细胞穿膜肽研究应用的新进展

细胞穿膜肽(Cell-penetrating peptides,CPPs)是一类能够穿过细胞膜或组织屏障的短肽。CPPs可通过内吞和直接穿透等机制运载蛋白质、RNA、DNA等生物大分子进入细胞内发挥其效应功能。相比于其他非天然的化学分子,CPPs具有生物相容性佳、对细胞造成的毒性小、完成入胞转运后可降解、并能与生物活性蛋白直接融合重组表达等优点,因此成为以胞内分子为靶标的药物递送技术发展的重要工具,并在生物医学研究领域具有良好的应用前景。文中针对CPPs的分类特点、入胞转运机制及其治疗应用的新近研究进展进行综述和讨论。     

细胞穿膜肽入胞检测方法概述

迄今为止,已有多达上百种的细胞穿膜肽(cell-penetrating peptides,CPPs)被发现报道,但这类多肽分子的入胞能力参差不齐,限制了其作为药物载体的应用。虽然已有多种实验方法可用于细胞穿膜肽入胞的检测,但由于缺乏通用的技术来确切证实CPPs的入胞能力,所以应当结合使用多种方法以降低误差。对不同的技术在检测CPPs入胞时的优缺点进行比较,并针对性地提出比较理想的解决方案,可为制订CPPs入胞标准化检测步骤提供一些参考。     

细胞穿膜肽介导蛋白/多肽类药物输送的研究进展

在当前药物研发中,蛋白/多肽类药物占据着重要地位。然而,此类药物大多需进入细胞内才能发挥作用,故细胞摄取率低的问 题成为制约其发展的关键因素。细胞穿膜肽是一类富含精氨酸的短肽,自身具有较强的生物膜穿透能力,可携带多种大分子甚至是纳米 粒入胞。因此,穿膜肽被广泛应用于药物输送,且基于穿膜肽介导药物胞内输送,成为解决蛋白/多肽类药物入胞问题的优选策略。主 要综述穿膜肽介导蛋白/多肽类药物输送用于不同疾病治疗的研究进展。     

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。     

细胞穿膜肽作为药物载体的研究进展

由于细胞膜的天然屏障作用,大多数具有治疗作用的极性分子、多肽、寡聚核苷酸很难进入细胞内发挥药效作用[1~3],因此很多具有良好治疗功能的大分子生物活性物质在实际应用中受到了极大的限制.为了克服这一治疗障碍和药物的转运,人们已经发展了各种穿透细胞膜的技术[4].目前通过细胞膜向细胞内转运的技术有病毒载体和非病毒载体.非病毒载体包括电穿孔、显微注射和脂质体转染[5,6].虽然这些方法广泛使用,但是存在很大的局限性,如转入效率低,细胞毒性大等.因此,寻找一种高效、无毒和能够穿过细胞的物质势在必行.     

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.     

细胞穿透肽应用的研究进展

细胞穿透肽是一类具有特殊细胞膜穿透作用的短肽,研究发现其为药物大分子物质的细胞内转运提供了有力工具,现已得到了广泛关注和大量研究.一方面,近年来发现,细胞穿透肽的应用面临着药物释放率、代谢降解、细胞系的分化状态和Rho-GTPases活性的依赖性等问题,从而阻碍了其研究开发的进程.另一方面,细胞穿透肽在肿瘤治疗、抗菌和抗炎靶向药物等方面的研究却取得了显著的进展,甚至进入了临床前和临床研究阶段.本文围绕细胞穿透肽在应用开发中所面临的挑战和机遇两方面,综述了近期的研究进展,并对细胞穿透肽在新领域中的应用进行了展望.     

Cell Penetrating Peptides and Cationic Antibacterial Peptides: TWO SIDES OF THE SAME COIN*

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.     

细胞穿透肽:一种新型非病毒载体

细胞膜的选择通透性对维持细胞内环境的稳定起着非常重要的作用,但细胞膜的这种特性限制了一些生物大分子和药物进入细胞内,不利于对一些细胞内疾病的诊断和药物靶向治疗的应用。如何将一些具有诊断和治疗潜力的生物大分子、药物通过细胞膜进入细胞内一直是医学界研究的热点和难点。细胞穿透肽是一类能够携带多肽、蛋白质、核酸、纳米颗粒、病毒颗粒及药物等穿过细胞膜进入细胞,导致完整载物内化的短肽,为生物大分子和药物进入细胞内部提供了有力的运载工具,其作为载体具有的高转导效率和低毒性特点,已经得到了广泛关注和大量研究。目前,细胞穿透肽作为生物分子和药物细胞内化的运载体已经在荧光成像,肿瘤治疗,抗炎治疗及药物靶向治疗中发挥了潜在的诊断和治疗作用,显示出其诱人的应用前景。     

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. d-Amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg₉-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg₉-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.     

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.     

Apolipoprotein E peptide-modified colloidal carriers: The design determines the mechanism of uptake in vascular endothelial cells

Supramolecular structures, particularly micelles and liposomes equipped with uptake-mediating address compounds, have attracted much attention as pharmaceutical formulations. Their development requires an understanding of the mechanism by which the carrier systems interact with and translocate into the target cells. We developed an apolipoprotein E-derived peptide, called A2, that efficiently translocates across cell membranes. Upon coupling of two palmitoyl chains (P2), the highly cationic sequence acquires detergent-like properties such as a strong tendency to self-associate and the ability to integrate into lipid bilayers. Confocal laser scanning microscopy and fluorescence activated cell sorting were used to compare the internalization of the fluorescence-labeled monomeric A2 with the uptake of the colloidal P2A2 micelles and P2A2-tagged liposomes into endothelial cells of blood vessels. Specific inhibitors of endocytosis were used to identify the underlying mechanisms. b.End3 and BAEC cells as example of endothelial cells of small capillaries and large vascular vessels, respectively, were examined. The uptake of monomeric A2 was characterized by poor cellular selectivity. A2 was efficiently internalized into both cell lines via at least two different mechanisms. Besides an endocytotic uptake route, a second passive pathway exists, that leads to a rapid distribution of A2 within the cytoplasm. Also liposomes tagged with P2A2 were non-selectively internalized into both b.End3 and BAEC cells. Their nonselective uptake was mediated by clathrin- and caveolin-independent endocytosis. In contrast, micellar P2A2 entered b.End3 cells via clathrin-mediated endocytosis, while no uptake of P2A2 into BAEC cells was observed. In conclusion, the specific clathrin-mediated uptake mode of P2A2 micelles might provide the basis for a blood brain barrier-specific targeting.     

新型细胞穿膜肽的鉴定方法与其在抗肿瘤治疗中的应用

如何将生物活性分子高效投递到靶标的细胞和组织仍然是生物治疗领域研究人员面临的难题之一。直到细胞穿膜肽(cell penetrating peptides, CPPs)的出现,其可介导多种外源性功能分子(核酸、多肽、蛋白质和化学药物)进入细胞,而且不影响外源活性分子的功能发挥。另外,CPPs在传递外源活性成分进入肿瘤组织和细胞方面表现出更具应用前景的优势。因此,通过对CPPs的分类、鉴定方法、穿膜机制、其在抗肿瘤治疗中的最新应用以及尚需要解决的问题进行综述,以期为新型CPPs的鉴定和其抗肿瘤治疗策略提供参考。     

分子内分子伴侣机制的研究进展

在原核生物、真核生物及病毒中,一些蛋白质的折叠不符合Anfinsen原则,即依靠自身的氨基酸序列是不够的,还需一段被称为分子内分子伴侣(IMC)的肽段来协助折叠．根据机制不同,IMC可分为两类：第一类IMC引导成熟肽折叠为具有空间结构的蛋白质;第二类IMC协助成熟肽的多聚化而使其获得生物学功能．IMC能提供比分子伴侣更契合的结构,更有效地引导成熟肽折叠,是一种更优的折叠策略．研究IMC分子机制,不仅能够确定IMC上哪些残基的协同作用引导成熟肽折叠,而且可通过改变或修饰其侧链来改造成熟肽,拓展传统的蛋白质工程．