A new bioproduction route for a novel antimicrobial peptide

Beta defensins are antimicrobial peptides (AMPs) with a broad spectrum antimicrobial behavior against pathogens while having minimal tendency to incur pathogen resistance. Human β-defensin 28 (hBD28) is a strongly cationic AMP and hence hypothesized to be highly effective in permeabilizing negatively-charged pathogen membranes. However, the scarcity of hBD28 in vivo has impeded detailed structure and antimicrobial studies of hBD28. Chemical synthesis of hBD28 rendered extremely poor yields due to inefficient cysteine oxidation. In this study, a rapid and scalable production route to produce bioactive hBD28 in Escherichia coli (E. coli) is reported. The design of a dual fusion tag expression construct was pivotal in enhancing soluble expression and easing purification of hBD28. The final hBD28 (purity >95%) displayed significant antimicrobial activity against E. coli K12 and showed dose-dependent killing kinetics. Circular dichroism spectroscopy confirmed the presence of both β-sheet and α-helix conformations in the secondary structure of hBD28.     

防御素的生物学特性及其抗病基因工程

防御素是一种富含半胱氨酸的小分子多肽,对细菌等微生物具有广谱抗性,且作用机制特殊。迄今为止,国内外在防御素方面进行了大量的研究,已经从各类生物体中分离出不同种类的防御素,并在基因工程和医药领域呈现广泛的应用前景。文章对防御素的分类、生物学特性,包括哺乳动物α-、β-、θ-防御素、昆虫以及植物防御素的分子结构及抗菌活性进行了综述,阐述了防御素的膜作用及与细胞内复合物结合的作用机制。总结和归纳了防御素基因的分离、表达研究进展及动、植物防御素基因在抗病基因工程领域的应用,并对防御素在未来的生物制药和植物抗病基因工程方面的应用前景进行了展望。     

α-Defensins Induce a Post-translational Modification of Low Density Lipoprotein (LDL) That Promotes Atherosclerosis at Normal Levels of Plasma Cholesterol

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def^+/+). Accelerated Def^+/+ mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def^+/+ to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def^+/+ mice prevented these outcomes. The same outcome was obtained by treating Def^+/+ mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis.     

The Urinary Proteomic Profile Implicates Key Regulators for Urologic Chronic Pelvic Pain Syndrome (UCPPS): A MAPP Research Network Study

Urologic chronic pelvic pain syndrome (UCPPS) is a condition of unknown etiology characterized by pelvic pain and urinary frequency and/or urgency. As the proximal fluid of this syndrome, urine is an ideal candidate sample matrix for an unbiased study of UCPPS. In this study, a large, discovery-phase, TMT-based quantitative urinary proteomics analysis of 244 participants was performed. The participants included patients with UCPPS (n = 82), healthy controls (HC) (n = 94), and disparate chronic pain diseases, termed positive controls (PC) (n = 68). Using training and testing cohorts, we identified and validated a small and distinct set of proteins that distinguished UCPPS from HC (n = 9) and UCPPS from PC (n = 3). The validated UCPPS: HC proteins were predominantly extracellular matrix/extracellular matrix modifying or immunomodulatory/host defense in nature. Significantly varying proteins in the UCPPS: HC comparison were overrepresented by the members of several dysregulated biological processes including decreased immune cell migration, decreased development of epithelial tissue, and increased bleeding. Comparison with the PC cohort enabled the evaluation of UCPPS-specific upstream regulators, contrasting UCPPS with other conditions that cause chronic pain. Specific to UCPPS were alterations in the predicted signaling of several upstream regulators, including alpha-catenin, interleukin-6, epidermal growth factor, and transforming growth factor beta 1, among others. These findings advance our knowledge of the etiology of UCPPS and inform potential future clinical translation into a diagnostic panel for UCPPS.     

Chemical Synthesis of Human β-Defensin (hBD)-1, -2, -3 and -4: Optimization of the Oxidative Folding Reaction

Reduced human β-defensin (hBD)-1, -2, -3 and -4 synthesized by Boc chemistry were subjected to oxidative folding reaction under optimal conditions. Among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione (GSH/GSSG), the buffer concentration and reaction temperature were essential for the predominant formation of the native disulfide structure. The homogeneity of the four synthetic hBDs was confirmed by analytical procedures using RP-HPLC, IEX-HPLC, capillary zone electrophoresis (CZE) and MALDI-TOF MS as well as sequencing, although high temperature (70 °C) was used for the RP-HPLC analysis of hBD-3 and hBD-4 to exclude the influence of equilibrium with the respective conformers having native disulfide pairing. All synthetic hBDs were shown to possess the native disulfide structure by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. Upon digestion of hBD-1 and hBD-4 with proline specific endopeptidase, the Cys-X bond was found to be reproducibly cleaved together with the Pro-X bond although the cleavage of Cys-X afforded the appropriate cystine segments for determining the disulfide structure of hBD-1 and hBD-4. With respect to antimicrobial activity against E. coli, the four synthetic hBDs of high homogeneity possessed the same potencies as those reported previously.Australian Peptide Conference Issue     

Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes

AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β (pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events. METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 μg/mL), and/or nigericin (Nig, 30 μmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies. RESULTS: We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1β, but not the processing of pro-IL-1β to IL-1β, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1β, but not the processing of IL-1β, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1β processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments. CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.     

菲律宾蛤仔（Ruditapes philippinesis）血浆中防卫素的纯化及其抑菌功能

经硫酸铵沉淀、凝胶排阻层析和离子交换层析 ,自菲律宾蛤仔 (Ruditapesphilippinnesis)血浆中分离纯化得到一种新的蛋白质———防卫素RPD 1(Ruditapesphilippinesisdefensin 1)。经SDS PAGE分析 ,其估计分子量为 2 4 .8kD ;经ABI4 73蛋白质自动测序仪测定 ,其N端部分氨基酸序列为AVPDVAFNAYG。在NCBI和EBI EMBL两个蛋白质数据库中未发现任何与该蛋白质有同源性的已知蛋白质。该蛋白质对革兰氏阳性菌和革兰氏阴性菌具有广谱抗性。对金黄色葡萄球菌 (Staphylococcusaureus)、枯草杆菌 (Bacillussubtilis)、四联微球菌 (Micrococcustetragenus)、大肠杆菌 (Escherichiacoli)、副溶血弧菌 (Vibrioparahaemolyticus)和鳗弧菌 (Vibrioanguillarum)的最小抑菌浓度分别为 9.6mg L、76 .8mg L、38.4mg L、76 .8mg L、19.2mg L和 19.2mg L。实验结果证明RPD 1为一种新的防卫素蛋白质。     

猪防御素PD基因表达载体的构建

目的：构建猪防御素PD基因表达载体。方法：化学合成经过适当改造的带有双酶切位点的PD基因,将该基因定向插入带有相同酶切位点的融合表达质粒PinPoin^TMXa-3的多克隆位点构建表达质粒。结果：构建的表达载体经双酶切电泳分析及插入基因片段序列分析,表明PD基因表达载体构建成功。结论：PD基因表达载体的构建,为进一步获得PD基因的表达产物,研究其抗菌活性、抗菌机理打下基础。     

Different dynamics and pathway of disulfide bonds reduction of two human defensins,a molecular dynamics simulation study

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human β defensin type 3 (hBD‐3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD‐3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD‐3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD‐3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD‐3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD‐3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD‐3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665–681. © 2016 Wiley Periodicals, Inc.     

Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth

Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development. A. Allen and A.K. Snyder contributed equally.