the major cockroach allergen which has the highest IgE response value of all cockroach allergens. Although several cockroach allergens have been identified and cloned, information regarding their B ell and T cell IgE-binding epitopes is limited. In order to analyze the IgE binding epitopes of Bla g 5, full-length and five peptide fragments (A, 1-100 amino acid residue; B, 91-201; Ba, 1-125; Bb, 1-150; Bc, 1-175) were expressed. Twelve (37.5%) of 32 sera from cockroach-sensitized subjects showed positive IgE reactivity to the recombinant Bla g 5 (rBla g 5). Six strong positive sera were selected for the epitope study. Recombinant proteins not containing 176-201 amino acid residues were unable to react to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 5, the pooled serum from 5 cockroach-sensitized patients did not show IgE reactivity to all synthetic peptides consisting of 15 residues covering 161-201 amino acids. These results suggest the possibility that Bla g 5 may have a conformational epitope in the C-terminal region. GST is the important target for the development of vaccines and drugs against allergic diseases because of high cross-reactivity among insect species. This study will aid recombinant allergen research for immunotherapy of cockroach allergens and other insect allergens.

Although cytoplasmic azoreductases have been purified and characterized from various bacteria, little evidence demonstrating that these azoreductases are directly involved in azo dye reduction in vivo is known. In order to evaluate the contribution of the enzyme to azo dye reduction in vivo, experiments were conducted to determine the effect of a recombinant cytoplasmic azoreductase (AzoA) from Enterococcus faecalis expressed in Escherichia coli on the rate of metabolism of Methyl Red, Ponceau BS and Orange II. The intact cells that contained IPTG induced AzoA had a higher rate of dye reduction with increases of 2 (Methyl Red), 4 (Ponceau BS) and 2.6 (Orange II)-fold compared to noninduced cells, respectively. Metabolites of Methyl Red isolated from induced cultures were identified as N,N-dimethyl-pphenylenediamine and 2-aminobenzoic acid through liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses. In conclusion, our data demonstrate that AzoA from Ent. faecalis is capable of increasing the reduction of azo dyes in intact E. coli cells and that cytoplasmic azoreductase is involved in bacterial dye degradation in vivo.

Ribosome display is a powerful cell-free technology to select a desired antibody together with its encoding mRNA. In this study, a human single-chain variable fragment (scFv) library was generated from the peripheral blood lymphocyte RNA of 10 ovarian cancer patients and then panned against bead-conjugated human PlGF, a protein that may contribute to the growth and metastasis of ovarian cancer. A selected scFv antibody was evaluated by Western blot and its affinity constant to the PlGF was determined by noncompetitive enzyme immunoassay. This study highlights the ribosomal display technology for the selection of human antibody from patient-derived gene pools.

A multi-scale parameterization approach, factor analysis scales of generalized amino acid information combined with auto cross covariance, was used to develop quantitative sequence-activity models of peptides using support vector machines. The results demonstrated that this approach could well characterize sequence features of the peptides studied.

Based on enzyme sequence, using composite vectors with amino acid composition, low-frequency power spectral density, increment of diversity by combining a different form of pseudo amino acid composition to express the information of sequence, a support vector machine (SVM) for predicting enzyme subclasses is proposed. By the jackknife test, success rates of our algorithm are higher than other methods.

We describe the recombinant production of the human Y1 receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y1 receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Protein transduction therapy is a promising alternative to gene therapy, but has not previously been investigated for osteosarcoma. We here demonstrate efficient growth inhibition of human osteosarcoma cells using a p53 peptide. Our result suggests that protein transduction therapy has significant potential as a novel therapeutic approach for osteosarcoma.

Based on template hexapeptides Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 and Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 analogues and corresponding deacylated homologues were synthesized substituting ornithine, diaminobutanoic (Dab) and diaminopropanoic (Dap) acids for lysine at position 6. The aim was to investigate the effect of the newly synthesized compounds on the neurogenic contractions of isolated rat vas deferens. Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 and its analogues manifested a strong inhibitory effect on the neurogenic contractions without effect on the muscle tone, which is characteristic effect of NOP receptor agonists. In contrast, Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 and its analogs manifested a strong inhibitory effect on the muscle tone and negligible effect on the neurogenic contractions which is characteristic effect of NOP receptor antagonists. The most active were the peptides in which Dab or Dap is the substitute. The study reveals that substitution of Lys with shorter amino acids could increase agonist or antagonist activity of the peptide.

The non-covalent interaction of reactive brilliant red (RBR) as a representative of sulfonic azo compounds with human serum albumin (HSA) was investigated by a combination of UV-VIS spectrometry, fluorophotometry, circular dichroism (CD) and isothermal titration calorimetry (ITC) technique. The thermodynamic characterization of the interaction was performed. The saturation binding numbers of RBR on peptide chains were determined and the effects of electrolytes and temperature were investigated. The ionic interaction induced a combination of multiple non-covalent bonds including hydrogen bonds, hydrophobic interactions and van der Waals force. A three-step binding model of RBR was revealed. The binding of RBR molecules might occur on the external surface of HSA via electric interaction when the mole ratio of RBR to HSA was less than 40 and RBR molecules entered the hydrophobic intracavity of HSA when the ratio was more than 40. Moreover, RBR binding resulted in a conformational change in the structure of HSA or even the precipitation of HSA and inhibited its function accordingly. The possible binding site and the conformational transition of HSA were hypothesized and illustrated. This work provides a new insight into non-covalent interaction between a sulfonic azo compound and protein, which may be further used to investigate the potential toxicity of azo dyes.

Establishing codon usage biases are crucial for understanding the etiology of central nervous system neurodegenerative diseases (CNSNDD) especially Alzheimer's disease (AD) as well as genetic factors. G and C ending codons are strongly biased in the coding sequences of these proteins as a result of genomic GC composition constraints. On the other hand, codons that identified as translationally optimal in the major trend all end in C or G, suggesting translational selection should also be taken into consideration additional to compositional constraints. Furthermore, this investigation reveals that three common codons, CGC (Arg), AGC (Ser), and GGC (Gly), are also critical in affecting codon usage bias. They not only can offer an insight into the codon usage bias of AD and its mechanism, but also may help in the possible cures for these diseases.

Two binary complexes (KAP2-C subunit and cAMP-bound KAP2) were built, to investigate molecular interaction. The binding sites of KAP2 include the acidic sequence motif (Asp73-Glu87), the inhibitor peptide/linker region (Arg93-Val118), and beta barrel of cAMP-binding domains (CBD-A/B). The binding surface on the C subunit anchoring KAP2 extends to the inhibitor binding site at the active site cleft (Glu127-Glu230), Pro243-Ser252 helix and the phosphorylated activation loop (Arg194-Thr201) of the large lobe besides some sites in the small lobe. KAP2 undergoes major conformational changes in comparison of the two complexes above, especially the linker region and Met251 at Arg234- Phe252 helix as an inflexion point of the turnaround. Additionally, the interaction between KAP2 and cAMP concentrates on two catalytic motifs (FGELAL and PRAA) of phosphate binding cassette regions and the cyclic-monophosphate and ribose of cAMP. On the other hand, WAVE1 of BAD complex maybe interacts with the D/D domain of KAP2 by each of three helical motifs (Asn24-Lys46, Pro492-Val514, and Glu525-Glu547). This is helpful for our research of molecular mechanism of PKA and further analysis of BAD complex how to modulate glycolysis and apoptosis.

The conformation of bovine serum fetuin (BSF) was examined over the pH 7.0-12.9 regions by circular dichroism, intrinsic fluorescence and ANS binding. We observed that at higher pH, BSF exists in alkaline unfolded state. Our results provided evidence that correlates simultaneous formation of secondary structure followed by accumulation of hydrophobic clusters.

Glutathione reductase was purified 34806-fold with a final yield of 85 % from the bovine kidney cortex. Some molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme obeys ‘branched’ mechanism: KmNADPH 18 ± 3 μM and KmGSSG 65 ± 5 μM were determined.

Most of gene product similarity measurements concentrate on the information content of Gene Ontology (GO) terms or use a path-based similarity between GO terms, which may ignore other important information contained in the structure of the ontology. In our study, we integrate different GO similarity measure approaches to analyze the functional relationship of genes and gene products with a new triangle-based visualization tool called GOVis. The purpose of this tool is to demonstrate the effect of three important information factors when measuring the similarity between gene products. One advantage of this tool is that its important ratio can be adjusted to meet different measuring requirements according to the biological knowledge of each factor. The experimental results demonstrate that GOVis can display diagrams of the functional relationship for gene products effectively.