FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1989855380> ?p ?o ?g. }

• W1989855380 endingPage "12635" @default.
• W1989855380 startingPage "12625" @default.
• W1989855380 abstract "The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8–10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins. The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8–10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins. The introduction of membrane-impermeable molecules into mammalian cells has become a key strategy for the investigation of intracellular processes (1Stephens D.J. Pepperkok R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4295-4298Crossref PubMed Scopus (111) Google Scholar). Peptide-mediated import has been attracting growing attention as a delivery technology during the last decade (for reviews, see Refs. 2Langel Ü. Cell Penetrating Peptides: Processes and Applications. CRC Press, Boca Raton, FL2002Crossref Google Scholar and 3Fischer P.M. Krausz E. Lane D.P. Bioconjug. Chem. 2001; 12: 825-841Crossref PubMed Scopus (132) Google Scholar). Linkage of so-called cell-penetrating peptides (CPPs) 1The abbreviations used are: CPP, cell-penetrating peptide; Antp, Antennapedia; DMF, N,N′-dimethylformamide; Fluo, 5(6)-carboxyfluorescein; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HBSS, Hank's buffered salt solution; HIV-1, human immunodeficiency virus, type 1; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; NDGA, nordihydroguaiaretic acid; PBS, phosphate-buffered saline; RT, room temperature; TGN, trans-Golgi network; TOF, time-of-flight. to other molecules mediates the non-invasive import of these cargo molecules into cells ex vivo as well as in whole animals (4Schutze-Redelmeier M.-P. Gournier H. Garcia-Pons F. Moussa M. Joliot A.H. Volovitch M. Prochiantz A. Lemonnier F.A. J. Immunol. 1996; 157: 655Google Scholar, 5Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2200) Google Scholar). Cargos have been peptides (6Prochiantz A. Curr. Opin. Neurobiol. 1996; 6: 629-634Crossref PubMed Scopus (197) Google Scholar, 7Hawiger J. Curr. Opin. Chem. Biol. 1999; 3: 89-94Crossref PubMed Scopus (133) Google Scholar), proteins as large as 120 kDa (5Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2200) Google Scholar, 8Rojas M. Donahue J.P. Tan Z. Lin Y.Z. Nat. Biotechnol. 1998; 16: 370-375Crossref PubMed Scopus (142) Google Scholar), oligonucleotides (9Astriab-Fisher A. Sergueev D. Fisher M. Shaw B.R. Juliano R.I. Pharm. Res. 2002; 19: 744-754Crossref PubMed Scopus (256) Google Scholar), plasmids (10Singh D. Bisland S.K. Kawamura K. Gariepy J. Bioconjug. Chem. 1999; 10: 745-754Crossref PubMed Scopus (56) Google Scholar), peptide nucleic acids (11Pooga M. Soomets U. Hällbrink M. Valkna A. Saar K. Rezaei K. Kahl U. Hao J.-X. Xu X.-J. Wiesenfeld-Hallin Z. Hökfelt T. Bartfei T. Langel Ü. Nat. Biotechnol. 1998; 16: 857-861Crossref PubMed Scopus (546) Google Scholar), and even nanoparticles (12Lewin M. Carlesso N. Tung C.-H. Tang X.-W. Cory D. Scadden D.T. Weissleder R. Nat. Biotechnol. 2000; 18: 410-414Crossref PubMed Scopus (1642) Google Scholar). Peptide cargos delivered by conjugation to CPPs include pseudosubstrates (13Theodore L. Derossi D. Chassaing G. Llirbat B. Kubes M. Jordan P. Chneiweiss H. Godement P. Prochiantz A. J. Neurosci. 1995; 15: 7158-7167Crossref PubMed Google Scholar), competitive inhibitors of an enzyme active site (14Nishikawa K. Sawasdikosol S. Fruman D.A. Lai J. Songyang Z. Burakoff S.J. Yaffe M.B. Cantley L.C. Mol. Cell. 2000; 6: 969-974Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), compartment-specific localization sequences (15Lin Y.-Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar), structural mimetics of interaction domains (16Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (829) Google Scholar), and epitopes for presentation by major histocompatibility complex class I molecules (4Schutze-Redelmeier M.-P. Gournier H. Garcia-Pons F. Moussa M. Joliot A.H. Volovitch M. Prochiantz A. Lemonnier F.A. J. Immunol. 1996; 157: 655Google Scholar, 17Pietersz G.A. Li W. Apostolopoulos V. Vaccine. 2001; 19: 1397-1405Crossref PubMed Scopus (53) Google Scholar). The advantages of peptide-based functional analyses in cell biology are the accessibility of large collections of different compounds by well established automated procedures (18Jung G. Beck-Sickinger A.G. Angew. Chem. Int. Ed. Engl. 1992; 31: 367-383Crossref Scopus (294) Google Scholar) and a rational approach to the generation of biologically active compounds based on available structural information of interaction domains. Structure-function relationships of intracellular interaction domains have been analyzed by testing a series of different peptides fused to cell-penetrating peptides (19Liu X.-Y. Timmons S. Lin Y.-Z. Hawiger J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11819-11824Crossref PubMed Scopus (106) Google Scholar). The delivery of exogenous antigens into the major histocompatibility complex class I processing pathway using CPPs has been presented in vitro and in whole animals (4Schutze-Redelmeier M.-P. Gournier H. Garcia-Pons F. Moussa M. Joliot A.H. Volovitch M. Prochiantz A. Lemonnier F.A. J. Immunol. 1996; 157: 655Google Scholar, 17Pietersz G.A. Li W. Apostolopoulos V. Vaccine. 2001; 19: 1397-1405Crossref PubMed Scopus (53) Google Scholar). On dendritic cells CPP-epitope constructs have been shown to enable prolonged antigen presentation (20Wang R.-F. Wang H.Y. Nat. Biotechnol. 2002; 20: 149-154Crossref PubMed Scopus (106) Google Scholar). CPPs therefore have the promise to represent a widely applicable means to enhance immune responses against cancer and infectious diseases. A total of about 20 different peptide delivery vectors have been described so far, the majority of which were identified as structural determinants mediating cellular internalization of proteins (3Fischer P.M. Krausz E. Lane D.P. Bioconjug. Chem. 2001; 12: 825-841Crossref PubMed Scopus (132) Google Scholar). While some of these peptides are purely cationic, others are amphipathic with a large fraction of basic residues, and again others are fully hydrophobic. These peptides vary in length from about 9 to more than 30 amino acids (3Fischer P.M. Krausz E. Lane D.P. Bioconjug. Chem. 2001; 12: 825-841Crossref PubMed Scopus (132) Google Scholar). Three highly basic import peptides, the Drosophila Antennapedia homeodomain-derived penetratin peptide (21Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar), the HIV-1 Tat-derived peptide (22Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar), and the oligoarginine peptides (23Futaki S. Int. J. Pharm. 2002; 245: 1-7Crossref PubMed Scopus (208) Google Scholar) have been used widely in many of the applications described above. The penetratin peptide and the HIV-1 Tat peptide have been identified as protein transduction domains of their respective proteins, while the oligoarginine peptides (among them the nona-arginine peptide R9) have been developed based on structure-activity relationships of the HIV-1 Tat peptide. Despite their broad acceptance as molecular carriers, the mechanism of internalization of CPPs and CPP-cargo constructs is not well understood. The uptake of the penetratin and the HIV-1 Tat peptide had originally been described to be insensitive to low temperature (21Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar, 22Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar, 24Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1443) Google Scholar) and to inhibitors of endocytosis (22Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar, 25Suzuki T. Futaki S. Niwa M. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2002; 277: 2437-2443Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Penetratin was also demonstrated to traverse a pure lipid bilayer (26Thoren P.E.G. Persson D. Karlsson M. Norden B. FEBS Lett. 2000; 482: 265-268Crossref PubMed Scopus (214) Google Scholar) without forming pores (26Thoren P.E.G. Persson D. Karlsson M. Norden B. FEBS Lett. 2000; 482: 265-268Crossref PubMed Scopus (214) Google Scholar, 27Persson D. Thoren P.E.G. Herner M. Lincoln P. Norden B. Biochemistry. 2003; 42: 421-429Crossref PubMed Scopus (84) Google Scholar). In summary, these results were consistent with the theory of a direct translocation of the cationic peptides through the plasma membrane (21Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar, 22Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar, 28Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar). Recent data demonstrated, however, that earlier interpretations of cell biological experiments may have suffered from artifactual uptake of CPPs caused by fixation of cells. Endocytosis is clearly involved in the internalization of the HIV-1 Tat peptide (29Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar). For Tat fusion proteins it was demonstrated that cellular internalization occurs through a temperature-dependent endocytic pathway that originates from lipid rafts and follows caveolar endocytosis (30Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Inhibitors of metabolism or endocytosis, such as cytochalasin D, were demonstrated to impair uptake of penetratin (31Drin G. Cottin S. Blanc E. Rees A.R. Temsamani J. J. Biol. Chem. 2003; 278: 31192-31201Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Recently the penetratin and the Tat peptide were shown to promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans (32Console S. Marty C. Garcia-Echeverria C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). With a picture of an endocytic uptake mechanism emerging, the implications of the endosomal pathway for functional cell biological studies using CPPs need to be readdressed. The biological activity exhibited by CPPs in cell biological applications (6Prochiantz A. Curr. Opin. Neurobiol. 1996; 6: 629-634Crossref PubMed Scopus (197) Google Scholar) is fully consistent with intact CPP-constructs reaching the cytosol. However, the accumulation of CPPs in the endocytic compartment (29Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar, 30Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 31Drin G. Cottin S. Blanc E. Rees A.R. Temsamani J. J. Biol. Chem. 2003; 278: 31192-31201Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar) raises the question to which degree and by which mechanism internalized CPPs reach the cytosol. A rational design of more effective CPPs can only be achieved if the mechanism of uptake is fully understood. In this study we addressed these questions by investigating the effect of drugs that interfere with distinct steps of the endosomal pathway and Golgi trafficking. To study a potential cell type dependence of the peptide trafficking, adherently growing MC57 fibrosarcoma cells and HeLa cells were examined. Both cell lines showed marked differences in the intracellular distribution of fluorescein-labeled CPPs. Materials—Standard chemicals for peptide chemistry were obtained from Fluka (Deisenhofen, Germany) and Merck; solvents were pure analytical grade. Fmoc-protected amino acids were purchased from Novabiochem, Senn Chemicals (Dielsdorf, Switzerland), and Orpegen Pharma (Heidelberg, Germany). The isomeric mixture of (5)6-carboxyfluorescein was from Fluka, and AlexaFluor 647-dextran (anionic; molecular mass, 10,000 Da) and Bodipy TR ceramide were obtained from Mobitech (Göttingen, Germany). Bafilomycin A1 was from Tocris Biotrend (Bristol, UK), chloroquine diphosphate was from Fluka, nordihydroguaiaretic acid (NDGA) was from Alexis Biochemicals (Grünberg, Germany), wortmannin was from Calbiochem, and brefeldin A was from Sigma. Peptide Synthesis—Automated peptide synthesis was performed by solid phase Fmoc/tert-butyl chemistry using an automated peptide synthesizer for multiple peptide synthesis (RSP5032, Tecan, Hombrechtlikon, Switzerland) in 2-ml syringes according to the following protocol. Fmoc-protected amino acids (12-fold excess) were coupled by in situ activation using N,N′-diisopropyl carbodiimide/1-hydroxybenzotriazol for 90 min followed by removal of the Fmoc protecting group by treatment with piperidine/DMF (1:4, v/v) twice for 8 min. The resin was washed with DMF (six times) after each coupling and deprotection step. The side chain of Tyr was tert-butyl-protected, the side chain of Arg was 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-protected, the side chains of Gln and Asn were trityl-protected, and the side chains of Lys and Trp were tert-butyloxycarbonyl-protected. Peptide amides were synthesized on Rink amide resin (Rapp Polymere, Tübingen, Germany). Peptides were cleaved off the resin by treatment with trifluoroacetic acid/triisopropylsilane/ethanedithiol/H2O (92.5:2.5:2.5:2.5, v/v/v/v) for 4 h. Crude peptides were precipitated by adding cold diethyl ether (-20 °C). The precipitated peptides were collected by centrifugation and resuspended in cold diethyl ether. This procedure was repeated twice. Finally peptides were dissolved in tert-butyl alcohol/H2O (4:1, v/v) and lyophilized. Labeling of Peptides with Carboxyfluorescein—Amino-terminal labeling of peptides with 5(6)-carboxyfluorescein was essentially performed as described previously (33Fischer R. Mader O. Jung G. Brock R. Bioconjug. Chem. 2003; 14: 653-660Crossref PubMed Scopus (93) Google Scholar). Fmoc-deprotected, resin-bound peptides were reacted with 5 eq of 5(6)-carboxyfluorescein, N,N′-diisopropyl carbodiimide, and 1-hydroxybenzotriazol, each in DMF for 16 h in 2-ml syringes on a shaker at RT. Reactions were stopped by washing the resins three times each with DMF, methanol, dichloromethane, and diethyl ether. Subsequently peptides were treated with piperidine/DMF (1:4, v/v) to remove ester-bound carboxyfluorescein (33Fischer R. Mader O. Jung G. Brock R. Bioconjug. Chem. 2003; 14: 653-660Crossref PubMed Scopus (93) Google Scholar). Completeness of amine acylation was confirmed using the Kaiser test (34Sarin V.K. Kent S.B. Tam J.P. Merrifield R.B. Anal. Biochem. 1981; 117: 147-157Crossref PubMed Scopus (991) Google Scholar). HPLC—Peptides and conjugates were analyzed by analytical reversed phase HPLC using a water (0.1% trifluoroacetic acid) (solvent A)/acetonitrile (0.1% trifluoroacetic acid) (solvent B) gradient on a Waters 600 system (Eschborn, Germany) with detection at 214 nm. The samples were analyzed on an analytical column (Nucleosil 100, 250 × 2 mm, C18 column, 5-μm particle diameter; Grom, Herrenberg, Germany) using a linear gradient from 10% B to 100% B within 30 min (flow rate, 0.3 ml/min). Peptides were purified by preparative reversed phase HPLC (Nucleosil 300, 250 × 20 mm, C18 column, 10-μm particle diameter; Grom) on a Waters 600 multisolvent delivery system (flow rate, 10 ml/min). Gradients were adjusted according to the elution profiles and peak profiles obtained from the analytical HPLC chromatograms. MALDI-TOF MS of Synthetic Peptides—1 μl of 2,5-dihydroxyacetophenone matrix (20 mg of 2,5-dihydroxyacetophenone, 5 mg of ammonium citrate in 1 ml of isopropyl alcohol/H2O (4:1, v/v)) was mixed with 1 μl of each sample (dissolved in acetonitrile/water (1:1) at a concentration of 1 mg/ml) on a gold target. Measurements were performed using a MALDI time-of-flight system (G2025A, Hewlett-Packard, Waldbronn, Germany). For signal generation 20–50 laser shots were added up in the single shot mode. Peptide Stock Solutions—Carboxyfluorescein-labeled peptides were dissolved in Me2SO at concentrations of 10 mm. These stock solutions were further diluted 1:20 in double distilled H2O. Peptide concentrations were determined by UV/visible spectroscopy of a further 1:100 dilution in 0.1 m Tris/HCl buffer (pH 8.8) with absorptions measured at 492 nm and assuming a molar extinction coefficient of 75,000 liters/(mol·cm). Cell Culture—The adherent MC57 fibrosarcoma cell line (35Hosaka Y. Yasuda Y. Seriburi O. Moran M.G. Fukai K. J. Virol. 1986; 57: 1113-1118Crossref PubMed Google Scholar) and HeLa cells were grown in a 5% CO2 humidified atmosphere at 37 °C in RPMI 1640 medium with stabilized glutamine and 2.0 g/liter NaHCO3 (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal calf serum (PAN Biotech), 100 units/ml penicillin, and 100 μg/ml streptomycin (Biochrom, Berlin, Germany). Both cell lines were passaged by trypsinization with trypsin/EDTA (0.05%:0.02%, w/v) (Biochrom) in PBS every 3rd–4th day. Flow Cytometry—MC57 or HeLa cells were seeded at a density of 50,000/well in 24-well plates (Sarstedt, Nümbrecht, Germany) in serum-containing RPMI 1640 medium. One day later, the cells were washed with serum-free RPMI 1640 medium and incubated in 200 μl of serum-free RPMI 1640 medium containing the appropriate inhibitors. After the indicated periods of time, peptides were added as described under “Results.” Each condition was tested in duplicate. After a 2-h incubation, cells were washed with PBS, detached by trypsinization for 10 min, suspended in ice-cold PBS containing 0.1% (w/v) bovine serum albumin, and measured immediately by flow cytometry. The fluorescence of 5000 vital cells was acquired. Vital cells were gated based on sideward scatter and forward scatter. For pulse/chase experiments, MC57 cells were washed three times with PBS after 2 h of peptide incubation and then incubated with 500 μl of serum-free RPMI 1640 medium for an additional 3 h. Cells were then washed with PBS, detached by trypsinization for 10 min, suspended in ice-cold PBS containing 0.1% (w/v) bovine serum albumin, and measured by flow cytometry as described above. To compare the pulse and the pulse/chase values, fluorescence intensities of fluorescent calibration beads (Mobitech) were also acquired for each series of measurements. Confocal Laser Scanning Microscopy—Confocal laser scanning microscopy was performed on an inverted LSM510 laser scanning microscope (Carl Zeiss, Göttingen, Germany) fitted with a Plan-Apochromat 63 × 1.4 numerical aperture lens. All measurements were performed with living, non-fixed cells. MC57 cells were seeded at a density of 10,000/well in 8-well chambered cover glasses (Nunc, Wiesbaden, Germany). Two days later, before addition of inhibitors or peptides, cells were washed once with serum-free RPMI 1640 medium. Bafilomycin A1 was added at a concentration of 300 nm in 200 μl of serum-free RPMI 1640 medium 30 min before addition of peptides. After 2 h of incubation with peptides, images were acquired immediately at RT with excess peptide in the medium. For double detection of fluorescein-labeled peptides and AlexaFluor 647-dextran the 488 nm line of an argon ion laser and the light of a 633 nm helium/neon laser were directed over an HFT UV/488/633 beam splitter, and fluorescence was detected using an NFT 545 beam splitter in combination with a BP 505–530 band pass filter for fluorescein detection and an LP 650 long pass filter for AlexaFluor 647 detection. Peptides and AlexaFluor 647-dextran were added as indicated under “Results.” After 2 h cells were washed three times with serum-free RPMI 1640 medium followed immediately by confocal microscopy at RT. For the induction of retrograde transport, after 2 h of incubation with peptide and dextran, cells were washed three times with serum-free RPMI 1640 medium followed by incubation with 25 μm NDGA for 3 h in RPMI 1640 medium. For double detection of fluorescein-labeled peptides and Bodipy TR ceramide the 488 nm line of an argon ion laser and the light of a 543 nm helium/neon laser were directed over an HFT UV/488/543 beam splitter, and fluorescence was detected using an NFT 545 beam splitter in combination with a BP 505–530 band pass filter for fluorescein detection and an LP 560 long pass filter for Bodipy detection. Staining of the Golgi complex in living cells with fluorescent ceramide was essentially performed as described by the supplier. Briefly MC57 and HeLa cells were seeded at a density of 10,000/well in 8-well chambered cover glasses. Two days later, cells were washed once with ice-cold Hank's buffered salt solution + 10 mm HEPES (pH 7.4) (HBSS/HEPES) and then incubated with HBSS/HEPES (containing 5 μm sphingolipid and 5 μm bovine serum albumin prepared as described by the supplier) for 30 min on ice and in the dark. Cells were rinsed three times with ice-cold HBSS/HEPES and incubated with the indicated peptide in serum-free medium for1hat37 °C in the incubator. Confocal images were then acquired immediately at RT with excess peptide in the medium. MALDI-TOF MS of Peptide-containing Cell Culture Supernatant—A confluent cell layer of MC57 cells in a 25-cm2 tissue culture flask was washed once with serum-free RPMI 1640 medium and incubated with 1 ml of serum-free RPMI 1640 medium containing 30 μm Fluo-Tat. After 2 h the cells were detached using 5 mm EDTA/PBS; transferred into a fresh tube; and washed twice with 10 ml of PBS. EDTA/PBS was used instead of trypsin/EDTA/PBS to exclude that peptide fragments were generated by residual amounts of trypsin during the chase period. Cells were then suspended in 500 μl of serum-free RPMI 1640 medium and incubated for 3 h at 37 °C in the incubator. The cells were centrifuged, and the cell-free supernatant was acidified using 100 μl of 0.1% HCl. The yellow sample (the yellow color originated from phenol red) was immediately desalted on a G1001A sample prep station (Hewlett-Packard) and concentrated to a volume of about 10 μl in a Speed-Vac concentrator. MS analysis was performed using a “Future” MALDI-TOF mass spectrometer (GSG, Mess-und Analysengeräte GmbH, Bruchsal, Germany). 1 μl of 2,5-dihydroxyacetophenone matrix was mixed with 1 μl of the sample on a gold target. For signal generation 35–50 laser shots were added up in the single shot mode (positive ion mode). Instrument calibration was performed using two synthetic peptides (calculated [M + H]+, 1156.7 and 2859.4 Da). Intracellular Peptide Stability—Intracellular peptide stability was investigated essentially as described elsewhere (36Elmquist A. Langel Ü. Biol. Chem. 2003; 384: 387-393Crossref PubMed Scopus (121) Google Scholar). A confluent cell layer of MC57 cells in a 25-cm2 tissue culture flask was washed once with serum-free RPMI 1640 medium and incubated with 1 ml of serum-free RPMI 1640 medium containing 15 μm Fluo-Antp. After 2 h the cells were washed twice with PBS, detached by trypsinization for 10 min at 37 °C, transferred into a fresh tube, and washed three times with 10 ml of PBS (to remove trypsin and extracellular trypsinized peptide). Cells were then lysed in 200 μl of 0.1% HCl for 10 min at RT. The cell lysate was then centrifuged for 10 min at 4 °C and 14,000 rpm. The peptide-containing supernatant was immediately desalted and concentrated to a volume of about 10 μl as described above. MS analysis of the sample was performed using a (Future) MALDI-TOF mass spectrometer as described above. Co-incubation of MC57 and HeLa Cells—For co-incubation experiments 5,000 each of MC57 and HeLa cells/well were seeded in an 8-well chambered cover glass. Two days later, cells were washed once with serum-free RPMI 1640 medium, and 200 μl of serum-free RPMI 1640 medium was added. Peptides were added as indicated under “Results.” After 2 h of peptide incubation, pictures were immediately acquired at RT, leaving excess peptide in the medium. Quantification of Cellular Internalization of Fluorescein-labeled CPPs by Fluorescence Emission Spectroscopy in Whole Cell Lysates— MC57 and HeLa cells were seeded in 6-well plates (Sarstedt) in serum-containing RPMI 1640 medium. Two days later, the confluent cell layer was washed with serum-free RPMI 1640 medium and incubated with 500 μl of serum-free RPMI 1640 medium ± bafilomycin A1 for 30 min. Then Fluo-Antp was added, and after 2 h of incubation, cells were washed twice with PBS, detached by trypsinization for 15 min, transferred into a fresh tube, and washed twice with 10 ml of PBS (to remove trypsin and trypsinized peptide). Cells were then lysed in 200 μl of Nonidet P-40 lysis buffer (0.5% (v/v) Nonidet P-40, 150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.0, containing protease inhibitor mixture (Roche Diagnostics)). The lysates were then sonicated and centrifuged for 30 min at 4 °C and 14,000 rpm. Fluorescein concentrations were determined in the supernatant using an LS50B spectrofluorometer (PerkinElmer Life Sciences) with excitation at 492 nm and detection of emission at 520 nm. The protein content of the lysates was determined using a commercially available Bradford protein assay kit (Bio-Rad). Each condition was tested in duplicate. Cellular Uptake of Penetratin, R9, and Tat Peptides—Considering two recent studies demonstrating the involvement of endocytosis in the uptake of the R9 peptide, the HIV-1 Tat peptide (29Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar), and Tat fusion pro" @default.
• W1989855380 created "2016-06-24" @default.
• W1989855380 creator A5026129752 @default.
• W1989855380 creator A5036916060 @default.
• W1989855380 creator A5057935520 @default.
• W1989855380 creator A5089361508 @default.
• W1989855380 date "2004-03-01" @default.
• W1989855380 modified "2023-10-10" @default.
• W1989855380 title "A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides" @default.
• W1989855380 cites W10640494 @default.
• W1989855380 cites W1510613202 @default.
• W1989855380 cites W1519843522 @default.
• W1989855380 cites W1539984517 @default.
• W1989855380 cites W1541488575 @default.
• W1989855380 cites W1568032381 @default.
• W1989855380 cites W1584683177 @default.
• W1989855380 cites W1614056212 @default.
• W1989855380 cites W1928166470 @default.
• W1989855380 cites W1963614280 @default.
• W1989855380 cites W1965261376 @default.
• W1989855380 cites W1965583590 @default.
• W1989855380 cites W1974302927 @default.
• W1989855380 cites W1977253956 @default.
• W1989855380 cites W1992095148 @default.
• W1989855380 cites W2004390233 @default.
• W1989855380 cites W2008082372 @default.
• W1989855380 cites W2010659535 @default.
• W1989855380 cites W2026405782 @default.
• W1989855380 cites W2026835141 @default.
• W1989855380 cites W2029027799 @default.
• W1989855380 cites W2029346749 @default.
• W1989855380 cites W2034492545 @default.
• W1989855380 cites W2035501441 @default.
• W1989855380 cites W2038720675 @default.
• W1989855380 cites W2042143311 @default.
• W1989855380 cites W2042143995 @default.
• W1989855380 cites W2043389898 @default.
• W1989855380 cites W2045243236 @default.
• W1989855380 cites W2046248975 @default.
• W1989855380 cites W2051565790 @default.
• W1989855380 cites W2055614158 @default.
• W1989855380 cites W2058907087 @default.
• W1989855380 cites W2058975342 @default.
• W1989855380 cites W2064242356 @default.
• W1989855380 cites W2066276063 @default.
• W1989855380 cites W2067099248 @default.
• W1989855380 cites W2076330454 @default.
• W1989855380 cites W2081684039 @default.
• W1989855380 cites W2082678397 @default.
• W1989855380 cites W2085965622 @default.
• W1989855380 cites W2087917270 @default.
• W1989855380 cites W2090161431 @default.
• W1989855380 cites W2093774343 @default.
• W1989855380 cites W2099089545 @default.
• W1989855380 cites W2099985438 @default.
• W1989855380 cites W2105216614 @default.
• W1989855380 cites W2105668301 @default.
• W1989855380 cites W2120365956 @default.
• W1989855380 cites W2121853695 @default.
• W1989855380 cites W2130268724 @default.
• W1989855380 cites W2151142281 @default.
• W1989855380 cites W2171846923 @default.
• W1989855380 cites W326302165 @default.
• W1989855380 cites W4239028853 @default.
• W1989855380 cites W91990295 @default.
• W1989855380 doi "https://doi.org/10.1074/jbc.m311461200" @default.
• W1989855380 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14707144" @default.
• W1989855380 hasPublicationYear "2004" @default.
• W1989855380 type Work @default.
• W1989855380 sameAs 1989855380 @default.
• W1989855380 citedByCount "263" @default.
• W1989855380 countsByYear W19898553802012 @default.
• W1989855380 countsByYear W19898553802013 @default.
• W1989855380 countsByYear W19898553802014 @default.
• W1989855380 countsByYear W19898553802015 @default.
• W1989855380 countsByYear W19898553802016 @default.
• W1989855380 countsByYear W19898553802017 @default.
• W1989855380 countsByYear W19898553802018 @default.
• W1989855380 countsByYear W19898553802019 @default.
• W1989855380 countsByYear W19898553802020 @default.
• W1989855380 countsByYear W19898553802021 @default.
• W1989855380 countsByYear W19898553802022 @default.
• W1989855380 countsByYear W19898553802023 @default.
• W1989855380 crossrefType "journal-article" @default.
• W1989855380 hasAuthorship W1989855380A5026129752 @default.
• W1989855380 hasAuthorship W1989855380A5036916060 @default.
• W1989855380 hasAuthorship W1989855380A5057935520 @default.
• W1989855380 hasAuthorship W1989855380A5089361508 @default.
• W1989855380 hasBestOaLocation W19898553801 @default.
• W1989855380 hasConcept C105702510 @default.
• W1989855380 hasConcept C12554922 @default.
• W1989855380 hasConcept C1491633281 @default.
• W1989855380 hasConcept C183882617 @default.
• W1989855380 hasConcept C185592680 @default.
• W1989855380 hasConcept C188027245 @default.
• W1989855380 hasConcept C2775862295 @default.
• W1989855380 hasConcept C55493867 @default.
• W1989855380 hasConcept C79879829 @default.

• next