Short peptide fragments with antiulcer activity from a collagen hydrolysate

A peptide acidic hydrolysate of collagen (PHC) was obtained under conditions (4 N HCl) ensuring the predominant formation of short peptides, glyprolines. They were separated and their antiulcer activity was studied. Thirty individual peptides with molecular masses of 174-420 amu were isolated from the PHC by HPLC. The PHC was shown to predominantly contain 2- to 4-aa peptides, including PG, GP, and PGP. Experiments on rats demonstrated that, on intragastric administration at a dose of 1 mg/kg, PHC enhances the stability of the gastric mucosa to the action of ulcerogenic factors, such as ethanol and stress, and exhibits a protecting antiulcer effect. Even a lesser dose (0.1 mg/kg), which reduced ulcer area twofold, was effective in the stress model of ulcer formation. The intraperitoneal and intragastric administration of PHC at a dose of 1 mg/kg was found to exhibit a therapeutic effect in the acetate model of ulcer formation.     

Purification and evaluation of a novel antioxidant peptide from corn protein hydrolysate

In the present study, corn protein hydrolysate (CPH) with antioxidant activity was obtained by enzymatic hydrolysis. Corn gluten meal (CGM) was hydrolyzed using two proteases (Alcalase and Protamex) to produce the antioxidant peptide. Extrusion and starch removal of corn protein were used as pretreatment procedures before proteolysis. Hydrolysis by Alcalase has more remarkable digesting efficiency on corn protein than that by Protamex. Therefore, the hydrolysate catalyzed by Alcalase was fractionated by ultrafiltration, and peptide with the highest antioxidant activity was purified from <6 kDa molecular weight fraction. The amino acid sequence of the novel peptide was Gln-Gln-Pro-Gln-Pro-Trp as identified by a quadrupole time-of-flight mass spectrometer (Q-TOF2), with molecular weight of 782.34 Da, which was matched to γ-zein f (50–55). The new peptide was further synthesized by Fmoc solid-phase method. It showed scavenging activity against DPPH, ABTS, and hydroxyl free radicals in dose dependent manner with EC₅₀ values of 0.95, 0.0112 and 4.43 mg/mL, respectively. It also exhibited notable reducing power of 0.54 at 2.0 mg/mL, but showed weaker Fe²⁺-chelating capacity with EC₅₀ value of 6.27 mg/mL. These results suggest that the hexapeptide is a potential natural antioxidant that can be used as drug or functional food ingredient.     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

Design of novel peptide analogs with potent fungicidal activity,based on PMAP-23 antimicrobial peptide isolated from porcine myeloid

PMAP-23 is a 23-mer peptide derived from porcine myeloid. To develop novel antifungal peptides useful as therapeutic drugs, it would require a strong fungicidal activity against pathogenic fungal cells. To this goal, several analogs, with amino acid substitutions, were designed to increase the net hydrophobicity by Trp (W)-substitution at positions 10, 13, or 14 at the hydrophilic face of PMAP-23 without changing the hydrophobic helical face. The Trp (W)-substitution (P6) showed an enhanced fungicidal and antitumor activities, with the fungicidal activity inhibited by salts and the respiratory inhibitor, NaN(3). The results suggested that the increase of hydrophobicity of the peptides correlated with fungicidal activity. The fungicidal effects of analog peptides were further investigated using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. In Candida albicans, the analog peptide (P6) exerted its fungicidal effect on the blastoconidia in 20% fetal bovine serum by disrupting the mycelial forms. Furthermore, P6 caused significant morphological changes, and these facts suggested that the fungicidal function of the novel analog peptide (P6) was by damaging the fungal cell membranes. Thus, this peptide may provide a useful template for designing novel antifungal peptides useful for the treatment of infectious diseases.     

AurH1: a new heptapeptide derived from Aurein1.2 antimicrobial peptide with specific and exclusive fungicidal activity

Due to the increasing incidence of fungal opportunistic infections and emergence of antibiotic‐resistant fungal strains, antimicrobial peptides (AMPs) are considered as ideal candidates for antifungal compounds. In silico methods can reduce the limitations of natural AMPs such as toxicity and instability and improve their antimicrobial properties and selectivity. In this study, we designed AurH1, a new truncated peptide, based on the six‐amino acid sequence of Aurein1.2. Further , the antimicrobial activities and toxicity effects of AurH1 on human skin fibroblast cells and red blood cells were investigated. Finally, field emission scanning electron microscopy (FE‐SEM) and flow cytometry were performed in order to study the mechanism of action of AurH1. The results indicated that AurH1 had only antifungal activity (at a minimal inhibitory concentration (MIC) of 7.3‐125 μg/mL) without any antibacterial effects on the selected bacteria, while Aurein1.2 had both antifungal and antibacterial activities as positive control. Furthermore, AurH1 did not show any toxicity on Hu02 cells and human red blood cells at its MIC range. In conclusion, it became clear that AurH1 is a selective peptide against fungi with no toxic effects on the selected bacteria and human cells.     

In vitro and in silico studies of novel synthetic ACE-inhibitory peptides derived from Saccharomyces cerevisiae protein hydrolysate

The structure-function relation of YR-10 (YGKPVAVPAR) was investigated by synthesizing four structural analogs of that including YHR-10 (YGKHVAVHAR), GA-8 (GKPVAVPA), GHA-8 (GKHVAVHA), and PAR-3 (PAR). GA-8 (GKPVAVPA) was synthesized on the basis of simulated enzymatic gastrointestinal digestion performed by bioinformatics tools (expasy-peptide cutter). This study explains the molecular mechanisms for the interaction of synthetic peptides with ACE. The IC₅₀ values of each were 139.554 ± 2.3, 61.91 ± 1.2, 463.230 ± 3.56, 135.135 ± 2.1, 514.024 ± 5.86 µM, respectively. Results indicated that Pro replacement with His in YR-10 and GA-8 increased ACE inhibitory activity respectively, by 55.63% and 70.82%. Removal of Tyr and Arg from respectively N and C terminal positions of YR-10, following in silico simulated gastrointestinal digestion caused the 3.31 fold decrease in ACE inhibitory activity. YHR-10 showed the best docking poses, and GHA-8 exhibited interaction with Zn²⁺. Lineweaver–Burk plots of most active peptides suggest that they act as noncompetitive inhibitors against ACE.     

Functionalization of peptide nucleolipid bioconjugates and their structure anti-cancer activity relationship studies

In the search for more potent peptide-based anti-cancer conjugates the generation of new, functionally diverse nucleolipid derived D-(KLAKLAK)₂-AK sequences has enabled a structure and anti-cancer activity relationship study. A reductive amination approach was key for the synthesis of alkylamine, diamine and polyamine derived nucleolipids as well as those incorporating heterocyclic functionality. The carboxy-derived nucleolipids were then coupled to the C-terminus of the D-(KLAKLAK)₂-AK killer peptide sequence and produced with and without the FITC fluorophore for investigating biological activity in cancer cells. The amphiphilic, α-helical peptide-nucleolipid bioconjugates were found to exhibit variable effects on the viability of MM.1S cells, with the histamine derived nucleolipid peptide bioconjugate displaying the most significant anti-cancer effects. Thus, functionally diverse nucleolipids have been developed to fine-tune the structure and anti-cancer properties of killer peptide sequences, such as D-(KLAKLAK)₂-AK.     

抗菌肽RIKL的分子设计及生物学活性分析

天然抗菌肽具有较强的杀菌能力,但高生物相容性抗菌肽的构建一直阻碍着该领域的发展.为了提高抗菌肽的选择特异性,通过分子动力学分析探讨了抗菌肽的结构特性,并检测其生物学活性.首先以(RXKY)2(YRY)2 (X代表Ile,Y代表Leu)为模板设计新型抗菌肽分子RIKL.通过圆二色谱(circular dichroism,...     

Immunosuppressory activity of ubiquitin fragments containing retro-RGD sequence

A peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) (U50-59) possesses a very high immunosuppressory activity, comparable to that of cyclosporine, both in the cellular and humoral immune responses. We found that the pentapeptide DGRTL (U52-56) is the shortest, effective immunosuppressory fragment of ubiquitin, although its potency is weaker than that of U50-59. Replacement of each consecutive residue with alanine in U52-56 allowed identification of essential amino acids involved in the immunosuppression. We also evaluated the roles of its N- and C-terminal groups by their acetylation and/or amidation, respectively. The active sequence is located in the external loop of the molecule and therefore it may serve as an important functional epitope for intermolecular binding. Based on the crystal structure of ubiquitin molecule, we designed and synthesized the cyclic analogue with a restricted conformation, cyclo(Glt-Gln-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Lys)-NH2 (Glt = glutaryl) by reacting the C-terminal Lys side chain with the glutarylated N-terminus. The peptide was designed to mimic the ubiquitin(48-59) loop, in order to obtain the ligand that may interact with hypothetical receptors of the loop. The cyclization product selectively but strongly suppresses the cellular immune response. The results indicate that the 48-59 loop may serve as an important functional epitope in the ubiquitin molecule for intermolecular binding.     

Antimicrobial activity and secondary structure of a novel peptide derived from ovalbumin

A novel antimicrobial peptide derived from ovalbumin has been discovered. First, the peptide fragment RKIKVYLPRMK (TK9.1) was identified based on computerized predictions of the secondary structure of peptides in a protein data bank. Using HeliQuest, the sequence was developed into RKIKRYLRRMI (TK9.1.3), which was synthesized using Fmoc‐solid phase peptide synthesis, and found to have strongly antimicrobial activity against Gram‐positive and Gram‐negative bacteria, and fungi but not cytotoxic to HeLa cells and hemolysis in mouse red blood cells. Although ovalbumin itself does not have an antibacterial activity, our results suggest that it may supply the organisms that consume it with antimicrobial peptides, in support of their immunodefence.     

The effect of N-terminal substitutions on the biological activity of MSH fragments.

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of -melanotropin, [Glp⁵]-MSH(5–10), [Glp⁵, -Phe⁷]-MSH(5–10), [Sar⁵, -Phe⁷]-MSH(5–10), [Nle⁴, -Phe⁷]-MSH(4–10), [N-carbamoyl]-MSH(5–10), and formyl and acetyl derivatives of -MSH(5–10), [Gly⁵]-MSH(5–10) and [Gly⁵, -Phe⁷]-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Co-refolding of two peptide fragments derived from Agrobacterium tumefaciens beta-glucosidase with catalytic activity

Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of β-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.     

PvD1 defensin,a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis

Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD₁ from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD₁ could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD₁ defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD₁ defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.     

Modeling the angiotensin‐converting enzyme inhibitory activity of peptide mixtures obtained from cheese whey hydrolysates using concentration–response curves

Three mathematical models, two logistic models (previously published in previous works) and one mechanistic, developed in this work and based on Michaelis–Menten kinetics, were compared to select the most adequate model in describing the angiotensin‐converting enzyme (ACE)‐inhibitory activity of bioactive peptide mixtures obtained from cheese whey protein. The significance of both the model and its parameters as well as the value of the regression coefficient was used as criteria to select the most adequate model for obtaining the IC₅₀ values corresponding to each bioactive peptides mixture. The best results were obtained with the Michaelis–Menten‐based model because it provided the best fits and in addition the values for its parameters were always significant. As parameters of this model have a physical meaning, it could be used for inhibition‐testing experiments in the development of novel bioactive peptides. The results obtained indicated that the peptide mixture derived from the neutrase hydrolysis exhibited strong ACE inhibition activity. The main active peptides were short, with molecular masses below 1 kDa (IC₅₀ = 40.37 ± 2.66 μg/mL) and represent 38% of the initial protein content in the hydrolysate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 kD,对革兰氏阳性菌(G+)和革兰氏阴性菌(G?)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体pET22b-ELP20-EK-YFGAP、pET22b-ELP40-EK-YFGAP和pET22b-ELP40-L-EK- YFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。     

Synthesis and structure-function study of artificial nucleases on the basis of hemin conjugates with peptide fragments of cell differentiation factor HLDF

A series of octa-hexapeptide fragments of HLDF and their conjugates with hemin were obtained by solid phase peptide synthesis. A relationship between the structure and the nuclease activity of the compounds was established. The effect of various factors (medium pH, the presence of metal ions, complexons, reducers, and buffer composition) on the DNA destruction with hemin peptides was studied. Preliminary information confirming an oxidative mechanism of this process was obtained. The cleavage of plasmid DNA under the action of hemin peptides was studied by the methods of electron microscopy, gel electrophoresis, and atomic force microscopy.     

鸡源抗菌肽Fowlicidin-2在大肠杆菌中的 重组表达及其生物学活性鉴定

【目的】对抗菌肽Fowlicidin-2基因进行克隆与表达,并鉴定其生物学活性。【方法】根据抗菌肽Fowlicidin-2氨基酸序列,依照大肠杆菌(E.coli)密码子的偏爱性,人工设计合成其编码基因。与质粒pET-32a连接,构建重组表达载体,转化表达宿主菌E.coliBL21(DE3),IPTG诱导表达,融合蛋白经溴化氰裂解后进行纯化,测定重组抗菌肽的抑菌活性。【结果】Fowlicidin-2融合蛋白以包涵体形式表达,经溴化氰裂解后,成功释放出Fowlicidin-2,获得的重组Fowlicidin-2对革兰氏阳性菌和革兰氏阴性菌均有明显的抑菌效果。【结论】实现了抗菌肽Fowlicidin-2的重组表达,为抗菌肽的重组量化制备提供了理论基础与技术手段。     

Identification and characterization of a bactericidal and proapoptotic peptide from Cycas revoluta seeds with DNA binding properties

Nowadays, novel pharmacies have been screened from plants. Among them are the peptides, which show multiple biotechnological activities. In this report, a small peptide (Ala–Trp–Lys–Leu–Phe–Asp–Asp–Gly–Val) with a molecular mass of 1,050 Da was purified from Cycas revoluta seeds by using reversed‐phase liquid chromatography. This peptide shows clear deleterious effects against human epidermoid cancer (Hep2) and colon carcinoma cells (HCT15). It caused inhibition of cancer cell proliferation and further disruption of nucleosome structures, inducing apoptosis by direct DNA binding. A remarkable antibacterial activity was also observed in this same peptide. Nevertheless, no significant lysis of normal RBC cells was observed in the presence of peptide. Additionally, an acetylation at the N‐termini portion is able to reduce both activities. Bioinformatics tools were also utilized for construction of a three‐dimensional model showing a single amphipathic helix. Since in vitro binding studies show that the target of this peptide seems to be DNA, theoretical docking studies were also performed to better understand the interaction between peptide and nucleic acids and also to shed some light on the acetyl group role. Firstly, binding studies showed that affinity contacts basically occur due to electrostatic attraction. The complex peptide‐ssDNA was clearly oriented by residues Ala¹, Lys³, and Asp⁶, which form several hydrogen bonds that are able to stabilize the complex. When acetyl was added, hydrogen bonds are broken, reducing the peptide affinity. In summary, it seems that information here provided could be used to design a novel derivative of this peptide which a clear therapeutic potential. J. Cell. Biochem. 113: 184–193, 2012. © 2011 Wiley Periodicals, Inc.     

鲮鱼皮胶原肽的制备及其抗氧化活性的检测

为了以细菌胞外蛋白酶酶解低值蛋白资源制备抗氧化活性肽以及挖掘新型蛋白酶,采用液体发酵培养的方法对假交替单胞菌Pseudoalteromonas sp.SHK1-2进行发酵产酶,获得胞外蛋白酶粗酶液用于酶解热水法提取的鲮鱼胶原蛋白,通过超滤、sephadex LH-20分子筛层析获得具有DPPH自由基清除能力(35.6%±7%)、氧自由基清除能力(Oxygen radical absorbance capacity,ORAC)及DNA氧化损伤抑制活性的小分子肽,液相色谱质谱联用鉴定该活性肽分子量为776.2 Da,预测氨基酸序列为Thr-Ala-Gly-His-Pro-Gly-Thr-His。ORAC检测验证了人工合成的多肽同样具有抗氧化活性。研究表明细菌胞外蛋白酶在低值资源高值化中具有一定的应用前景,对于新型蛋白酶及其应用的挖掘具有一定的参考意义。