A motif detection and classification method for peptide sequences using genetic programming

An exploration of common rules (property motifs) in amino acid sequences has been required for the design of novel sequences and elucidation of the interactions between molecules controlled by the structural or physical environment. In the present study, we developed a new method to search property motifs that are common in peptide sequence data. Our method comprises the following two characteristics: (i) the automatic determination of the position and length of common property motifs by calculating the physicochemical similarity of amino acids, and (ii) the quick and effective exploration of motif candidates that discriminates the positives and negatives by the introduction of genetic programming (GP). Our method was evaluated by two types of model data sets. First, the intentionally buried property motifs were searched in the artificially derived peptide data containing intentionally buried property motifs. As a result, the expected property motifs were correctly extracted by our algorithm. Second, the peptide data that interact with MHC class II molecules were analyzed as one of the models of biologically active peptides with buried motifs in various lengths. Twofold MHC class II binding peptides were identified with the rule using our method, compared to the existing scoring matrix method. In conclusion, our GP based motif searching approach enabled to obtain knowledge of functional aspects of the peptides without any prior knowledge.     

Hidden Markov model-based prediction of antigenic peptides that interact with MHC class II molecules

Elucidating the interaction between major histocompatibility complex (MHC) molecules and antigenic peptides is fundamental to better understanding of the processes involved in immune responses and for the development of innovative immunotherapies. In the present study, hidden Markov models (HMM) were combined with the successive state splitting (SSS) algorithm for optimization of the HMM structure, to predict peptide binders to the human MHC class II molecule HLA-DRB1*0101. The predictive performance of our model (S-HMM) was compared with fully connected HMM and artificial neural network (ANN) methods using the relative operating characteristic (ROC) analysis. The S-HMM predictions had values of ROC > or = 0.85 which was at least as good, or better than the comparison methods. In addition, S-HMM is trained on positive data only and does not require exhaustive data preprocessing, such as peptide alignment. Our results demonstrated that S-HMM combines the high accuracy of predictions with the simplicity of implementation and is therefore useful for analyzing MHC class II binding peptides. In particular the S-HMM may be trained using only positive data and, the preprocessing of training data, such as peptide alignment and the selection of binding cores, is not required in this method.     

Prediction of peptide binding to major histocompatibility complex class II molecules through use of boosted fuzzy classifier with SWEEP operator method

To treat autoimmune diseases, it is important to identify which peptides bind to major histocompatibility complex (MHC) class II molecules (HLA-DRs). Predicting the peptides that bind to MHC class II molecules can effectively reduce the number of experiments required for identifying helper T cell epitopes. In our previous study, we applied fuzzy neural networks (FNNs) to solve this problem. However, an FNN requires a long calculation time and a large number of peptides; this means performing several experiments. In this study, we applied a boosted fuzzy classifier with the SWEEP operator method (BFCS) to solve this problem. For comparison, two other conventional modeling methods, namely, support vector machine and FNN combined with the SWEEP operator method (FNN-SWEEP) instead of using solely an FNN, were employed. Compared with FNN, FNN-SWEEP is extremely fast and has an almost identical prediction accuracy. The model constructed by BFCS showed an accuracy approximately 5%-10% higher than that constructed by FNN-SWEEP. In addition, BFCS was 30,000-120,000 times faster than FNN-SWEEP. This result suggests that BFCS has the potential to function as a new method of predicting peptides that bind to various protein receptors.     

Screening of peptides with a high affinity to bile acids using peptide arrays and a computational analysis

Bile acid binding peptides have attracted attention for the improvement and prevention of hypercholesterolemia. In this study, screening of bile acid high affinity peptides was investigated using computationally-assisted peptide array analysis. Starting with the screening data obtained from a limited, random 6-mer library (2212 sequences), the peptides with a high affinity to bile acid were characterized by comparison of high- and low-affinity peptides using fuzzy neural network (FNN) analysis. The physical properties of amino acids at specific positions that contribute to bile acid binding activity were extracted as the structural rule; optimization was carried out using three repeated screening cycles of the rule extraction. The extracted structural rule indicates that Trp, Tyr, Phe, Leu, Ile and Val are enriched in bile acid binding peptides. The yields of bile acid binding peptides with an affinity of above the VAWWMY peptide (soystatin, control sequence) were significantly higher in the optimized structural rule (32.5%) compared to that of the random library (3.1%), and 6 peptides were obtained with above 2.0-fold increased binding activity.     

In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype

Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe2O3) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metal-reducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.     

抗菌肽结构优化与改造策略研究进展

抗菌肽（AMPs）是一类具有抗菌活性的生物活性肽,由于热稳定性好、细胞选择性强、对哺乳动物细胞毒副作用少和不易产生耐药性等优势,成为近年来生物活性肽领域重要的研究热点。该文简要介绍了抗菌肽的分类,综述了抗菌肽的一级结构与空间结构特点,并从改变净正电荷数量与分布、调整抗菌肽α-螺旋度、提高抗菌肽蛋白酶耐受性、改变疏水性及平均疏水矩大小、改变两亲性、改变氨基酸残基位置及肽链长度、构建杂合抗菌肽、降低哺乳动物细胞毒性和制备金属抗菌肽提升抗菌活性与拓展抗菌谱等方面阐述抗菌肽的结构改造策略,并对抗菌肽结构优化策略研究进行了展望,旨在为抗菌肽结构优化策略的选择提供理论参考。     

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.     

Impact of feline zona pellucida glycoprotein B-derived synthetic peptides on in vitro fertilization of cat oocytes

Although immunocontraception based on porcine zona pellucida (ZP) proteins is widely applied in many species, it is not suitable for cat contraception due to the lack of cross-reactivity. Since the first ZP gene expressed during oocyte growth in domestic cats is ZPB, we assumed that immunization with feline ZPB (fZPB)-derived synthetic peptides may cause irreversible infertility, which would be preferable in stray cats. Thus, the present study evaluated the immunogenicity and the contraceptive potential of synthetic fZPB peptides. Antigenic epitope sequences were detected via epitope mapping using specific rabbit anti-fZP antibodies. Six peptides representing the recognized epitopes were synthesized subsequently. Two out of six peptides (ZPB amino acid residue 130-149 = P3 and 175-193 = P6) cross-reacted with anti-fZP antiserum in dot blot analysis and ELISA. Coupled to BSA, both peptides were utilized to produce specific antibodies in rats. Despite several booster injections the antibody titers monitored by ELISA did not exceed 1:5000. Both rat antisera were tested for contraceptive potential in cat in vitro maturation/in vitro fertilization (IVF). Antiserum against peptide P3 significantly inhibited sperm binding and fertilization of cat oocytes in vitro (57.3% of sperm binding; 41.5% of fertilization), whereas the inhibition by anti-P6 was not significant. Pre-incubation of sperm cells with both peptides before IVF failed to affect either sperm binding or fertilization (22.3 +/- 3.7 sperm/egg vs 25.5 +/- 5.8 for P3 and 20.7 +/- 4.0 for P6, respectively). In conclusion, antibodies directed against one of the two identified antigenic determinants of fZPB inhibited sperm binding and IVF and therefore showed promising results as a contraceptive. However, the specific immune response and anti-fertile properties of this synthetic vaccine have to be examined in vivo to verify the suitability of its components.     

Meta‐Analysis for Correlating Structure of Bioactive Peptides in Foods of Animal Origin with Regard to Effect and Stability

Amino acid (AA) sequences of 807 bioactive peptides from foods of animal origin were examined in order to correlate peptide structure with activity (antihypertensive, antioxidative, immunomodulatory, antimicrobial, hypolipidemic, antithrombotic, and opioid) and stability in vivo. Food sources, such as milk, meat, eggs, and marine products, show different frequencies of bioactive peptides exhibiting specific effects. There is a correlation of peptide structure and effect, depending on type and position of AA. Opioid peptides contain a high percentage of aromatic AA residues, while antimicrobial peptides show an excess of positively charged AAs. AA residue position is significant, with those in the first and penultimate positions having the biggest effects on peptide activity. Peptides that have activity in vivo contain a high percentage (67%) of proline residues, but the positions of proline in the sequence depend on the length of the peptide. We also discuss the influence of processing on activity of these peptides, as well as methods for predicting release from the source protein and activity of peptides.     

复合酶解米渣制备蛋白肽的理化与功能性质研究

以米渣为原料,采用复合酶解法制备高水溶性米渣蛋白肽,并对其理化与功能性质进行了分析。结果表明:米渣蛋白肽中总肽含量为88.93%,短肽含量为13.05%,总糖含量为3.14%;氨基酸组成合理,能很好地满足体内营养需要;水中溶解度在pH9.0时最大,为99.15%;乳化性及乳化稳定性均呈现较好变化趋势,乳化性在pH9.0时最大,乳化稳定性在pH11.0时最大。     

米渣肽抗疲劳作用及分离鉴定

米渣中蛋白质量分数在60%以上,但目前米渣仅用于附加值低的饲料行业。为了提高米渣的功能特性,拓展其应用范围,采用碱性蛋白酶对米渣进行酶解,酶解的产物经大孔树脂纯化得到具有高蛋白含量的米渣肽。超滤分离得到具有不同相对分子质量范围的米渣肽。与灌胃生理盐水、米渣肽和高相对分子质量米渣肽组相比,灌胃低相对分子质量(<1 000)米渣肽可以显著延长小鼠游泳的疲劳时间和小鼠血糖含量,并显著降低血乳酸含量,说明小分子肽具有显著的抗疲劳作用。经过高效液相色谱分离,得到一个抗疲劳肽,其序列为Gln-Ser-Pro-Glu-Ile。     

基于模拟胃肠道消化的云南民族乳制品蛋白肽研究

乳扇、乳饼和奶渣是云南民族传统乳制品,具有历史悠久、特色鲜明、蛋白质含量高等特点.本研究基于模拟胃肠道消化,采用液相色谱-串联质谱、氨基酸分析仪研究乳扇、乳饼和奶渣的肽谱及游离氨基酸含量,并借助蛋白肽数据库分析潜在的生物活性肽.结果表明,乳扇蛋白肽221条,分子质量集中在500～750Da,潜在的功能活性肽包括血管紧张...     

Cheesemaking with a Lactococcus lactis strain expressing a mutant oligopeptide binding protein as starter results in a different peptide profile

Lactic starters used for cheese manufacture play an important role in the production of bitter peptides and their degradation to non-bitter products. The oligopeptide transport system (Opp) of lactococci is essential for milk peptide utilization. The periplasmic substrate binding protein serves to capture the substrate with high affinity and to deliver it to a membrane-bound complex that translocates it inside the cell. Prt(+)- and Lac(+)-derivatives of MG1363 DeltaoppA strains expressing a wild-type MG1363 OppA or a mutant OppA with a single point mutation at residue 471 (OppA(D471R)) from a plasmid were constructed. These strains were used as lactic starters in cheese manufacture to improve flavour quality by removing hydrophobic peptides from the cheese matrix, through their preferential transport by OppA(D471R). Cheeses made with these strains were not significantly different from control cheeses after 1 day of ripening with respect to bacterial counts, pH and proteolysis, and only slight differences were recorded after 9 and 20 days of ripening. HPLC chromatograms of the hydrophilic and hydrophobic peptides present in the water-soluble fraction of experimental cheeses showed significant differences in peptide content as well as in peak profiles. These results suggest a different peptide utilization in the strain expressing OppA(D471R) and make it suitable for use as starter to improve cheese quality.     

活性肽与MHC结合能力预测的免疫信息学方法研究进展

综述活性肽与MHC(主要组织相容性复合体,major histocompatibility complex)结合能力预测的免疫信息学方法的最新进展,介绍常用的肽与MHC分子结合预测的相关工具及方法,分析了各类方法的特点、研究重点和难点,以期为寻找免疫活性肽提供更快捷的方法。     

两种血管紧张素转化酶抑制肽作用于靶标的分子机理

采用脱脂乳为原料,利用四步反相高压液相色谱从Lactobacillus helveticus 和Lactobacillus casei subsp. casei制作的酸乳中分离到两种血管紧张素转化酶Ⅰ(angiotensin Ⅰ -converting enzyme,ACE)抑制肽VPP 和IPP,测得它们抑制ACE 活性的IC50 分别为8.89μmol/L 和5.17μmol/L。根据氨基酸序列分析的结果,构建VPP 和IPP 的分子结构,通过分子柔性对接方法研究它们与ACE 相互作用的分子机理,确定它们的作用位点、作用力类型及相互作用能。结果表明：VPP、IPP 和ACE 的活性口袋之间均形成3 个氢键,且存在疏水,亲水等作用力,IPP 与ACE之间的结合较VPP 更稳定,与IPP 比VPP 具有较低的IC50 值的实验结果相一致。     

基于分子对接技术研究鱼源抗冻多肽与鱼肌球蛋白的相互作用

冻藏期间蛋白质冷冻变性是引起解冻后鱼糜凝胶化能力下降的主要原因之一,而肌球蛋白重链（Myosin Heavy Chain,MHC）是鱼糜凝胶形成的主要贡献者。本研究使用不同生物酶酶解制备抗冻多肽,通过计算机模拟酶解技术筛选抗冻多肽。利用蛋白质同源建模构建海鲈鱼肌球蛋白空间结构,通过分子对接和分子动力学模拟解析抗冻多肽与海鲈鱼肌球蛋白重链的作用位点及可能的作用机制。结果表明,胰蛋白酶酶解物具有高抗冻活性,对菌体低温胁迫保护达到80.35%±4.39%,并具有良好的热滞活性及抑制重结晶作用。模拟胰蛋白酶酶解获得的肽段GPR与GPAGGK可以与肌球蛋白重链通过分子间作用力结合,其中GPAGGK的结合更为稳定。这种相互作用可以阻碍肌球蛋白在温度变化中的结构改变,其机理可能是抗冻多肽结合在肌球蛋白重链的结构空腔上,影响了结合水解离后引起的疏水键及二硫键等化学键的形成,阻碍蛋白侧链聚集、结构改变和冰晶的位移等,这有利于鱼糜及鱼糜制品在冷冻贮藏中的品质保持。本研究结果为抗冻多肽应用于鱼糜及其制品在冷冻贮藏过程中品质保持的应用提供科学依据。     

基于生物信息学稻米半胱氨酸蛋白酶抑制剂来源生物活性肽的虚拟筛选及分子对接研究

稻米半胱氨酸蛋白酶抑制剂(Oryzacystatin,OC)是除贮藏蛋白外一类重要的稻米蛋白,为筛选稻米OC来源的生物活性肽,以稻米OC蛋白质序列为对象进行生物信息学分析和计算机辅助酶解,建立OC蛋白虚拟模拟胃肠道消化产物的肽数据库,预测其生物活性肽序列.进一步结合分子对接虚拟筛选具有结合潜力的活性肽并探讨其与靶蛋白的...     

Effect of mutagenesis at the region upstream from the G(Q/E) motif of three types of geranylgeranyl diphosphate synthase on product chain-length

(All-E) geranylgeranyl diphosphate synthases have been classified into three types based on the characteristic sequences around the first aspartate rich motif, which is highly conserved among the enzymes. In type I geranylgeranyl diphosphate synthases, which consist of archaeal enzymes, a bulky amino acid residue at the 5th position upstream from the motif plays a main role in the product determination, by blocking further elongation of prenyl chain as the bottom of the reaction cavity. On the other hand, type III geranylgeranyl diphosphate synthases, which consist of the enzymes from eukaryotes except for plants, use a bulky amino acid residue at the 2nd position upstream from the conserved G(Q/E) motif for product chain-length determination. Thus we introduced mutations into the region upstream from the G(Q/E) motif of geranylgeranyl diphosphate synthases of the three different types to confirm the importance of the region for the product chain-length determination. The results of the mutational analyses indicated that not only the 2nd but also the 3rd position upstream from the G(Q/E) motif is involved in the product chain-length determination mechanism in types I and III geranylgeranyl diphosphate synthases, while the amino acid substitution in this region did not affect the chain-length of the products of type II geranylgeranyl diphosphate synthase, which consist of the enzymes from bacteria and plants. The region upstream from the G(Q/E) motif possibly contributes to the product determination in the wide range of geranylgeranyl diphosphate synthases, as well as that around the first aspartate rich motif.     

Improving surface functional properties of tofu whey-derived peptides by chemical modification with fatty acids

Effect of acylation with saturated fatty acids on surface functional properties of tofu whey-derived peptides was investigated. Tofu whey (TW) and soy proteins (7S, 11S, and acid-precipitated soy protein [APP]) were hydrolyzed by Protease M 'Amano' G, and resulting peptide mixtures were acylated with esterified fatty acids of different chain length (6C to 18C) to form a covalent linkage between the carboxyl group of fatty acid and the free amino groups of peptide. Acylation significantly (P < 0.05) increased emulsifying properties of 7S, 11S, and APP peptides independent of fatty acid chain length. Acylation decreased water binding capacity although oil binding capacity of acylated tofu whey ultra filtered fraction (UFTW < 3 kDa), 7S- and 11S-peptides were improved compared to native peptides. 7S peptides acylated with long chain fatty acids had shown significant higher surface hydrophobicity as in contrast with acylated UFTW < 3 kDa and APP peptides. Fluorescence spectra studies revealed structural conformation of acylated soy peptides as compared to native peptides. This study shows that chemical modification with fatty acids can further affect functional properties of soy proteins.     

Low molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native flaxseed protein

Flaxseed protein isolate (FPI) contains high amount of arginine, which plays important physiological roles especially as nitric oxide precursor in the vascular endothelium. Arginine-rich peptides can be generated from FPI and used as a source of nitric oxide, which can produce in vivo vasodilatory effects during hypertension. Enzymatic hydrolysis of FPI with trypsin and pronase resulted in a hydrolysate that was fractionated using electrodialysis-ultrafiltration (EDUF). EDUF experiment resulted in migration of peptides to the anionic and cationic recovery compartments. Compared to FPI with 11% arginine, about one-third of the cationic fraction was composed of arginine. Thirteen potential peptide sequences were identified to be present in the cationic compartment of which 12 contained at least one arginine residue. None of the peptides identified from the anionic compartment contained arginine. Oral administration of the cationic peptides (200 mg/kg body wt.) to spontaneously hypertensive rats resulted in a more rapid decrease in systolic blood pressure when compared to similar amounts of FPI or the amino acid form of arginine. It was concluded that the rapid effect of the arginine-rich peptide product suggests faster rate of peptide absorption than amino acids and this may be exploited to provide fast relief from hypertension.