TAT蛋白转导域：蛋白质治疗的新曙光

TAT蛋白转导域是源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能够将与之共价连接的多肽、蛋白、核酸等生物大分子快速而高效地转导入细胞内部,在药物转运和疾病治疗等领域有着巨大的应用潜力.TAT蛋白转导域首先通过电荷相互作用吸附于细胞膜,然后通过脂筏介导的巨胞饮作用进入细胞.随着体外研究的不断成熟,应用TAT蛋白转导域治疗人类肿瘤、卒中、炎症等疾病的动物模型也获得了成功,TAT蛋白转导域进入临床指日可待.     

HIV-1 TAT蛋白转导肽的研究进展

TAT蛋白转导肽是人类免疫缺陷病毒1型(human immunodeficiency virus type 1, HIV-1)编码的一段富含碱性氨基酸、带正电荷的多肽,属于蛋白转导域家族的一员。长期研究发现其全长及11个碱性氨基酸富集区的核心肽段(YGRKKRRQRRR)不仅能够在包括蛋白质、多肽及核酸等多种外源生物大分子的跨膜转导过程中具有重要作用,而且能够携带这些外源生物大分子通过活体细胞的各种生物膜性结构(如细胞膜和血脑屏障等)并发挥生理功能,但其跨膜转导机制仍不明确。新近研究还发现TAT核心肽段在促进外源蛋白高效表达过程中也具有重要作用,能够显著增加外源蛋白高效、可溶性表达的水平,显示了TAT蛋白转导肽的新功能。以TAT蛋白转导肽跨膜转导作用的长期研究背景为基础,分别从TAT蛋白转导肽的结构特点、其跨膜转导作用的影响因素及其作用机制等方面进行了系统综述,进一步结合TAT蛋白转导肽的最新研究进展分别从药物研发、机制探索及新功能的开发等方面展望了后续研究方向与应用价值,不仅为深入阐述TAT蛋白转导肽的跨膜转导作用的功能意义提供了参考依据,而且为TAT蛋白转导肽在微生物工程及蛋白质工程等领域的潜在应用价值提供了重要参考信息。     

HIV-Tat蛋白转导域在医学研究中的应用

HIVTat蛋白转导域(proteintransductiondomain,PTD)是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,它能将与其共价连接的多肽、蛋白质及DNA等分子跨膜导入几乎所有的组织和细胞,甚至可以通过血脑屏障,转导效率很高而且对细胞没有损伤。TAT融合蛋白系统被认为是一种很有前途的运载工具,在基础医学研究和临床治疗方面都有着非常广泛的应用前景 。     

病毒感染复数影响腺病毒感染T淋巴细胞效率的初步研究

以往研究发现,增加感染复数(multiplicity of infection,MOI)可以有效提高5型腺病毒(Ad5)感染T细胞的效率,但其具体机制并未十分清楚。选取T淋巴瘤细胞为靶细胞,通过荧光定量PCR及透射电镜观察等方法探讨不同MOI对重组腺病毒Ad5-GFP复制周期中病毒结合及病毒进入两个环节的影响,发现高MOI条件下不会影响T细胞结合的腺病毒数量,却可显著增加经胞吞进入T细胞的腺病毒数量。这些结果表明,增加MOI有利于腺病毒经胞吞进入T细胞,从而可提高腺病毒感染效率。这为进一步研究腺病毒感染T淋巴细胞的机制提供了理论基础。     

TAT-EDAG融合蛋白的原核表达及其转导活性的研究

HIV-TAT蛋白转导域（PTD）是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,它能将与之连接的多肽、蛋白质及DNA等分子跨膜导入几乎所有的组织和细胞,转导效率高而对细胞没有损伤.构建了TAT-EDAG、TAT-GFP融合蛋白原核表达载体,在大肠杆菌BL21（DE3）细胞中实现了两种融合蛋白的可溶性原核表达,在非变性条件下进行蛋白纯化,获得了纯度在90%以上的融合蛋白.脱盐处理后,利用TAT-GFP转染体外培养的鼠成纤维细胞证实了TAT转导肽的生物活性;利用TAT-EDAG转染体外培养的HL-60细胞,Western blotting分析表明：TAT-EDAG可以导入HL-60细胞中.这为下一步应用于体外造血干细胞扩增研究奠定了基础.     

蛋白转导域透膜作用的研究进展

HIV-TAT蛋白转导域（Protein transduction domain,,PTD）是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能与多肽、蛋白质及DNA等分子连接并跨膜导入绝大部分的组织细胞或透过血脑屏障,转导效率高且对细胞无损伤。TAT-PTD与细胞膜之间的电荷作用,吸附于膜表面,依赖脂筏介导的巨胞饮作用进入细胞。TAT融合蛋白系统是一种极有价值的运载工具,在基础医学研究和临床治疗方面有着广泛的应用前景。     

硫利达嗪联合溶瘤腺病毒ZD55-TRAIL对HeLa细胞抑制作用及其机制探讨

靶向基因-病毒治疗方法是近年来产生的一种较为有效的癌症生物治疗方法。但其癌症治疗效果仍需进一步提高。在该研究工作中,通过联合使用临床神经治疗药物硫利达嗪（thior_idazine）与溶瘤腺病毒ZD55-TRAIL来增强对HeLa细胞的杀伤作用。通过MTT实验、倒置显微镜观察、结晶紫染色实验观察了联合使用thioridazine和ZD55-TRAIL对子宫颈癌细胞株HeLa细胞的毒性作用;使用Hoechst33342染色、流式细胞实验和Western blot实验检测了联合使用thioridazine和ZD55-TRAIL在引起HeLa细胞发生凋亡上的作用。结果表明,小分子药物thioridazine与病毒ZD55-TRAIL联合使用可以增强对HeLa细胞的杀伤作用,通过下调抗凋亡蛋白XIAP的水平,更显著地促进HeLa细胞发生凋亡。该研究首次报道了联合使用精神疾病药物thioridazine和ZD55-TRAIL对子宫颈癌细胞HeLa的抑制作用,可能是子宫颈癌治疗的一种有效方法。     

P37腺病毒表达载体的构建及对乳癌细胞的促迁移作用

胃癌组织中存在着较高的猪鼻支原体感染率,而P37是猪鼻支原体的主要免疫原.以往研究表明,P37能抑制肿瘤细胞的黏附,促进肿瘤细胞浸润和转移.为了更好地研究P37在肿瘤发生和转移中的功能,通过基因克隆的方法,利用Ad-easy体系,在细菌BJ5183中同源重组后,转染293细胞,成功包装出重组P37腺病毒.它能有效感染乳腺癌细胞BICR.通过RT-PCR和蛋白质印迹检测表明,感染重组P37腺病毒后的BICR细胞能大量表达并分泌P37蛋白.运用该腺病毒体系进行细胞迁移实验表明,P37能显著增强BICR细胞的体外迁移能力.     

TAT蛋白转导肽介导的秀丽线虫体内外源蛋白的跨膜转导研究

TAT蛋白转导肽是HIV-1病毒编码的一段富含碱性氨基酸序列的多肽,能够高效介导多种外源生物大分子通过多种膜性结构,如细胞质膜和血脑屏障等。为探索TAT蛋白转导肽介导的秀丽线虫体内外源蛋白跨膜转导作用,以EGFP为报告基因结合常规分子克隆技术构建了原核表达载体pET28b-EGFP和pET28-TAT-EGFP,继而利用诱导剂IPTG(终浓度1mmol/L)诱导表达了靶蛋白并结合荧光显微观察、SDS-PAGE和Western blot等鉴定技术获得表达靶蛋白的大肠杆菌BL21(DE3)细胞,最后将其涂布到含有Kana⁺的LB固体培养基上直接饲喂野生型N2株系线虫,利用荧光显微镜观察绿色荧光信号在线虫体内的分布。结果证明,TAT-EGFP融合蛋白较之于EGFP可高效、可溶性表达,而且通过直接饲喂秀丽线虫表达靶蛋白的大肠杆菌48小时后,TAT-EGFP荧光信号明显分布于线虫肠壁细胞,而EGFP荧光信号则分布在秀丽线虫肠腔,空载体对照组未见任何荧光信号,说明TAT蛋白转导肽能够高效介导外源蛋白在秀丽线虫体内跨膜转导。同时,通过比较空载体对照组与实验组线虫微分干涉图像,未见线虫出现明显的细胞形态变化,说明TAT蛋白转导肽介导的外源蛋白跨膜转导作用是安全的,为在秀丽线虫体内直接研究外源蛋白的功能以及进行蛋白药物的研发提供了重要参考。     

TAT蛋白介导外源物质进入细胞的作用机制探讨

来源于人类免疫缺陷病毒HIV-1的反式激活因子(trans-activator,TAT)蛋白能够有效的介导多肽、蛋白质、基因以及一些其它的物质进入细胞。它以低亲和力与细胞上的受体相结合,通过破坏细胞质膜,以非内吞途径转导外源物质进入细胞内部。     

Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis

The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.     

Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein

Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.     

Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.     

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.     

带有蛋白转导结构域的Bcr/Abl癌蛋白片段的表达及其跨膜转运

HIV 1编码的反式激活蛋白TAT具有将细胞外蛋白转导进入细胞的基序 ,称为蛋白转导结构域 (PTD) .为研究PTD介导的PTD Bcr Abl融合蛋白的跨膜转运 ,合成了编码PTD的基因片段 ,并与PCR扩增的慢性粒细胞白血病癌蛋白bcr abl基因片段融合 .在大肠杆菌中表达纯化了融合蛋白 ,将纯化的融合蛋白加入培养的HL60细胞和C2C12细胞后 ,发现PTD基序可以介导Bcr Abl蛋白自由从细胞外跨膜转导进入细胞内 .研究结果可能为用外源蛋白负载 (loading)免疫活性细胞如抗原提呈细胞提供新的途径 .     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.     

HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors

Several novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal stretch of 11 basic amino acid residues (47-57) from the human immunodeficiency virus type 1 (HIV-1) TAT protein transduction domain (PTD) for protein translocation and cellular localization. The vectors also contained a six-histidine (His(6)) tag at the N- or C-terminus for convenient purification and detection, and a multiple cloning site for easy insertion of foreign genes. Some heterologous genes including HSV-TK, Bcl-rambo, Smac/DIABLO and GFP were fused in-frame to TAT PTD and successfully overexpressed in Escherichia coli. The purified TAT-GFP fusion protein was able to transduce into the mammalian cells and was found to locate mainly in the cytosol when exogenously added to the cell culture medium. However, using a transfection system, mammalian-expressed TAT-GFP predominantly displayed a nuclear localization and nucleolar accumulation in mammalian cell lines. This discrepancy implies that the exact subcellular localization of transduced protein may depend on cell type, the nature of imported proteins and delivery approach. Taken together, our results demonstrate that a TAT PTD length of 11 amino acids was sufficient to confer protein internalization and its subsequent cellular localization. These novel properties allow these vectors to be useful for studying protein transduction and nuclear import.     

The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.