Cellular Uptake of Exogenous Human PDCD5 Protein
2006; Elsevier BV; Volume: 281; Issue: 34 Linguagem: Inglês
10.1074/jbc.m600183200
ISSN1083-351X
AutoresYing Wang, Dan Li, Hui Fan, Linjie Tian, Yingcheng Zhong, Yingmei Zhang, Lan Yuan, Caining Jin, Caihua Yin, Dalong Ma,
Tópico(s)Ubiquitin and proteasome pathways
ResumoPDCD5 (human programmed cell death 5) plays a significant role in apoptotic and paraptotic cell deaths. However, it was found that recombinant PDCD5 added exogenously to culture medium could also enhance programmed cell death triggered by certain stimuli. Here we show that PDCD5 has a remarkable role in intercellular transport in various cells (endogenous caveolin-1-positive and -negative cells) through a clathrin-independent endocytic pathway that originates from heparan sulfate proteoglycan binding and lipid rafts. These conclusions are supported by the studies of slow internalization kinetics of PDCD5 endosomes, by the resistance of endosomes to nonionic detergents, by the overexpression of the clathrin dominant negative mutant form, which did not block PDCD5-fluorescein isothiocyanate uptake, and by PDCD5 localization in lipid rafts by immunofluorescence, electron microscopy techniques, and sucrose density centrifugation. This is further supported by the findings that certain drugs that disrupt lipid rafts, compete with cell membrane heparan sulfate proteoglycans, or block the caveolae pathway, impair the PDCD5 internalization process. The translocation activity of PDCD5 may possess physiological significance and be a potential mechanism for its programmed cell death-promoting activity. PDCD5 protein also has the ability to drive the internalization of large protein cargo, depending on the residues 109-115 mapped by deletion mutagenesis, and can introduce the Mdm-2 binding domain of human p53 into living cells to induce cell death in human cancer cells, indicating that PDCD5 may serve as a vehicle and thus have potential in the field of protein delivery to the cells. This is the first evidence of such findings. PDCD5 (human programmed cell death 5) plays a significant role in apoptotic and paraptotic cell deaths. However, it was found that recombinant PDCD5 added exogenously to culture medium could also enhance programmed cell death triggered by certain stimuli. Here we show that PDCD5 has a remarkable role in intercellular transport in various cells (endogenous caveolin-1-positive and -negative cells) through a clathrin-independent endocytic pathway that originates from heparan sulfate proteoglycan binding and lipid rafts. These conclusions are supported by the studies of slow internalization kinetics of PDCD5 endosomes, by the resistance of endosomes to nonionic detergents, by the overexpression of the clathrin dominant negative mutant form, which did not block PDCD5-fluorescein isothiocyanate uptake, and by PDCD5 localization in lipid rafts by immunofluorescence, electron microscopy techniques, and sucrose density centrifugation. This is further supported by the findings that certain drugs that disrupt lipid rafts, compete with cell membrane heparan sulfate proteoglycans, or block the caveolae pathway, impair the PDCD5 internalization process. The translocation activity of PDCD5 may possess physiological significance and be a potential mechanism for its programmed cell death-promoting activity. PDCD5 protein also has the ability to drive the internalization of large protein cargo, depending on the residues 109-115 mapped by deletion mutagenesis, and can introduce the Mdm-2 binding domain of human p53 into living cells to induce cell death in human cancer cells, indicating that PDCD5 may serve as a vehicle and thus have potential in the field of protein delivery to the cells. This is the first evidence of such findings. The plasma membrane, consisting of a lipid bilayer into which proteins and glycoproteins are inserted, is generally impervious to proteins. Some larger proteins termed translocatory proteins, however, can enter the cell by endocytosis into endocytic vesicles through as yet unknown exact mechanisms. The mounting interest in the cell-penetrating capacity of these translocatory proteins is due to their ability to drive the internalization of large protein cargoes that are chemically coupled or fused to them. This group of proteins includes human immunodeficiency virus Tat (1Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2330) Google Scholar, 2Green M. Loewenstein P.M. Cell. 1988; 55: 1179-1188Abstract Full Text PDF PubMed Scopus (1280) Google Scholar), Drosophila Antennapedia (3Joliot A.H. Pernelle C. Deagostini-bazin H. Prochintz A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1864-1868Crossref PubMed Scopus (554) Google Scholar), and herpes simplex virus VP22 (4Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar), all of which are most commonly used in drug delivery and gene therapy studies. These translocatory proteins share several common features: they are released from cells by a pathway distinct from the recognized secretory routes involving a secretion signal (5Helland D.E. Welles J.L. Caputo A. Haseltine W.A. J. Virol. 1991; 65: 4547-4549Crossref PubMed Google Scholar, 6Joliot A. Maizel A. Rosenberg D. Trembleau A. Dupas S. Volovitch M. Prochiantz A. Curr. Biol. 1998; 8: 856-863Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar); they bind to target cells in a receptor-independent manner; and each of them has a highly basic region that appears to mediate the ability of these proteins to bind to polyanions, such as heparin/heparan sulfate (HS), 2The abbreviations used are: HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MβCD, methyl-β-cyclodextrin; PBS, phosphate-buffered saline; PTD, protein transduction domains; TRITC, tetramethylrhodamine isothiocyanate; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; GM1, Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1′-ceramide. polysialic acid, and nucleic acids. Deletion analyses of the first two proteins, Tat and Drosophila Antennapedia, have mapped the apparent intercellular transfer function to the short runs of highly basic residues, termed protein transduction domains (PTDs), or cell-permeable peptides. Early mechanistic studies showed that Tat-mediated transduction occurs through a rapid, temperature- and energy-independent process, suggesting direct penetration across the lipid bilayer. Because of the strong cell surface binding characteristics of the Tat PTD, measurements of protein internalization by flow cytometry or after fixation led to the above incorrect early assumptions regarding cellular uptake. Many studies have shown that Tat and Tat fusion proteins are rapidly internalized by lipid raft-dependent endocytosis or macropinocytosis after a cell surface interaction of Tat with cell membrane heparan sulfate proteoglycans (HSPGs) (7Rusnati M. Tulipano G. Spillmann D. Tanghetti E. Oreste P. Zoppetti G. Giacca M. Presta M. J. Biol. 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Sci. 2003; 4: 125-132Crossref PubMed Scopus (210) Google Scholar, 14Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1415) Google Scholar). PDCD5 (programmed cell death 5), formerly designated as TFAR19 (TF-1 cell apoptosis-related gene 19), is cloned as a gene whose expression is increased during the apoptotic process of TF-1 cells induced by cytokine withdrawal using a cDNA-RDA method (cDNA representational differences analysis) (15Liu H.T. Wang Y. Zhang Y.M. Song Q.S. Di C.H. Chen G. Tang J. Ma D.L. Biochem. Biophys. Res. Commun. 1999; 254: 203-210Crossref PubMed Scopus (166) Google Scholar). Previous studies have shown that PDCD5, when transiently or stably overexpressed in TF-1, MGC-803, and HeLa cells, facilitates programmed cell death triggered by certain stimuli, such as growth factor withdrawal or serum withdrawal from culture medium (15Liu H.T. Wang Y. Zhang Y.M. Song Q.S. Di C.H. Chen G. Tang J. Ma D.L. Biochem. Biophys. Res. Commun. 1999; 254: 203-210Crossref PubMed Scopus (166) Google Scholar) and enhances TAJ/TROY-induced paraptotic cell death (16Wang Y. Li X. Wang L. Ding P. Zhang Y.M. Han W.L. Ma D.L. J. Cell Sci. 2004; 117: 1525-1532Crossref PubMed Scopus (167) Google Scholar). Moreover, PDCD5 translocated from the cytoplasm to the nuclei and the up-regulated expression during apoptosis (17Chen Y.Y. Sun R.H. Han W.L. Zhang Y.M. Song Q.S. Di C.H. Ma D.L. FEBS Lett. 2001; 509: 191-196Crossref PubMed Scopus (108) Google Scholar, 18Yang Y.H. Zhao M. Li W.M. Chen Y.Y. Kang B. Lu Y.Y. Apoptosis. 2006; 11: 993-1001Crossref PubMed Scopus (73) Google Scholar) and the introduction of anti-PDCD5 antibody could suppress the etoposide-induced apoptotic effects of PDCD5 in HeLa cells (19Rui M. Chen Y.Y. Zhang Y.M. Ma D.L. Life Sci. 2002; 71: 1771-1778Crossref PubMed Scopus (63) Google Scholar). Recently, the decreased expression of PDCD5 has been reported in various human tumors, such as breast cancer (20Hedenfalk I. Duggan D. Chen Y. Radmacher M. Bittner M. Simon R. Meltzer P. Gusterson B. Esteller M. Kallioniemi O.P. Wilfond B. Borg A. Trent J. N. Engl. J. Med. 2001; 344: 539-548Crossref PubMed Scopus (1387) Google Scholar), hepatocellular carcinoma (21Xu X.R. Huang J. Xu Z.G. Qian B.Z. Zhu Z.D. Yan Q. Cai T. Zhang X. Xiao H.S. Qu J. Liu F. Huang Q.H. Cheng Z.H. Li N.G. Du J.J. Hu W. Shen K.T. Lu G. Fu G. Zhong M. Xu S.H. Gu W.Y. Huang W. Zhao X.T. Hu G.X. Gu J.R. Chen Z. Han Z.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15089-15094Crossref PubMed Scopus (325) Google Scholar), cervical cancer (22Liu Z.H. Zhang D. Li K.M. Liao Q.P. J. Peking Univ. (Health Sci. 2004; 36: 407-410Google Scholar), gastric tumor (18Yang Y.H. Zhao M. Li W.M. Chen Y.Y. Kang B. Lu Y.Y. Apoptosis. 2006; 11: 993-1001Crossref PubMed Scopus (73) Google Scholar), lung cancer (23Spinola M. Meyer P. Kammerer S. Falvella F.S. Boettger M.B. Hoyal C.R. Pignatiello C. Fischer R. Roth R.B. Pastorino U. Haeussinger K. Nelson M.R. Dierkesmann R. Dragani T.A. Braun A. J. Clin. Oncol. 2006; 24: 1672-1678Crossref PubMed Scopus (93) Google Scholar), and chronic myelogenous leukemia (24Ma X. Ruan G. Wang Y. Li Q. Zhu P. Qin Y.Z. Li J.L. Liu Y.R. Ma D.L. Zhao H.S. Clin. Cancer Res. 2005; 11: 8592-8599Crossref PubMed Scopus (35) Google Scholar). These observations suggest that PDCD5 plays a significant role in both apoptotic and nonapoptotic programmed cell death and may participate in the pathophysiologic course of diseases involving abnormal programmed cell death. We have found that exogenously added human recombinant PDCD5 to culture medium of TF-1 cells or HL-60 cells can also enhance programmed cell death triggered by growth factor deprivation in TF-1 cells 3Y. Zhang and D. Ma, unpublished observations. or serum deprivation in HL-60 cells (25Zhang Y.M. Xu X.Z. Liu H.T. Song Q.S. Ma D.L. J. Chinese Immunol. 2000; 16: 8-11Google Scholar). An interesting question that remains to be addressed is how the exogenous PDCD5 enters the cells and promotes programmed cell death. Here we show that human recombinant PDCD5 protein makes use of clathrin-independent endocytosis to enter the cells. PDCD5 also has the ability to drive the internalization of large protein cargo EGFP that is fused to it. Furthermore, we have mapped a specific region of PDCD5 is necessary to drive translocation via mutagenesis assays. To determine whether the interaction of PDCD5 with the cell surface leads to the PDCD5 internalization, we tried to deliver a biologically functional peptide through PDCD5 into the cells. Others have found that the biologically functional peptide from the Mdm-2 binding domain of human p53, residues 17-26 (ETFSDLWKLL), fused to penetratin of the antennapedia to enable transport across the cell membrane, resulting in human cancer cell death (26Kussie P.H. Gorina S. Marechal V. Elenbass B. Moreau J. Levine A. Pavletich N.P. Science. 1996; 174: 948-953Crossref Scopus (1793) Google Scholar, 27Kanovsky M. Raffo A. Drew L. Rosal R. Do T. Friedman F.K. Rubinstein P. Visser J. Robinson R. Brandt-Rauf P.W. Michl J. Fine R.L. Pincus M.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12438-12443Crossref PubMed Scopus (93) Google Scholar). Our study shows that PDCD5 can also introduce human p53 peptide into cells and induce their death. Compared with transferrin internalized by the clathrin pathway and Tat involved in lipid rafts/caveolae endocytosis (11Fittipaldi 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), human PDCD5 protein is a molecule similar to Tat. Therefore, it may be useful for exploiting PDCD5 as a vehicle for transcellular delivery of various molecules. The translocation activity of PDCD5 also suggests that human PDCD5 may promote programmed cell death via a novel mechanism that involves its reentry into the cells. Antibodies and Fluorescent Markers—Antibodies against clathrin heavy chain, transferrin receptor and caveolin-1 were from Transduction Laboratories (BD Biosciences). Anti-HSP70 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-histone H3 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). IRDye 800- or IRDye 700-conjugated anti-mouse and anti-rabbit IgG secondary antibody and IRDye 800-conjugated anti-GFP antibody were obtained from Rockland. Secondary TRITC-labeled goat anti-mouse antibody, TRITC- and FITC-labeled transferrin, and Alexa Fluor 594-labeled cholera toxin subunit B and anti-EGFP antibody were all from Molecular Probes, Inc. (Eugene, OR). Protease inhibitor mixture tablets were obtained from Roche Applied Science. Hoechst 33342 was from Sigma. pDsRed-Hub Plasmid Construction—The cDNA encoding Homo sapiens clathrin heavy chain residues 1073-1675 (28Liu S.H. Marks M.S. Brodsky F.M. J. Cell Biol. 1998; 140: 1023-1037Crossref PubMed Scopus (116) Google Scholar), clathrin hub fragment, was cloned into the BamHI and XhoI sites of the pDsRed-C3 vector (Clontech) to generate pDsRed-Hub for transfection. The plasmid was confirmed by DNA sequencing. Cell Cultures and Transfection—HEK293 and U937 cells were maintained in RPMI 1640 (Invitrogen), and HT-29 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), penicillin (100 units/ml), streptomycin (100 μg/ml), and l-glutamine (2 mm). 4 × 106 HEK293 cells in 400 μl were transiently transfected by electroporation with 10 μg of the expression plasmid at 120 V for 20 ms, using an electric pulse generator (Electro Square Porator ECM 830, BTX, San Diego, CA). Cells were generally assayed 36-48 h after transfection. Recombinant PDCD5 Protein—Recombinant PDCD5 protein was purified as described previously (15Liu H.T. Wang Y. Zhang Y.M. Song Q.S. Di C.H. Chen G. Tang J. Ma D.L. Biochem. Biophys. Res. Commun. 1999; 254: 203-210Crossref PubMed Scopus (166) Google Scholar, 29Wang L. Fan H. Mo X.N. Zhang Y.M. Wei Z.R. Zhong Y.C. Huang D.W. J. Peking Univ. (Health Sci.). 2003; 35: 360-363Google Scholar). Briefly, Escherichia coli pop2136 harboring the prokaryotic expression vector pMTY4-PDCD5 was heated to induce the expression of the MS2-PDCD5 fusion protein. After denaturing, renaturing, and cleavage with thrombin, PDCD5 was purified by ion exchange chromatography with DEAE-Sepharose Fast Flow (Amersham Biosciences) and by gel filtration over Sephacyl S-200 HR (Amersham Biosciences). Fluorescein isothiocyanate (FITC) labeling of recombinant PDCD5 protein was prepared as described previously (30Masseyeff R.F. Albert W.H. Staines N.A. Methods Immunol. Anal. 1993; 3: 237-238Google Scholar). Subcloning and Deletion Mutagenesis—PDCD5 sequence was cloned into the EcoRI site of pEGFP-C3 vector (Clontech), and then EGFP and EGFP-PDCD5 sequence were cloned into the NotI site of pGEX-4T-2 vector (Amersham Biosciences) to generate pGEX-EGFP and pGEX-EGFP-PDCD5, respectively. To facilitate expression vector construction, a NotI recognition site was introduced at both ends of the open reading frame by PCR with primers. The C-terminal deletion mutants of PDCD5 (amino acids 1-115 and 1-108, respectively) were constructed using PCR amplification of the relevant portions of PDCD5 cDNA, followed by restriction digestion and subsequent subcloning into pGEX-4T-2 vector to generate pGEX-EGFP-PDCD5Δ116-125 and pGEX-EGFP-PDCD5Δ109-125, respectively. PDCD5 deletion constructs are designated PDCD5Δx-y, where x and y indicate the first and last deleted PDCD5 residue according to the complete human PDCD5 sequence (15Liu H.T. Wang Y. Zhang Y.M. Song Q.S. Di C.H. Chen G. Tang J. Ma D.L. Biochem. Biophys. Res. Commun. 1999; 254: 203-210Crossref PubMed Scopus (166) Google Scholar). All constructs were sequenced using an ABI 3100 DNA sequencer. The pGEX plasmids containing the required clone were transformed into the E. coli strain BL21 (DE3) to express the corresponding GST fusion proteins. GST fusion proteins were respectively bound to glutathione-Sepharose 4B resin (Amersham Biosciences). After on-column thrombin cleavage, the EGFP, EGFP-PDCD5, EGFP-PDCD5Δ116-125, and EGFP-PDCD5Δ109-125 proteins were released from GST, because the GST moiety remains bound to the Sepharose resin while the desired protein is eluted with PBS buffer. p53N Peptide and p53N-PDCD5 Expression—The peptide ETFSDLWKLL from the Mdm-2 binding domain of p53, denoted as p53N, was synthesized by solid phase synthesis and purified by HPLC to 95% purity in GL Biochem (Shanghai) Ltd. The oligonucleotides encoding the residues 17-26 (ETFS-DLWKLL) from the amino-terminal of p53 Mdm-2-binding domain of p53 were synthesized and subcloned into the pGEX-4T to express the p53N peptide fused to the N terminus of PDCD5 (i.e. p53N-PDCD5 protein). The p53N-PDCD5 protein was expressed in BL21 (DE3) and purified with glutathi-one-Sepharose 4B resin (Amersham Biosciences). FITC labeling of recombinant p53N-PDCD5 protein was prepared as previously described previously (30Masseyeff R.F. Albert W.H. Staines N.A. Methods Immunol. Anal. 1993; 3: 237-238Google Scholar). Protein Subcellular Fractionation Analysis—5 × 106 HEK293 cells or U937 cells were incubated with recombinant EGFP-PDCD5 for 5 h and washed in PBS, and cells were fractionated into cytosol, membranes, nuclei, and cytoskeleton using the Qproteome cell compartment kit from Qiagen according to the manufacturer's instructions, and then one-fifth of each fraction was subjected to Western blotting. The fractions were analyzed to detect EGFP-PDCD5 protein and HSP70 and histone 3 proteins as the controls for cross-contamination during the cell fractionation procedure were sequentially reprobed with corresponding antibodies, respectively, on the same membrane. Isolation of Triton X-100-insoluble Membranes—Isolation of Triton X-100-insoluble membranes was performed essentially as described previously (31Hinrichs J.W. Klappe K. Hummel I. Kok J.W. J. Biol. Chem. 2004; 279: 5734-5738Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 32Rakheja D. Narayan S.B. Pastor J.V. Bennett M.J. Biochem. Biophys. Res. Commun. 2004; 317: 988-991Crossref PubMed Scopus (31) Google Scholar), with some modifications. 5 × 106 HEK293 cells or 1 × 107 U937 cells were incubated with recombinant EGFP-PDCD5 for 5 h, washed, and left in 1 ml of ice-cold 1% (v/v) Triton X-100 in MNE buffer (25 mm MES (pH 6.5), 150 mm NaCl, 5 mm EDTA, and 1× protease inhibitor mixture (Roche Applied Science)) and incubated on ice for 20 min. The cell suspension was then homogenized with a Dounce homogenizer at 20 strokes. The homogenate was then adjusted to 40% sucrose by the addition of 1 ml of 80% (w/v) sucrose in MNE buffer and loaded onto the bottom of a 5-ml ultracentrifuge tube. On top of this, 2 ml of 30% (w/v) sucrose in MNE buffer was overlaid, and then 1 ml of 5% (w/v) sucrose in MNE was overlaid. After centrifugation for 18 h at 100,000-150,000 × g in a Beckman MLS50 swing-out rotor, 10 0.5-ml fractions were collected from the top of the gradient. To concentrate the raft fraction, each fraction was diluted 10-fold with ice-cold MNE and centrifuged in a Beckman MLS50 swing-out rotor at 150,000 × g for 2 h at 4°C. The pellets were solubilized in sample buffer, separated by SDS-PAGE, and subjected to Western blotting. The same membrane was reprobed with corresponding antibodies, respectively. Western Blotting—Proteins were transferred to nitrocellulose membranes (Hybond™ and ECL™; Amersham Biosciences). After blocking in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBS-T) and 5% (w/v) nonfat dry milk for 1 h at room temperature, membranes were incubated with IRDye 800-conjugated anti-GFP antibody or with corresponding primary antibody overnight at 4 °C. Membranes were then washed with TBS-T three times for 10 min and directly detected or incubated with corresponding IRDye 800 or IRDye 700-labeled IgG secondary antibody in the dark for 1 h at room temperature. Following another three washes with TBS-T for 10 min, the membranes were scanned in the appropriate channels (800 nm for IRDye800 antibody, 700 nm for IRDye700 antibody) of the LI-COR Infrared Imaging System (Odyssey, Lincoln, NE) and analyzed with Odyssey software. Drug Treatments—Cells were pretreated with the different drugs, 0.1-100 μg/ml heparin, 1.25-10 mm methyl-β-cyclodextrin (MβCD), 5 μm cytochalasin D, 20 μm nocodazole, 10-250 μm genistein, genistin, or 100 nm to 2.5 μm staurosporine, respectively, for 30 min in RPMI 1640 medium supplemented with 10% fetal bovine serum, after which time recombinant protein in fresh medium containing the same inhibitor was added. Cells were then processed after treatment with inhibitor and recombinant protein for fluorescence microscopy or flow cytometry. All these drugs were from Sigma. Treatment with Triton X-100—Cells were incubated with recombinant PDCD5-FITC together with transferrin-TRITC for 5 h, washed, and left in ice-cold 1% Triton X-100 in PBS for 20 min before fixation with 2% paraformaldehyde. Fluorescence Microscopy—For different fluorescent protein treatments, HEK293 cells were grown in specialized glass-bottom microwell dishes (MatTek Corp.) to about 50% confluence, and then fresh, 10% fetal bovine serum medium containing different fluorescent molecules was added. Final concentrations of fluorescent molecules were 1 μm recombinant PDCD5-FITC, p53N-PDCD5-FITC, Alexa Fluor 594-labeled cholera toxin B and/or transferrin-TRITC, or 1 μm protein of EGFP, EGFP-PDCD5, EGFP-PDCD5Δ116-125, or EGFP-PDCD5Δ109-125. After 5 h of treatment with recombinant proteins, cells were rinsed twice with PBS buffer and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. For immunostaining, fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed and blocked with PBS containing 2% bovine serum albumin (BSA) for 30 min at room temperature, and incubated with a 1:200 dilution of primary antibodies in PBS supplemented with 2% BSA. Cells were rinsed three times with PBS and incubated with a 1:100 dilution of secondary antibodies for 30 min in 2% BSA in PBS. Cells were rinsed three times, all fluid was removed, and samples were mounted with 90% glycerin (Sigma) in PBS. For live cell recording, cells plated on 35-cm glass bottom dishes (MatTek Corp.) were placed in a humidified Plexiglas chamber and maintained at 37 °C throughout the experiment. For the fast dynamics recording and co-localization experiments, cells were imaged using a TCS-SP laser-scanning confocal microscope with a ×40 or ×63 oil immersion lens (Leica Microsystems, Mannheim, Germany). Electron Microscopy—To study the internalization of PDCD5 coupled to gold particles (PDCD5-gold), a pre-embedding procedure was performed. Briefly, according to the Colloidal Gold Conjugation Protocol (Schleicher & Schuell), PDCD5 gold labeling was done at pH 6.2 (for PDCD5, the pI is pH 5.9 (29Wang L. Fan H. Mo X.N. Zhang Y.M. Wei Z.R. Zhong Y.C. Huang D.W. J. Peking Univ. (Health Sci.). 2003; 35: 360-363Google Scholar)). HEK293 or HT-29 cells cultured on 12-well cell culture plates for 20 h were washed once with Dulbecco's modified Eagle's medium plus 25 mmol/liter HEPES containing 0.1% BSA at 8 °C and then incubated for 1 h with PDCD5-gold (9 nm, 3.6 μg/ml) at 8 °C and shifted to 37 °C for 1 h to induce the internalization. After several washes, cells were scraped, pelleted for 5 min at 3000 rpm in an Eppendorf centrifuge, and fixed with 3% glutaraldehyde in PBS for 2 h at 4°C. Fixed cells were prepared for electron microscopy as previously described (33Tran D. Carpentier J.L. Sawano F. Gorden P. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7957-7961Crossref PubMed Scopus (198) Google Scholar). Thin sections were cut, mounted on grids, and viewed in a JEM-1230 transmission electron microscope (JEOL, Japan) after contrasting with uranyl acetate. Flow Cytometry—Quantification of internalized FITC-labeled PDCD5 recombinant protein was performed. Briefly, cells were plated in 24-well plates to about 70% confluence and incubated with 5 μg/ml recombinant PDCD5-FITC for the times indicated in Fig. 1, or cells were pretreated with the different concentrations of heparin (0.1-100 μg/ml) or the different concentrations of MβCD (1.25-10 mm) for 30 min and then incubated with 5 μg/ml PDCD5-FITC or p53N-PDCD5-FITC recombinant protein in fresh medium containing the same inhibitor. Cells were then washed, trypsinized, centrifuged, again washed twice with PBS, and finally analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences). Programmed Cell Death Induction and Detection of Phosphatidylserine Externalization—HT-29 cells were seeded into each well of 24-well tissue culture dishes containing 1 ml of culture medium. Cells were allowed to adhere for 24 h when the medium was replaced with media containing a 0.75-3 μm concentration of the recombinant p53N-PDCD5 protein, recombinant PDCD5 protein, or p53N peptide to be tested. Another set of wells (controls) in each experiment was processed identically but with peptide and protein-free media. Cells were fed every 24 h with 0.3 ml of their respective peptide- or protein-containing media. The cultures were examined daily for changes in cell growth and morphology. Cells were released and collected after 2 days of treatment. 1 × 105 washed cells were resuspended in 200 μl of binding buffer (PBS containing 1 mm calcium chloride). To detect phosphatidylserine (PS) externalization, FITC-conjugated annexin V (0.5 μg/ml final concentration) was added to the suspended cells according to the manufacturer's instructions (Biosea, China). After incubation for 20 min at room temperature, 400 μl of binding buffer was added again, and samples were immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences) with excitation using a 488-nm argon ion laser. Caspase-3-like Activity Assay—Briefly, after treatment with a 3 μm concentration of the recombinant p53N-PDCD5 protein, recombinant PDCD5 protein, or p53N peptide for 2 days, incubation medium was removed, and the cells were lysed for 10 min in ice-cold lysis buffer containing 10 mm Tris-HCl, pH 7.5, 10 mm Na2HPO4/NaH2PO4, 130 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride. Cell lysates were clarified by centrifugation at 18,000 × g for 20 min at 4 °C. Cell lysates containing 15 μg of protein were incubated at 37 °C in buffer containing 25 mm HEPES, pH 7.5, 1 mm EDTA, 100 mm NaCl, 0.1% CHAPS, and 10 mm dithiothreitol with the fluorogenic substrate Ac-DEVD-7-amino-4-methylcoumarin (ALEXIS Biochemicals Industriestrasse). Fluorescence was measured with the use of a FLUOStar fluorometer (BMG Labtechnologies) equipped with an excitation filter of 380 nm and emission filter of 460 nm. Results were calculated as a proportion of the control over 90 min (T90/T0). Samples were prepared in triplicate. Kinetics of PDCD5 Internalization—The kinetics of cellular internalization of PDCD5-FITC in HEK293 cells and U937 cells is shown comparatively in Fig. 1. The PDCD5-FITC protein or FITC with the equal absorbance to PDCD5-FITC at 495 nm was added to the cell culture medium of human HEK293 or U937 cells, and cellular fluorescence was quantitatively assessed at different time points by flow cytometry. Analysis of the flow cytometry profiles and of the mean cellular fluorescence values shown in Fig. 1, A-D, clearly indicates that the PDCD5-FITC protein internalization kinetics in different cell types is similar. However, internalization of PDCD5-FITC was blocked at 4 °C in both cell types (Fig. 1G), suggesting that the internalization is an energy-dependent process. A microscopic analysis of cells treated with PDCD5-FITC protein indicated that fluorescence was localized to discrete compartments in the cytoplasm (Fig. 1E) with a very few labeled proteins reaching the nuclei (Fig. 1F), suggesting internalization by an endocytic process in this case.
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