穿膜肽-LacI HPM载体的构建及其DNA结合活性测定

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,构建新型基因转导载体。方法：PCR扩增LacI、Lacl基因前头肽序列、前头肽序列突变体、TAT序列的编码基因,构建前头肽序列突变体和TAT的原核表达载体,可溶性表达TAT-LacI HPM融合蛋白并纯化,在缇冲液中氧化获得TAT-LacI HPM二聚体并浓缩,PCR检测二聚体融合蛋白与质粒的体外结合能力。结果：获得了pET-28a（-4-）-LacI HPM及pET-28a（＋）-TAT-LacI HPM表达质粒,表达纯化并获得二聚化融合蛋白,体外结合实验确定TAT-Lac／HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论：构建了穿膜呔TAT-LacI HPM,为进一步研究其作为新型DNA转运载体的可行性奠定了基础。     

细胞穿膜肽的穿膜活性与序列特征的关系

细胞穿膜肽(cell penetrating peptides,CPPs)是一种小分子多肽,能够容易地穿过细胞膜.这类分子,尤其是具有靶向功能的CPPs为高效率投送药物到靶细胞带来希望.因此,对其展开研究对于生物医学有着一定的意义.本工作主要从序列水平对具有不同穿膜活性的CPPs进行研究,试图找出影响CPPs穿膜活性的因素,以及不同活性CPPs与非穿膜肽(Non CPPs)序列上的差异,并引入一种分析生物序列的方法.我们基于CPPsite数据库和不同的文献获取CPPs和Non CPPs序列,并进一步从CPPs序列中提取具有高、中、低穿膜活性的穿膜肽(HCPPs、MCPPs、LCPPs)用于构建数据集.基于这些数据集,开展了以下研究:首先,利用方差分析的方法,对不同活性的CPPs以及Non CPPs的氨基酸及二级结构组成进行分析,发现氨基酸的静电与疏水相互作用对CPPs的穿膜活性起到了重要影响,同时螺旋结构和无规卷曲也会影响CPPs的穿膜活性;其次,使用理化性质与长度将不同活性的CPPs展示在二维平面上,发现在某些特殊的性质下不同活性的CPPs与Non CPPs可以产生聚簇现象,HCPPs、MCPPs以及LCPPs和Non CPPs被分成了三簇,这种现象显示了它们之间的差异;最后,本文引入了生物序列理化质心的概念,将组成序列的残基看作质点,进而把序列抽象成质点系进行研究,并将此方法应用到CPPs的分析中,通过PCA方法将不同活性的CPPs投射到三维平面上,结果发现绝大部分CPPs聚在一起,部分LCPPs与Non CPPs聚在一起.此工作对于CPPs的设计,以及理解不同活性CPPs序列上的差异具有一定的意义.另外,本文引入的生物序列理化质心的分析方法也可以用于其他生物问题的分析,同时它们可以作为某些生物分类问题的输入参数,在模式识别中起到一定的作用.     

穿膜肽的结构特点和穿膜机制

穿膜肽（penetratin）是果蝇的触角足同源异形域的DNA结合结构域第三个片段的商品名,由16个氨基酸残基组成,它可以介导多种疏水大分子进入活体细胞质内而不破坏细胞膜的完整性;其最大的特点是可以介导多种大分子进入细胞内,并且无需外源能量,分子作为整体插入细胞内,穿膜过程不需解折叠。该发现开辟了药物进入细胞的新的介导途径。该文介绍穿膜肽的结构特点、穿膜机制、应用及局限性。     

穿膜肽的内化机制及其应用

穿膜肽是一类具有特殊穿膜功能的多肽分子,能携带其它分子甚至超分子颗粒穿膜进入细胞内部．早期研究认为,其进胞是一种无需受体、也不存在饱和状态的非经典胞吞行为．近年研究表明,其穿膜机制可能与其含有的氨基酸种类有很大关系．现在,穿膜肽的穿膜过程称为巨型胞饮行为,它与传统的胞吞形式很相似．当然,还可能存在着其它的进胞方式而没有被证明或发现．关于穿膜肽的应用也是人们最感兴趣的,在很多领域的研究都在进行并不断取得进展．不论是生物界还是医学界,穿膜肽都被认为将是一类非常有发展潜力的多肽分子．     

膜穿透肽的应用与穿膜机制研究

膜穿透肽(membrane penetrating peptide,MPP)能引导大分子物质穿透细胞膜.应用MPP为载体,引导神经营养分子通透血脑屏障进入神经元,能有效治疗中枢神经系统疾病;在基因治疗方面, MPP引导干扰小RNA进行基因治疗,避免了使用病毒载体等一些传统基因治疗方法的毒副作用.穿膜机制研究证实 MPP通透细胞膜的过程分为三个阶段:与细胞表面结合;细胞巨胞饮摄取 MPP;MPP从胞饮体中逃逸入胞质,其中最后阶段是限速步骤.随着对多肽片段的深入研究和穿膜机制的逐渐明晰,MPP的应用将会更为深入和广泛.     

穿膜肽的研究现状及应用

以细胞内物质为靶标的药物(大分子、蛋白质、多肽及核酸)只有穿透细胞膜才能进一步发挥其药效。细胞穿透多肽(穿膜肽)是由少于30个氨基酸残基组成的小肽,它们能够通过与细胞膜相互作用而穿透细胞膜这一天然屏障。穿膜肽大致分为宿主防御肽、基于信号序列的穿膜肽和富含精氨酸的穿膜肽;穿膜肽进入细胞的机制尚未完全阐明,存在倒置微团模型、地毯式模型及打孔模型等假说。穿膜肽能够携带各种物质进入细胞的特性受到人们的关注。我们就穿膜肽的种类、穿膜机制,及其在生物影像学和生物递送系统中的应用做一综述。     

细胞穿膜肽的研究进展

细胞穿膜肽是一类能携带大分子物质进入细胞的短肽,其穿膜能力不依赖经典的胞吞作用。经过对天然存在的细胞穿膜肽的生物化学性质研究,已经逐渐掌握了细胞穿膜肽的一些共有特性,这类物质均为带有正电荷的长短不等的多肽片段,其中富含精氨酸、赖氨酸等碱性氨基酸残基,二级结构皆具有α-螺旋的空间构象。利用这些特性,目前已人工合成了穿透力更强、效率更高的穿膜肽PEP-1、MPG,并且成功地携带大分子物质进入细胞发挥生物学活性。     

穿膜肽介导的新型基因转运载体的功能研究

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,建立-种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a（＋）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为-种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。     

穿膜肽TAT和R9的生物学效应的体内体外研究

本文比较了两种常用穿膜肽TAT和R9的体外穿膜效率、对细胞影响以及小鼠活体内不同器官的穿膜效率。非特异性穿膜肽TAT和R9分别为11个氨基酸和9个氨基酸的短肽,能够有效地穿过细胞膜进入细胞。本研究纯化制备出融合蛋白TAT-EGFP及R9-EGFP多肽,用相同浓度的活性TAT-EGFP和R9-EGFP处理肺癌细胞,观察比较二者的穿膜活性及效率;设置不同浓度梯度检测两种穿膜肽是否对细胞产生影响;通过小鼠腹腔注射TAT-EGFP和R9-EGFP活性肽段,研究二者在活体能够穿膜到达的靶位置,并比较分析二者穿膜的效果器官分布。结果显示制备获得的两种融合蛋白TAT-EGFP和R9-EGFP均能有效穿过实现穿膜功能,两种穿膜肽对乳腺癌细胞影响较小。活体试验证明两种穿膜肽在心、肝、脾、肺、肾器官均有分布,R9主要富集在小鼠的肾和肝脏中,TAT在6种被研究的器官都有富集,两种细胞穿膜肽都能透过血脑屏障到达脑部。相对来说,TAT在小鼠活体中的穿膜效果更好,荧光富集多,R9整体穿膜效果较弱。本研究分析比较了两种穿膜肽的细胞安全性浓度和活体的穿膜能力,为TAT和R9两种穿膜肽在后续试验中的合理有效的选择和使用,提供了基础数据和指导性依据。     

细胞穿膜肽在肿瘤靶向治疗及疾病诊断中的应用

近年来细胞穿膜肽(cell-penetrating peptides,CPP)在生物医药领域被广泛应用,它为生物分子的胞内递送提供了有效的策略。关注CPP在肿瘤治疗及疾病诊断中的作用,并重点介绍其在肿瘤靶向治疗和医学影像诊断中的应用及优势。同时,根据CPP在药物传递系统中的特点,改进CPP存在的不足,扩大其联合用药的可能性,这也成为CPP研究的热点。对CPP及其在肿瘤等疾病的诊断及治疗中的应用作一综述,并简述其优化及改进策略,以期促进CPP在临床中的应用。     

Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.     

Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes

A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk l-α-phosphatidylcholine (EYPC)/egg yolk l-α-phosphatidyl-dl-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-d-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.     

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.     

Enhanced intracellular peptide delivery by multivalent cell-penetrating peptide with bioreducible linkage

Multivalent cell-penetrating peptides (CPPs) have been reported to show enhancement in cellular uptake and endosomolytic activity. However, its application was limited to trans-delivery of cargo which is lower in cellular uptake efficiency of cargo than cis-delivery. Here, we tried the cis-delivery of cargo with multivalent CPP by preparing bioreducible dimeric CPP–cargo with apoptotic activity using TatBim peptide, a fusion of Tat CPP and Bim peptide derived from Bim apoptosis-inducing protein. Dimeric TatBim was almost twice as highly internalized by cells and significantly induced apoptosis compared to monomeric TatBim. Contribution of bioreducible linkage of dimeric TatBim towards apoptotic activity was also confirmed.     

A new peptide with membrane-permeable function derived from human circadian proteins

Recently, the small regions of some proteins suchas human immunodeficiency virus type 1 (HIV-1) Tat-(48-60) and Drosophila Antennapedia-(43-58) have beenreported to have the ability to translocate through the cellmembranes and carry the exogenous molecules into cyto-plasm and nucleus, and are called membrane-permeablepeptides (MPPs) [1–14]. Delivery of bioactive peptidesacross the blood-brain barrier is generally restricted to small(6 amino acids or less) or highly lipophilic peptides. But…     

抗菌肽P7抑制大肠杆菌的非膜作用机制北大核心CSCD

【目的】研究抗菌肽P7抑制大肠杆菌的非膜作用机制。【方法】P7与溴化乙锭竞争结合大肠杆菌基因组DNA的荧光光谱,分析P7与DNA的结合方式;流式细胞术分析P7与大肠杆菌基因组DNA结合对细菌细胞周期的影响;采用磁珠富集和PCR扩增相结合的方法分析P7特异结合的DNA序列;通过实时荧光定量PCR分析P7对大肠杆菌DNA复制和SOS损伤修复基因表达的影响;核酸染料的荧光分析研究P7对大肠杆菌DNA和RNA合成的影响。【结果】P7以嵌插的方式作用于大肠杆菌基因组DNA碱基对并形成肽-DNA复合物,使溴化乙锭-DNA复合体系的荧光强度减弱。P7可以显著增加大肠杆菌细胞周期中S期细胞数目,抑制大肠杆菌DNA复制。P7特异性结合rnh A使该基因表达水平显著下调2.24倍。同时,在肽的影响下参与大肠杆菌DNA复制相关的ssb、dna G、lig B和rnh A基因的表达水平显著下调(P<0.05),DNA损伤修复的rec A和rec N基因显著上调(P<0.05)。P7可降低大肠杆菌DNA和RNA的合成。【结论】P7特异性地结合rnh A序列引起大肠杆菌DNA的损伤并抑制大肠杆菌的DNA复制。在P7的影响下,参与大肠杆菌DNA复制相关的基因的表达水平下调,DNA损伤修复基因显著上调,同时抑制大肠杆菌DNA和RNA的合成。     

Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay

Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.     

A cell-penetrating peptide derived from mammalian cell uptake protein of Mycobacterium tuberculosis

A Mycobacterium tuberculosis membrane protein called Mycobacterium cell entry protein (Mce1A) was previously shown to mediate the uptake of nonpathogenic Escherichia coli and latex beads by nonphagocytic mammalian cells. Here we characterize further the in vitro invasive activity of Mce1A using colloidal gold nanoparticles and fluorescent latex microspheres. Mce1A-coated colloidal gold particles induced plasma membrane invagination and entered membrane-bound compartments inside HeLa cells. Few of the protein-coated particles were also found in the cytosol compartment. Cytochalasin D and nocodazole inhibited the uptake by HeLa cells, indicating that rearrangement of both microtubules and microfilaments was necessary for the uptake. The functional domain of Mce1A for invasion was narrowed to a highly basic 22-amino acid sequence termed Inv3. A synthetic Inv3 peptide stimulated uptake of colloidal gold particles as well as latex microspheres by HeLa cells. A chimeric protein composed of Inv3 sequence at the N terminus of beta-galactosidase appeared to stain the nuclear membrane, suggesting that it entered the HeLa cell cytoplasm. These observations suggest that the cell uptake activity of Mce1A is confined to a small peptide domain located in the core region of the protein. Inv3 could be used to ferry any protein in fusion with it into mammalian cells and may serve as a potent nonviral delivery system.     

Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 +/- 0.0122 m mol(-1) s(-1), higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells.     

Secondary conformational conversion is involved in glycosaminoglycans-mediated cellular uptake of the cationic cell-penetrating peptide PACAP

Glycosaminoglycans (GAGs) contribute to the cellular uptake of cationic cell-penetrating peptides (CPPs). However, molecular details about the contributions of GAGs in CPP internalization remain unclear. In this study, we examined the cellular uptake mechanism of the arginine-rich CPP pituitary adenylate-cyclase-activating polypeptide (PACAP). We observed that the uptake efficacy of PACAP is dependent on the expression of cell surface GAGs. As the binding of PACAP to sulfated GAGs induced a random coil-to-α-helix conformational conversion, we investigated the role of the helical formation in PACAP internalization. Whereas this secondary structure was not crucial for efficient internalization in GAGs-deficient cells, PACAP α-helix was essential for GAGs-dependent uptake.