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1.
Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat2) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat2 showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat2. Cellular internalization of Tat and Tat2 was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 °C) remarkably increased cellular internalization of Tat as well as Tat2 but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 μM), chloroquine (100 μM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat2. These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat2 suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines. 相似文献
2.
Internalization of fluorescently labeled CPPs, pVEC, transportan and scrambled pVEC, in a range of plant cells was investigated. Cellular uptake of the peptides was found to be tissue dependent. pVEC and transportan were distinctly internalized in triticale mesophyll protoplasts, onion epidermal cells, leaf bases and root tips of seven-day old triticale seedlings but showed negligible florescence in coleoptile and leaf tips as observed under a fluorescence microscope. Further, pVEC and transportan uptake studies were focused on mesophyll protoplasts as a system of investigation. In fluorimetric studies transportan showed 2.3 times higher cellular internalization than pVEC in protoplasts, whereas scrambled pVEC failed to show any significant fluorescence. Effect of various factors on cellular internalization of pVEC and transportan in protoplasts was also investigated. The cellular uptake of both the peptides was concentration dependent and nonsaturable. The cellular uptake of pVEC and transportan was enhanced at low temperature (4 degrees C). The presence of endocytic/macropinocytosis inhibitors did not reduce the cellular uptake of the peptides, suggesting direct cell penetration, receptor-independent internalization of pVEC and transportan into the plant cells. 相似文献
3.
Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore
we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast
agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane
translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide
and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting,
magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373
glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20%
of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation
of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake
of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake. 相似文献
4.
Richard JP Melikov K Vives E Ramos C Verbeure B Gait MJ Chernomordik LV Lebleu B 《The Journal of biological chemistry》2003,278(1):585-590
Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids. 相似文献
5.
Bullok KE Gammon ST Violini S Prantner AM Villalobos VM Sharma V Piwnica-Worms D 《Molecular imaging》2006,5(1):1-15
Rapid and efficient delivery of imaging probes to the cell interior using permeation peptides has enabled novel applications in molecular imaging. Membrane permeant peptides based on the HIV-1 Tat basic domain sequence, GRKKRRQRRR, labeled with fluorophores and fluorescent proteins for optical imaging or with appropriate peptide-based motifs or macrocycles to chelate metals, such as technetium for nuclear scintigraphy and gadolinium for magnetic resonance imaging, have been synthesized. In addition, iron oxide complexes have been functionalized with the Tat basic domain peptides for magnetic resonance imaging applications. Herein we review current applications of permeation peptides in molecular imaging and factors influencing permeation peptide internalization. These diagnostic agents show concentrative cell accumulation and rapid kinetics and display cytosolic and focal nuclear accumulation in human cells. Combining methods, dual-labeled permeation peptides incorporating fluorescein maleimide and chelated technetium have allowed for both qualitative and quantitative analysis of cellular uptake. Imaging studies in mice following intravenous administration of prototypic diagnostic permeation peptides show rapid whole-body distribution allowing for various molecular imaging applications. Strategies to develop permeation peptides into molecular imaging probes have included incorporation of targeting motifs such as molecular beacons or protease cleavable domains that enable selective retention, activatable fluorescence, or targeted transduction. These novel permeation peptide conjugates maintain rapid translocation across cell membranes into intracellular compartments and have the potential for targeted in vivo applications in molecular imaging and combination therapy. 相似文献
6.
Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery. 相似文献
7.
8.
Complexes of plasmid DNA with basic domain 47-57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways 总被引:5,自引:0,他引:5
Ignatovich IA Dizhe EB Pavlotskaya AV Akifiev BN Burov SV Orlov SV Perevozchikov AP 《The Journal of biological chemistry》2003,278(43):42625-42636
9.
Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains 总被引:6,自引:0,他引:6
Tkachenko AG Xie H Liu Y Coleman D Ryan J Glomm WR Shipton MK Franzen S Feldheim DL 《Bioconjugate chemistry》2004,15(3):482-490
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. 相似文献
10.
Eric Vivès Claude Granier Paul Prevot Bernard Lebleu 《International journal of peptide research and therapeutics》1997,4(4-6):429-436
Summary Tat, a 86-amino acid protein involved in the replication of Human Immunodeficiency Virus type 1 (HIV-1), is able to translocate
efficiently through the plasma membrane and to reach the nucleus to transactivate the viral genome. The region 37–72 of the
Tat protein, centered on a cluster of basic amino acids, has been assigned to this translocation activity. Recent data in
our group have attributed this membrane translocating activity to a peptide extending from residues 48 to 60, which contains
a cluster of eight basic amino acids within a linear sequence of nine residues. Internalization of this peptide into cells
occurred within minutes at concentrations as low as 100 nM. In order to define more precisely the involvement of these basic
amino acids in peptide translocation, several analogues carrying deletions or substitutions of one, or several, of the basic
residues were synthesized and tested for their cellular uptake and nuclear translocation. A direct correlation between the
overall charge of the peptide and its cell internalization was found. In addition, the covalent linkage of this short basic
peptide allows the efficient translocation of a non-membrane permeant peptide. 相似文献
11.
12.
13.
Protamine, a mixture of positively charged proteins from salmon roe used in diverse pharmaceutical applications, was reacted with the N-hydroxysuccinimide ester of tetramethylrhodamine to yield tetramethylrhodamine-labeled protamines (Pro(Rh)) containing one mole of fluorochrome per mole of protein. The internalization of tetramethylrhodamine-labeled protamine (Pro(Rh)) and the fluorescein-labeled tat peptide (Tat(Fl)) showed a similar dependence on time and concentration. Pro(Rh) and Tat(Fl) showed strong nuclear localizations, evident with both live cells and fixed cells co-stained with DAPI, a nuclear stain. The loss of fluorescence when cells were loaded with Pro(Rh) or Tat(Fl) was similar, further supporting a strong similarity between these two materials. Finally, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced. All experiments were performed with HeLa and CaCo-2 cells with similar results. These observations imply that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides. In addition, the fluorescent protamines developed here might be used to further our understanding of this important pharmaceutical. 相似文献
14.
Quantitative analysis of permeation peptide complexes labeled with Technetium-99m: chiral and sequence-specific effects on net cell uptake 总被引:2,自引:0,他引:2
Gammon ST Villalobos VM Prior JL Sharma V Piwnica-Worms D 《Bioconjugate chemistry》2003,14(2):368-376
This study investigated sequence-specific cell uptake characteristics of Tat basic domain and related permeation peptides with an emphasis on residue chirality, length, and modified side chains. Effects on cell permeation of defined basic domain sequences within a library of 42 different peptides were evaluated using transport of radiolabeled peptides into human Jurkat leukemia cells. All other factors being equal, when the chirality of the peptide sequence was changed from l to d, uptake values increased up to 13-fold. Control experiments showed that the quantitative difference in uptake could not be attributed to increased decomposition of an l- versus a d-peptide by cellular or serum proteases. Furthermore, length, sequence, and type of chelation domain impacted peptide uptake into cells. The highest level of uptake was found with the following peptides: (23) d-Tat-Orn [Ac-rkkrr-orn-rrr-AHA-kgc-amide] and (33) d-poly-Arg(9) [Ac-rrrrrrrrr-AHA-kgc-amide]. The best of these peptide sequences could be employed as in vivo imaging and drug delivery agents to translocate substrates into cells. 相似文献
15.
The effect of androgens on the nuclear uptake of both tritiated estradiol (3H-E2) and the estrogen receptor was studied in immature rat uteri. It was demonstrated that in vitro preincubation of immature rat uteri with various androgens (1 muM to 50 muM) followed by incubation with 3H-E2 (20 nM) resulted in a greatly decreased specific nuclear uptake of 3H-E2. Non-androgenic steroids had no effect. It was also confirmed that 5alpha-dihydrotestosterone (DHT) causes the accumulation of the estrogen receptor in the nuclei of uterine tissue. In vitro incubations of rat uteri with DHT (1muM and 50muM) were found to cause maximal nuclear estrogen receptor accumulation to the same degree as caused by estradiol, i.e. the nuclear uptake of approximately 100% of the estrogen receptor. Antiandrogens, which block the binding of androgens to the testosterone receptor in various tissues, did not inhibit the DHT - induced decrease in the 3H-E2 uptake by the uterine nuclei or the DHT - caused accumulation of the estrogen receptor in nuclei. These results seem to indicate that the uterine testosterone receptor has no role in the androgen - induced nuclear uptake of the estrogen receptor. However, the non-steroidal antiestrogens inhibited the DHT - induced nuclear accumulation of the estrogen receptor. This would seem to indicate that the estrogen - and androgen - induced nuclear accumulation of the estrogen receptor share a common mechanism. 相似文献
16.
Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression 总被引:11,自引:0,他引:11
The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression. By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined. These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization. Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain. This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR. To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain. A number of these proteins showed a trans-dominant phenotype. These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR. Thus, an intact TAR element was not required for this inhibition. We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein. The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization. These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression. 相似文献
17.
18.
Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 +/- 0.11 mM(-1) sec(-1) at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals. 相似文献
19.
Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores 总被引:1,自引:0,他引:1
Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals,
in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat,
Tat2, M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate
stage). We demonstrated that Tat (RKKRRQRRR) and Tat2 (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores,
respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31%
of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat2-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal
laser microscopy was carried out to recommend a ratio of 4:1 Tat2-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat2 can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS
protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently
has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing
simple, time saving, cost effective plant genetic engineering technologies. 相似文献
20.
Molecular determinants for cellular uptake of Tat protein of human immunodeficiency virus type 1 in brain cells. 总被引:13,自引:0,他引:13
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We measured the cellular uptake of 125I-labeled full-length Tat (amino acids 1 to 86) (125I-Tat(1-86)) and 125I-Tat(1-72) (first exon) in human fetal astrocytes, neuroblastoma cells, and human fetal neurons and demonstrated that the uptake of 125I-Tat(1-72) without the second exon was much lower than that of 125I-Tat(1-86) (P < 0.01). This suggests an important role for the C-terminal region of Tat for its cellular uptake. 125I-Tat uptake could be inhibited by dextran sulfate and competitively inhibited by unlabeled Tat but not by overlapping 15-mer peptides, suggesting that Tat internalization is charge and conformationally dependent. Interestingly, one of 15-mer peptides, Tat(28-42), greatly enhanced 125I-Tat uptake. These findings are important for understanding the neuropathogenesis of human immunodeficiency virus type 1 infection and in the potential application of Tat for drug delivery to cells. 相似文献