Protein and Peptide Letters - Volume 19, Issue 9, 2012

Phase I metabolism is an important consideration in drug discovery because it profoundly affects the toxicity and activity profile of a drug candidate. In these metabolic processes, CYP450 family is responsible for the majority of biotransformation events. However, it is still an important challenge to predict sites of metabolism (SOM) of a new chemical entity due to the complex reaction mechanism and variety in CYP450 enzymes. SOMEViz is an online service designed for predicting and visualizing human cytochromes P450 (CYP450)-mediated sites of metabolism (SOM) of a molecule, on the basis of a previously reported model [1]. The service provides an access for predicting sites of metabolism of molecules with reasonable accuracy, and predicted results are shown in a user-friendly as well as interactive way, which may help chemists explore metabolism properties of chemicals in the early stage of drug discovery. The web-based GUI of SOMEViz offers user a straightforward way to manage and visualize the sites of metabolism (SOM) prediction results. The service and examples are available free of charge at http://www.dddc.ac.cn/some.

In systems biology, regulatory pathway is one of the most important research areas. However, regulatory pathway is so complicated that we still poorly understand this system. On the other hand, with rapid accumulated information on different organisms, it becomes more and more possible to in-depth investigate regulatory pathway. To understand regulatory pathway well, figuring out the components of each pathway is the most important step. In this study, a network- based method was proposed to classify human genes into corresponding pathways. The information of proteinprotein interactions retrieved from STRING was used to construct a network and jackknife test was employed to evaluate the method. As a result, the first order prediction accuracy was 87.91%, indicating that interactive proteins always have similar biological regulatory functions. By comparing the predicted results obtained from other methods based on blast and amino acid composition, respectively, it implies that our prediction method is quite promising that may provide an opportunity to understand this complicated pathway system well.

It is important to understand the cause of amyloid illnesses by predicting the short protein fragments capable of forming amyloid-like fibril motifs aiding in the discovery of sequence-targeted anti-aggregation drugs. It is extremely desirable to design computational tools to provide affordable in silico predictions owing to the limitations of molecular techniques for their identification. In this research article, we tried to study, from a machine learning perspective, the performance of several machine learning classifiers that use heterogenous features based on biochemical and biophysical properties of amino acids to discriminate between amyloidogenic and non-amyloidogenic regions in peptides. Four conventional machine learning classifiers namely Support Vector Machine, Neural network, Decision tree and Random forest were trained and tested to find the best classifier that fits the problem domain well. Prior to classification, novel implementations of two biologically-inspired feature optimization techniques based on evolutionary algorithms and methodologies that mimic social life and a multivariate method based on projection are utilized in order to remove the unimportant and uninformative features. Among the dimenionality reduction algorithms considered under the study, prediction results show that algorithms based on evolutionary computation is the most effective. SVM best suits the problem domain in its fitment among the classifiers considered. The best classifier is also compared with an online predictor to evidence the equilibrium maintained between true positive rates and false positive rates in the proposed classifier. This exploratory study suggests that these methods are promising in providing amyloidogenity prediction and may be further extended for large-scale proteomic studies.

C-terminal domain of peptidoglycan hydrolase enterolysin A (EnlA) is involved in specific recognition and binding to the target cell envelopes and represents true cell wall binding (CWB) domain. Sensitivity/resistance to EnlA is dependent on binding ability/disability of its CWB domain. We assume that main mechanism of resistance against EnlA is absence of the specific receptor on the cell surface, which is necessary for binding of the enzyme molecule. Using competitive and enzymatic assays we have uncovered the chemical nature of the EnlA receptor, which is a lipoteichoic acid.

The effect of additional amino acid residue(s) fused to the N- and/or C-terminal on properties of the heterogeneously expressed protein is usually difficult to be predicted. Recombinant proteins expressed without any fused sequence should be the most desired materials for related studies, such as protein drug preparation, biochemistry investigations. Here, we report a very simple and universal method enabling the expression of protein in its unfused form between the same two restriction enzyme sites (at a higher level) if a plasmid can support the fused expression. The method provided an assessable solution for unfused expression without increase in experimental resource; the necessary material is an additional primer. The method is especially useful for making whole-cell biocatalyst in which no purification steps are required.

Saturation Transfer Difference NMR (STD NMR) is used for the detection of the binding constant of the decapeptide RRRDDDSDDD with CK2α, the catalytic subunit of the proteinkinase CK2. For this work a valid irradiation frequency of the CK2α had to be found ensuring that no peptide resonance is affected by the irradiation. This is the principle problem for investigations of protein peptide interactions by STD NMR due to the similarity of protein and peptide resonances. It is shown that by careful selection of the irradiation point a KD value averaging to 1 mM can be found. In addition, preferred binding sites of the peptide are detected and it is shown that the side chains of serine and aspartate are closest to the protein surface.

Obtaining soluble proteins in sufficient concentrations is a major obstacle in various experimental studies. How to predict the propensity of targets in large-scale proteomics projects to be soluble is a significant but not fairly resolved scientific problem. Chaos game representation (CGR) can investigate the patterns hiding in protein sequences, and can visually reveal previously unknown structure. Fractal dimensions are good tools to measure sizes of complex, highly irregular geometric objects. In this paper, we convert each protein sequence into a high-dimensional vector by CGR algorithm and fractal dimension, and then predict protein solubility by these fractal features together with Chou's pseudo amino acid composition features and support vector machine (SVM). We extract and study six groups of features computed directly from the primary sequence, and each group is evaluated by the 10-fold cross-validation test. As the results of comparisons, the group of 445-dimensional vector gets the best results, the average accuracy is 0.8741 and average MCC is 0.7358. The resulting predictor is also compared with existing methods and shows significant improvement.

Both fluorescence spectroscopic and molecular docking methods were used to investigate the interaction between bovine serum albumin (BSA) and a known Bcl-xl/Bcl-2 inhibitor HA 14-1. Based on the spectral overlap between the emission of BSA and absorption of HA 14-1, Forster energy transfer was proposed to be the possible quenching mechanism. The Stern-Volmer constants are 2.49 x 104, 2.04x 104 and 0.90 x 104 M-1 at 293, 303 and 318 K, respectively, indicating that a static quenching process dominates. Thermodynamic parameters were further obtained. The derived negative Δ H (-27.51 kJ mol-1) and Δ S (-11.11 J mol-1K-1) values suggest hydrogen bond interaction and van der Waals force are the main binding force. The docking study was performed on BSA model. According to the docking score and the number of hydrogen bonds, the potential binding site for HA 14-1 is proposed to be the site IIA, also known as drug site 1.

Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer related death. The epidermal growth factor receptor (EGFR) pathway is over expressed in HNSCC. EGFR regulates the HNSCC by inducing signalling events responsible for regulating key tumorigenic processes such as proliferation, inhibition of apoptosis, cell adhesion/ motility, growth and survival. Present study evaluates the potential of N-(3-Ethynylphenyl)-6, 7-bis (2-methoxyethoxy) quinolin-4-amine as a new inhibitor for EGFR. We have explored the binding and inhibitory potential of the compound using molecular docking, structural interactions fingerprinting and molecular dynamics studies. The inhibitor exhibits extensive interactions with the EGFR catalytic site in the form of hydrogen bonds, pi-pi bond and salt bridges. It shows high specificity and binding affinity towards the protein. The compound can further be explored for its potential to serve in the diagnosis and treatment of HNSCC. The quantitative prediction provides a scope for future experimental testing, facilitating the understanding of the crosstalks between signalling pathways.

Corneal angiogenesis and lymphangiogenesis are induced by vascular endothelial growth factors (VEGFs) signaling through its receptors VEGFR-1, -2, and -3. Endostatin is a peptide antagonist of these receptors that causes inhibition of bFGF-induced corneal angiogenesis and lymphangiogenesis. Here we show that binding of VEGF-C and endostatin to recombinant VEGFR-3 is competitive. Alignments of the primary amino acid sequences of VEGF-C and the C-terminal endostatin peptide (mEP: LEQKAASCHNSYIVLCIENSFMTSFSK) identified two conserved cysteine residues separated by seven amino acids. Peptides of VEGF-C and mEP containing these conserved residues bound toVEGFR-3. However, substitution of alanine for either of the cysteines in the mEP peptide perturbed the secondary structure, and this mutated peptide was unable to bind to VEGFR-3. Analysis by surface plasmon resonance demonstrated that the binding of the mEP peptide for recombinant VEGFR-3 had a Ka of 1.41x107M-1s-1, Kd of 0.6718 s-1, and a KD of 4.78x10-8M. Characterization of the mechanism of endostatin binding to VEGFR-3 may lead to the development of novel therapies for lymphangiogenesis-related disorders, such as transplant rejection, lymphedema, and cancer metastasis.

Marine algae can serve as sources of bioactive compounds and currently have been shown their potential biological and pharmaceutical applications. Marine algae lectins have been shown to be effective at controlling inflammatory processes. This work aimed to analyze the immunostimulatory properties of lectins from the marine algae Solieria filiformis (SfL), Pterocladiella capillacea (PcL) and Caulerpa cupressoides (CcL). This analysis was performed on BALB/c mouse splenocytes by measuring cytokine and nitric oxide production and cellular damage using tests of cytotoxicity and cell viability. These lectins were not cytotoxic (1-100 μg/mL), and were not able to induce IFN-γ and IL-2 production. IL- 10 production was induced at high levels by all lectins tested. Treatment with SfL induced IL-6 production at higher levels at all experimental times, whereas treatment with PcL and CcL induced higher levels only in 24 and 72 h. Treatment with SfL did not result in nitrite oxide production, whereas treatment with PcL or CcL was able to induce nitrite release at high levels (after 24, 48 and 72 h). Lesser cellular damage (5%) was observed in splenocytes treated with these lectins (10 μg/mL). Thus, the lectins from these algae were not cytotoxic, promoted increased in cell viability and induced Th2 immune responses in mouse splenocytes, indicating that they have anti-inflammatory effects.

Amyloidosis resulting from the deposition of aggregated protein has been linked to many debilitating degenerative diseases which include most notably Alzheimer's and Parkinson's. The tendency for a protein to alternatively form highly ordered amyloid fibrils is dependent on many biological factors. Mutations, temperature, concentration, translational motion and pH play a pivotal role in inducing fibril aggregate assembly in vitro. The key feature appears to be the need to destabilize the native state structure as a required first step. In this paper we report on the detailed conversion of the death domain of the human Fas-associated death domain, an all α-helical protein with a Greek-key topology, into an all β-sheet amyloid fibril, using a comprehensive range of spectroscopic techniques that provide insight into this process. This transition from α-helical to β-sheet seems to require destabilization but not complete loss of the secondary structure to explore alternative conformations. This is a fascinating transition that supports the hypothesis that all proteins have the innate ability to form a fibril-like structure. Thus, the primary structure can encode two alternative three-dimensional structures: the native, functional state and the β-amyloid state. The Fas-associated death domain does not appear to naturally form amyloid fibrils in vivo. Our results clearly indicate that proteins evolved to avoid amyloid fibril formation because we find that the conditions required for formation in our model system are very specific and far from physiological.

The hydrophobic core in Bcl-xL composed of Trp137, Ile140, Trp181, Ile182, Trp188 and Phe191 is highly conserved and essential for protein folding, protein stability and binding affinity with BH3-peptide. 9 mutants of Ile140 residue were constructed and characterized in order to get better understanding of the effect of the hydrophobic core. Binding assay demonstrated that binding affinities between 4 charged mutants and BH3-peptide were significantly weakened or lost, suggesting that the integrity of the hydrophobic core has close relationship with binding. The CD spectroscopy results indicated that disruption of the hydrophobic core may affect local conformation within the protein and result in intrinsic inactivity. Further chemical-induced protein folding results on these 4 mutants revealed that the conserved hydrophobic core is also important for the protein stability.

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ∼ 4.7 mg L-1. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.

As per Bentham Science’s policy, the following article has been retracted at the request of the Editor-in-Chief and its Authors published in ‘Protein & Peptide Letters“ due to their use of text obtained from another paper published in the Biochemical Journal......