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.     

金属硫蛋白及其结构域突变体的紫外和圆二色光谱学研究

采用 p GEX- 4T- 1融合表达载体高效表达所得的金属硫蛋白及其结构域突变体 ,包括 α结构域 ,β结构域 ,α- KKS- α和 β- KKS- β( KKS为金属硫蛋白结构域之间的天然连接区氨基酸 ) ,经纯化和纯度鉴定后 ,利用紫外和圆二色光谱进行结构研究 .在脱金属的上述蛋白中 ,固定 p H为中性 ,改变加入 Cd2 + 的比例 ,或固定 Cd2 + 浓度 ,逐渐调节 p H至中性 ,观察紫外和圆二色光谱中镉硫金属簇吸收峰的形成 .研究结果表明 :镉硫金属簇的形成依赖于加入金属的比例和 p H值 ,所有蛋白均于p H3.1 5以上开始形成明显的吸收峰 .紫外图谱中的吸收峰位于 2 54nm附近 ,但在圆二色图谱中不同蛋白形成的峰的位置不同 ,MT,α结构域和 α- KKS- α在 2 2 5nm和 2 58nm处有吸收 ,β结构域在 2 60 nm处有吸收 ,而 β- KKS- β在 2 4 5nm处有吸收 ;向 α结构域 ,β结构域 ,MT,α- KKS- α和 β-KKS- β中分别加入 4eq,3eq,7eq,8eq和 6eq( eq:equivalent,当量 )的 Cd2 +时 ,吸收峰可达到最大值 .同时发现 α结构域的吸收峰强于 β结构域 ,而且双结构域突变体的镉硫金属簇则明显强于相应的单结构域突变体 ,这表明吸收峰的强弱与金属结合力的大小相关 ,而且结构域之间存在相互作用 ,从而影响与金属的结合 .     

Efficient intracellular delivery of an exogenous protein GFP with genetically fused basic oligopeptides.

Several oligopeptides, derived from certain proteins, translocate as a form fused to small molecules or exogenous proteins across the plasma membrane into cells. Some of these oligopeptides, the so-called protein-transduction domains (PTDs), contain a high proportion of basic residues. The translocation of some of these basic PTDs, such as oligoarginines, has been studied as chemically fused forms to other organic compounds. In this study, we also tested to determine whether or not oligoarginines, when fused genetically to an exogenous protein such as GFP, are also able to translocate efficiently across the plasma membrane. The oligoarginine Rn (n = 5,6,7,8,9)-GFP fusion proteins were translocated quite efficiently, and the transduction efficiency increased in proportion to the number of arginine residues. However, the cellular uptake of the oligolysine-GFP fusion proteins was less efficient than that of the corresponding oligoarginine-GFP fusion proteins. When fused to GFP, the translocation efficiency of R5 was similar to that of Tat(49-57)(RKKRRQRRR). This finding suggests that the arginine homo-oligopeptide is more efficient than other PTDs which contain a mixture of basic residues. On the other hand, both the K9- and Tat(49-57)-GFP fusion proteins were transduced with similar efficiencies. It appears that basic oligopeptides may be useful for the efficient translocation of diverse exogenous proteins as genetically fused forms.     

TAT蛋白质转导结构域与SV40启动子特异的三锌指结构融合蛋白的表达与纯化

利用蛋白质转导结构域(PTDs)可以将与之融合表达的蛋白质直接送入细胞中。将通过筛选噬菌体展示锌指库得到的特异作用于SV40启动子上9bp序列的三锌指结构的序列插入含有TAT蛋白的蛋白质转导结构域的表达载体pET—TAT-NLS中,构建融合蛋白的表达载体pET-TAT-NLS—clone3。融合蛋白在E．coli BL21(DE3)中得到了可溶性表达,含量约占总蛋白的18％;并通过镍亲和凝胶层析柱得到了较好的纯化融合蛋白。     

金属硫蛋白α和 β结构域的结构功能比较研究

金属硫蛋白具有α和β两个独立的结构域,它们结构不同,并能独立的行使功能。为了进一步研究这两个结构域之间的区别,分别采用镉和铜重组金属硫蛋白并继以枯草杆菌蛋白酶水解的方法制备α和β结构域,以及利用pGEX-4T-1这种融合表达载体表达α和β结构域。所得产物经凝胶过滤层析分离纯化后,进行了氨基酸组成,巯基和金属含量以及分子量测定,以上性质均与天然的金属硫蛋白α和β结构域相同。然后利用紫外吸收光谱和圆二色吸收光谱来研究它们的巯基金属簇结构,从UV和CD图谱可以看出,通过蛋白水解和基因表达制备的α和β结构域都具有独立的镉硫金属簇结构,但β结构域的镉硫金属簇不如α结构域紧密,其在254nm的吸收峰不象α结构域那么明显。利用DTNB的竞争反应测定了α和β结构域对镉和锌的结合力,实验结果表明,α结构域倾向于结合Cd2+,β结构域倾向于结合Zn2+。以上研究对于进一步了解α和β结构域的生理功能和分子进化提供了有利的证据。     

Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor-α induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.     

肺癌中金属硫蛋白的表达及其与细胞增殖、凋亡的关系

目的　探讨肺癌中金属硫蛋白 (Metallothionein ,MT)的表达及其与临床病理学参数、Ki 6 7、P5 3和凋亡的关系。方法　免疫组织化学SP法对 5 6例石蜡包埋的肺癌组织分别进行MT、Ki 6 7、P5 3蛋白的检测并对 11例石蜡包埋的癌旁正常支气管组织进行MT蛋白的检测。TUNEL法对 19例MT蛋白阴性和弱阳性表达及 2 8例MT蛋白中等阳性和强阳性表达的肺癌标本进行原位细胞凋亡检测。结果　MT蛋白在肺癌中的表达高于其在正常支气管组织中的表达 ,但无显著差异 (P =0 5 6 1) ;肺癌中MT蛋白的表达与患者的性别、肿瘤的组织学类型及分化程度有关 (均为P <0 0 5 ) ,与患者的年龄、肿瘤大小、TNM分期和淋巴结转移与否无关 (均为P >0 0 5 ) ,与Ki 6 7蛋白的表达呈正相关 (r=0 2 78,P <0 0 5 ) ,与凋亡呈负相关 (r=- 0 319,P <0 0 5 ) ,与P5 3蛋白的表达无显著相关 (r=0 16 7,P >0 0 5 )。结论　MT蛋白的表达与肺癌的分化程度、Ki 6 7及凋亡显著相关 ,MT可作为判断肺癌细胞分化程度、增殖活性及凋亡等的指标。     

Transtactin: a universal transmembrane delivery system for Strep‐tag II‐fused cargos

The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD‐fused Strep‐Tactin, which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of Strep‐tag II‐linked small molecules, peptides and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin‐introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for Strep‐tag II‐fused cargos.     

Shenfu injection suppresses apoptosis by regulation of Bcl-2 and caspase-3 during hypoxia/reoxygenation in neonatal rat cardiomyocytes in vitro

Shenfu injection (the major components of which are ginsenosides compound, extract of Panax ginseng shown to have antioxidant properties) is a well-known important Chinese traditional medicine used for the treatment of various diseases especial for cardiac diseases. The precise mechanism of the biological actions of this plant is not fully understood, in order to elucidate the protection of cardiomyocytes. The aim of the present study was to investigate the effect of Shenfu injection on hypoxia/reoxygenation (H/R)-induced apoptosis and the expression of bcl-2 and caspase-3 in cultured neonatal rat cardiomyocytes in vitro. Ventricular myocytes were isolated from neonatal rat hearts and were exposed to 4 h of hypoxia followed by 16 h of reoxygenation. The results indicated that treatment with different doses of Shenfu injection protected cardiacmyocyte cultures from hypoxia/reoxygenation-induced apoptosis. Caspase-3 activation was decreased in hypoxic/reoxygenationed cardiomyocytes co-treated with Shenfu injection when compared to hypoxia/reoxygenation alone treated cultures. Expression of the Bcl-2 proteins was increased in Shenfu injection-treated cardiomyocytes subjected to hypoxia/reoxygenation. In conclusion, ginsenosides compound has obviously protective effects on cardiacmyocytes against apoptosis induced by hypoxia/reoxygenation injury, whose mechanisms probably involve the inhibition of down-regulation of Bcl-2 protein levels and sequential activation of caspase-3.     

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.     

Hypoxia-Induced Modulation of Apoptosis and BCL-2 Family Proteins in Different Cancer Cell Types

Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM_EL. BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and –independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.     

Full quantum-mechanical structure of the human protein Metallothionein-2

Metallothioneins (MT) are small, metal-binding proteins with diverse functions related to metal ion homeostasis. This paper presents the full 384-388-atom structures of the two native Zn(II)- and the Cd(II)-containing domains of human MT2, optimized with density functional theory. The presented structures are accurate to ~ 0.03 Å for bond lengths and thus provide new physical insight into the detailed electronic structures of MTs, in particular with accurate accounts of bridging vs. terminal bonds not available from NMR or EXAFS. The MT protein enhances the asymmetry, as compared to the protein-free clusters, causing a hierarchy in binding that most likely allows MTs to transfer ions to multiple targets in vivo. The protein polarization is substantial and occurs primarily via the terminal sulfurs, a key mechanism in providing domain-specific electronic structures. The β-domain polarizes its smaller cluster less on average, due to its less polarizable, higher negative charge density, as reflected in longer MS bond lengths and smaller bond orders. This may explain why MT2β is more reactive and dynamic and why MTs have evolved two different-size, asymmetric domains with different metal binding affinities fit for different molecular targets of metal ion transfer.     

Enhanced uptake of a heterologous protein with an HIV-1 Tat protein transduction domains (PTD) at both termini

Poor membrane permeability of proteins is a major limitation of protein therapy. In a previous study, we showed that the minimal sequence required for efficient transduction of Tat-GFP is the basic domain from 49-57 of HIV-1 Tat called the protein transduction domain (PTD. Here we have generated HIV-1 Tat PTD GFP fusion proteins in which HIV-1 Tat PTD is fused with the N- and/or C-termini of GFP. The various GFP fusion proteins were purified from Escherichia coli and characterized for their ability to enter mammalian cells using Western blot analysis, confocal microscopy and flow cytometry. The GFP fusion protein with Tat PTD at its C-terminus was taken up as efficiently as the GFP fusion protein with Tat PTD at its N-terminus. However, the same protein with PTDs at its both termini was taken up even more efficiently. All the GFP fusion proteins were present in both the nucleus and cytosol of the transduced cells. Uptake was lower at 4 degrees C than at 37 degrees C. The availability of the expression vectors developed in this study may help to devise novel strategies in the rational development of protein-based drugs.     

Noncovalent protein transduction in plant cells by macropinocytosis

* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.     

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.     

Influence of protein transduction domains on target-specific chimeric proteins

Direct targeting to the cytoplasm and nucleus using protein transduction domains (PTD) has been described to be efficient but non-cell-type-specific, and only has clinical relevance when the molecule is active exclusively in the diseased cell. The use of PTDs is an attractive mechanism to improve drug delivery. In this work, we designed recombinant proteins that contain epidermal growth factor as ligand to render uptake target cell-specific. We evaluated the potential of several PTDs to induce the cytosolic uptake of the catalytic domain of diphtheria toxin by measuring cytotoxicity. Although PTD-dependent membrane transfer is very low, the proteins exhibited concentration-dependent cytotoxic activity. Higher binding at 4 degrees C compared to 37 degrees C suggests that uptake by the PTDs MTS and TLM occurs via an endocytic pathway. Non-specific binding is predominantly a function of the PTD and greatly increases by substitution of a non-polar glycine with a negatively charged glutamate in the PTD HA2.     

Levosulpiride, (S)-(-)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl) methyl]-2-methoxybenzamide, enhances the transduction efficiency of PEP-1-ribosomal protein S3 in vitro and in vivo

Many proteins with poor transduction efficiency were reported to be delivered to cells by fusion with protein transduction domains (PTDs). In this study, we investigated the effect of levosulpiride on the transduction of PEP-1 ribosomal protein S3 (PEP-1-rpS3), and examined its influence on the stimulation of the therapeutic properties of PEP-1-rpS3. PEP-1-rpS3 transduction into HaCaT human keratinocytes and mouse skin was stimulated by levosulpiride in a manner that did not directly affect the cell viability. Following 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced inflammation in mice, levosulpiride alone was ineffective in reducing TPA-induced edema and in inhibiting the elevated productions of inflammatory mediators and cytokines, such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor- α. Anti-inflammatory activity by PEP-1-rpS3 + levosulpiride was significantly more potent than by PEP-1-rpS3 alone. These results suggest that levosulpiride may be useful for enhancing the therapeutic effect of PEP-1-rpS3 against various inflammatory diseases. [BMB reports 2011; 44(5): 329-334].