Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma

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Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma Cells Hyo Young Kim, Sabin Kim, Hae Jun Pyun, Jeehye Maeng, and Kyunglim Lee Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500547f • Publication Date (Web): 19 Nov 2014 Downloaded from http://pubs.acs.org on November 22, 2014

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Molecular Pharmaceutics

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Cellular Uptake Mechanism of TCTP-PTD in Human Lung

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Carcinoma Cells

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Hyo Young Kim, Sabin Kim, Hae Jun Pyun, Jeehye Maeng, and Kyunglim Lee*

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Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans

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University, Seoul 120-750, Republic of Korea

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Corresponding Author

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Address: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans

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University, Seodaemoon-Gu, Ewhayeodae-gil 52, Seoul 120-750, Korea. Telephone: +82-2-

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3277-3024. Fax: +82-2-3277-2851. E-mail: [email protected]

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Running head: Transduction mechanism of TCTP-PTD in A549 cells

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ACS Paragon Plus Environment


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ABSTRACT

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We reported previously that human translationally controlled tumor protein (TCTP) contains,

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at its NH2-terminus, a protein transduction domain (PTD), which we called TCTP-PTD, with

5

the amino acid sequence, MIIYRDLISH. In this report we describe how TCTP-PTD

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penetrates A549 human lung cancer cell membranes and promotes protein internalization.

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Cellular uptake of fluorescent TCTP-PTD and a recombinant fusion protein consisting a

8

TCTP-PTD and GFP (green fluorescent protein) was analyzed by confocal fluorescence

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microscopy and flow cytometry. Inhibitor assays using several agents that perturb the

10

internalization process revealed that TCTP-PTD transduces the cells partly via lipid

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raft/caveolae-dependent

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dynamin/actin/microtubule-dependent pathway. To trace the pathway followed by the

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penetration of TCTP-PTD, the localization of PTDs was investigated in the lipid-raft,

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subcellular, and ER fractions. We found that after entry, TCTP-PTD is localized in the

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cytoplasm and cytoskeleton, but not in the nucleus, and is transported into endoplasmic

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reticulum (ER). Expression levels of caveolin-1 in A549 and HeLa cells are different and

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these differences appear to contribute to the sensitivity of TCTP-PTD uptake inhibition,

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against lipid raft depleter, nystatin. This elucidation of the underlying mechanism of TCTP-

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PTD translocation, may help design of approaches that employ TCTP-PTD in the cellular

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delivery of bioactive molecules.

endocytosis

and

partly

by

macropinocytosis

in

a

21

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KEYWORDS: Endocytosis pathway, Mechanism, Lipid-rafts/caveolae, Protein transduction

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domain (PTD), Subcellular fractionations, Translationally controlled tumor protein (TCTP) 2


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1. INTRODUCTION

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A highly conserved translationally controlled tumor protein (TCTP) exists in many species.

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Since the cloning, 20 years ago, of the human 1 and murine 2 TCTP’s disparate functions have

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been an area of active investigative interest. Human TCTP cDNA encodes a 172 amino acids-

5

long protein

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growth, apoptosis, protein synthesis, tumor reversion, histamine-releasing activity and in late

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phase allergic reaction

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transduction domain (PTD) with the sequence, MIIYRDLISH, at its NH2-terminus8. We

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designated this peptide TCTP-PTD, because it facilitates the transduction of other proteins

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1

with a molecular mass of 23 kDa and has been shown to be involved in cell

3-7

. We have previously showed that TCTP contains a protein

into cells 8.

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Mounting evidence from studies with cationic PTDs such as TAT-PTD and polyArg 9, 10,

12

indicates that initial electrostatic interaction between PTDs and cell surface proteoglycans

13

leads to reconstruction of the cytoskeleton actin network and the activation of the small

14

GTPases such as RhoA 11, 12. Also, interaction of PTDs with the components of extracellular

15

matrix is known to trigger the internalization of PTDs through energy dependent processes 13,

16

14

17

cells

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type which determines the membrane characteristics, (ii) the type of the PTD, (iii) type and

19

concentration of the cargo, and (iv) the interaction of PTD with various cell surface

20

molecules 17.

. Cationic PTDs having positive charges can bind to the negatively charged surface of the 9, 15

. The subsequent cellular uptake is determined by factors

16

including: (i) the cell

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We showed previously that TCTP-PTD acts as a protein transduction vehicle both when

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fused to a protein and also when linked to an adenoviral antigen, apoptotic peptide and 3



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insulin, whether covalently or non-covalenly 8, 18, 19. Unlike TAT-PTD which binds to HSPGs

2

of cell surface for internalization, TCTP-PTD utilizes mechanisms that mainly involve lipid-

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raft endocytosis and partial macropinocytosis for internalization8. However, the exact

4

mechanisms underlying the internalization of proteins into cells by TCTP-PTD are unclear

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and the goal of this study is to attempt fill this gap.

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Toward this goal, we examined how TCTP-PTD promotes internalization in A549 lung

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carcinoma cells through biochemical approaches. We employed specific endocytic inhibitors,

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such as chlorpromazine (CPZ), dynasore, methyl-β-cyclodextrin (MβCD), cytochalasin D, 5-

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(N-ethyl-N-isopropyl) amiloride (EIPA), nocodazole, chloroquine, and nystatin. We isolated

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lipid-rafts, subcellular fractions, and endoplasmic reticulum (ER) from TCTP-PTD-treated

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A549 cells, and attempted to determine the pathway of TCTP-PTD entry into cells and also

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where TCTP-PTD localizes inside the cells following the cellular entry.

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2. MATERIALS AND METHODS

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2.1. Constructs and Reagents. TAT-PTD region of pTAT-HA-EGFP vector (kindly provided

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by S.F. Dowdy, UCSD, CA, USA)

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protein was fused at the C-terminus of TCTP-PTD and three glycine residues were inserted

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between TCTP-PTD and GFP. Synthetic TCTP-PTD peptides were conjugated to TAMRA

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and rhodamine, respectively, for use in quantitative assessment of cellular uptake. High purity

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(> 90%) peptides obtained from Peptron (Daejeon, Korea) were freeze-dried and

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reconstituted in high-purity DMSO (10 mM) and stored at -70°C until use. The endocytic

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inhibitors, cytochalasin D, chlorpromazine hydrochloride (CPZ), 5-(N-ethyl-N-isopropyl)

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amiloride (EIPA), dynasore, methyl-β-cyclodextrin (MβCD), chloroquine, and nystatin were

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from Sigma Aldrich (St. Louis, MO, USA) except for nocodazole which was from Tocris

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Bioscience (Minneapolis, MN, USA). Anti-GFP, -GAPDH, and -tubulin antibodies were

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obtained from Santa Cruz Biotechnology (Delaware, CA, USA). Anti-cytochrome c, -

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caveolin-1, -calnexin and -PARP antibodies were obtained from Cell Signaling Technology

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(Beverly, MA, USA). OptiPrep was from Sigma Chemical Corp (St. Louis, MO, USA).

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was removed or substituted by TCTP-PTD. The GFP

17

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2.2. Cells and Cell Culture. HeLa (human cervical cancer cells) and HepG2 (human liver

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cancer cells) were maintained in Dulbecco’s Modified Eagle Media (DMEM) (Invitrogen)

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supplemented with 10% fetal bovine serum (FBS). A549 (Human lung cancer cells) were

21

cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Invitrogen)

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supplemented with 10% FBS. Both the cell lines were purchased from American Type

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Culture Collection (Rockeville, MD, USA). 5



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2.3. Expression and Purification of TCTP-PTD-GFP Fusion Protein. The recombinant

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plasmids were transformed into E. coli BL21 (DE3) pLysS to express fusion proteins. The

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bacteria were grown to reach an OD600 of 0.5–0.8 prior to induction. For the expression of

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TCTP-PTD-GFP and GFP proteins, cells were treated with 1 mM isopropyl-β-D-

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thiogalactoside (IPTG), and incubated for 3 h. The cells were then harvested, suspended in

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binding buffer (20 mM Tris–HCl, pH 7.9, 0.5 M NaCl) and sonicated. The fusion proteins

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were purified by Ni2+ affinity chromatography using a Sepharose resin column (ELPiS

8

Biotech). After washing the column with 100 mM imidazole in binding buffer, the fusion

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protein was eluted with 1 M imidazole in binding buffer. After removing imidazole using PD-

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10 desalting column (GE Healthcare Bioscience), the purified fusion protein was used

11

immediately or stored frozen in 10% glycerol at −70°C.

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2.4. Cellular Uptake Analysis and Flow Cytometry. For the transduction analysis of TCTP-

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PTD-GFP fusion proteins, cells were cultured to 70–80% confluence in 6-well microtiter

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plates. Just before the transduction experiment, the culture medium was replaced with 0.5 ml

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fresh medium, to which TCTP-PTD-GFP fusion proteins were added. After incubation for the

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indicated intervals, the cells were washed with PBS at least four times to eliminate the

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tethered proteins at the surface of the cells. Cells were then examined directly by confocal

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fluorescence microscopy (Zeiss LSM 700) and were analyzed by western blotting.

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For the quantification of uptake, cells were grown to 60-70% confluence in cover glass-

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bottomed dishes prior to incubation with TCTP-PTD (TAMRA-TCTP-PTD, Rhodamine-

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TCTP-PTD, FITC-TCTP-PTD, and TCTP-PTD-GFP) at a concentration of 10 µM for 2 h. 6


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The cells were then washed at least twice with cold PBS and treated with trypsin/EDTA for

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10 min to completely remove the cell surface-bound proteins. They were then examined for

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internalization by flow cytometry using a FACS Calibur flow cytometer (BD Biosciences).

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Internalization of TCTP-PTD was quantified using the area under a curve of each histogram.

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The relative internalization was presented as the percentage of the value of TCTP-PTD

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uptake.

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2.5. Treatment with Endocytic Inhibitors. Cells were treated with various inhibitors (10-50

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µM CPZ, 40-160 µM dynasore, 5-10 mM MβCD, 10-50 µM each EIPA, nocodazole, and

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cytochalasin D, 50 µM chloroquine, 50 µg/ml nystatin) for 30 min in a serum-free medium

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and then TCTP-PTD fusion protein or TAMRA-TCTP-PTD or FITC-TCTP-PTD was added.

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After 2 h, the cells were analyzed by flow cytometry, confocal fluorescence microscopy and

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western blotting.

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2.6. Preparation of Lipid-rafts. MacDonald’s method

16

used for preparing lipid-rafts. Cells were treated with GFP and TCTP-PTD-GFP for various

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times, in four 100-mm plates. They were then washed and scraped out in 1 ml wash buffer

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(10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA), centrifuged for 10 min at 1,000 g,

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and the pellets were resuspended in 0.2 ml of wash buffer containing a protease inhibitor

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cocktail (Roche, MA, USA). The cells were then lysed by passage through a 22 gauge needle

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(15 times) and centrifuged at 15,000 g for 15 min. The supernatant was transferred to a new

22

tube. An equal volume (0.2 ml) of wash buffer containing 50% OptiPrep density gradient

modified as described below, was

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medium was then added to the post-nuclear supernatants which were then placed at the

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bottom of a 4 ml centrifuge tube. Gradients of 10% to 40% OptiPrep density gradient

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medium in wash buffer (0.2 ml) were poured on top of the lysate centrifuged for 5 h at

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150,000 g in a SW-60Ti rotor in a Beckman ultracentrifuge. The distribution of caveolin-1

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among the fractions was assessed by western blotting.

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2.7. Localizations of TCTP-PTD in Subcellular Fractions. The cytoplasm, organelle

8

membranes (including ER and mitochondria), nucleus, and cytoskeletal matrix fractions were

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purified using a ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem) with slight

10

modifications22. Each fraction was analyzed by western blotting using antibodies specific for

11

tubulin for cytoplasm, PARP for the nucleus, cytochrome c for organelle membranes,

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calnexin for ER, and actin for cytoskeleton.

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2.8. Isolation of ER after Penetration of TCTP-PTD. For isolation of ER after TCTP-PTD

15

entry in A549 cells, the cells were plated in 100-mm dish and treated with TCTP-PTD for 2 h,

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and then washed twice with PBS. One ml isosmotic homogenization buffer (50 mM HEPES,

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pH 7.4, 1.25 mM sucrose, 125 mM KCl) was then added. The cells were homogenated, and

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centrifuged at 1,000 g for 10 min at 4 °C. The supernatant was then transferred to a clean tube,

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centrifuged at 12,000 g for 15 min at 4 °C and the pelleted mitochondria and cell debris were

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discarded. The supernatant was then transferred to a new tube and centrifuged at 90,000 g for

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60 min at 4 °C. The supernatant was discarded and the pellet which contains the ER fraction

22

was resuspended in suspension buffer (1X isosmotic buffer with 5 mM EGTA) containing 1X 8


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protease inhibitor cocktail (Roche, MA, USA) and examined for the presence of calnexin

2

using western blotting.

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3. RESULTS

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3.1. Cellular uptake of TCTP-PTD fusion proteins and peptides. To study the molecular

4

pathway involved in the penetration of extracellular TCTP-PTD into cells, TCTP-PTD was

5

fused at the N-terminus of GFP protein, as shown in Fig. 1A. TCTP-PTD (residues 1-10 of

6

TCTP protein) was inserted into pTAT-HA vector and then constructs were expressed in

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bacterial expression system. After purification, the purity of recombinant proteins was

8

analyzed by SDS-PAGE and Coomassie Staining (Fig. 1A). A549 cells were treated with

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TCTP-PTD-GFP and the penetration of the protein was detected by western blotting using

10

anti-GFP antibody. Fig. 1B shows that TCTP-PTD-GFP internalized into A549 cells, but GFP

11

did not. To confirm the penetration of TCTP-PTD into cells using flow cytometry and

12

confocal fluorescence microscopy, TCTP-PTD peptides were labeled with fluorescent dyes

13

including TAMRA, FITC and Rhodamine, respectively, at the N-terminus of the peptide. We

14

found that both fluorescent labeled-TCTP-PTDs penetrated into the cells in FACS analysis

15

(Fig. 1C) as well as in confocal fluorescence microscopy (Fig. 1D). Absence of potential

16

artifacts from membrane-tethered peptides, was confirmed by the z-stack confocal images of

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internalized PTDs (Fig. S1 in Supporting Information). Clearly TCTP-PTD can internalize

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into the cells both fluorescent-labeled peptides and GFP fused proteins.

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3.2. Effect of endocytic inhibitors on TCTP-PTD uptake. As previously shown with most

21

PTDs, TCTP-PTD penetrated HeLa cells via energy-dependent endocytosis using lipid raft-

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mediated pathway and partly macropinocytosis 8. To understand the mechanism of cellular

23

internalization of TCTP-PTD in A549 cells, effects of various endocytic pathway-specific 10


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inhibitors on the internalization of TCTP-PTD were investigated using FACS analysis and

2

western blotting.

3

We first examined whether TCTP-PTD internalizes into A549 cells via clathrin-

4

dependent pathway. We tested the effect of chlorpromazine (CPZ), a cationic amphipathic

5

inhibitor of clathrin, on the TCTP-PTD uptake in A549 cells. The cells were treated with 10,

6

20, and 50 µM CPZ for 30 min, respectively. We then added TAMRA-TCTP-PTD to the

7

cells and measured TCTP-PTD penetration by flow cytometry. We found that CPZ did not

8

interrupt TCTP-PTD penetration (Fig. 2A). This was confirmed by confocal fluorescence

9

microscopy analysis (Fig. 2B) and the flow cytometry dot plots of internalized FITC-TCTP23

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PTD (Fig. S2 in Supporting Information). However, as demonstrated by others

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transport of TAT-PTD was inhibited by CPZ treatment (Fig. S3 in Supporting Information).

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Increase in the cellular uptake of TAMRA-TCTP-PTD at 50 µM CPZ treatment (Fig. 2A)

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suggests that TCTP-PTD internalizes the cells via non-endocytic pathway in the presence of

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high doses of CPZ, as shown in other PTDs 24. These results led us to exclude the clathrin-

15

mediated endocytosis (CME) as the major entry route of TCTP-PTD into A549 cells (Fig. 2).

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Next, we investigated whether TCTP-PTD penetrates the cells by caveolae-dependent

17

and lipid-raft mediated pathways. Caveolae, invaginated regions of lipid rafts domains, are

18

constituted by cholesterol and sphingolipids and are formed by the action of caveolins

19

We examined the effect of cholesterol depletion on the TCTP-PTD uptake using methyl-β-

20

cyclodextrin (MβCD). MβCD induces the depletion of cholesterol in the plasma membrane

21

and disrupts the lipid-rafts. As shown in Fig. 3A, the penetrating ability of TAMRA-TCTP-

22

PTD was decreased by approximately 70% by 5 mM MβCD, implying the involvement of

23

lipid raft-dependent endocytosis. 11


, cellular

25

.


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1

Because dynamin, a GTPase that plays a role in the scission of vesicles, is necessary

2

for both clathrin- and caveolae-mediated endocytosis, we studied its effect on the

3

penetration TCTP-PTD in A549 cells, using dynasore, an inhibitor for dynamin GTPase

4

activity. Treatment of A549 cells with 40 µM dynasore decreased TAMRA-TCTP-PTD

5

penetration by 50% (Fig. 3A). Analysis using confocal fluorescence microscopy (Fig. 3B)

6

and flow cytometry dot plots (Fig. S2 in Supporting Information) confirmed that both

7

inhibitors inhibited the internalization of FITC-TCTP-PTD into cells. Then we tested using

8

western blot analysis whether the uptake of TCTP-PTD-GFPs also depends lipid-rafts and

9

dynamin, we found that MβCD as well as dynasore reduced the amount of internalized

10

fusion proteins (Fig. 3C). These results suggest that, TCTP-PTD penetration employs intact

11

dynamin and lipid-rafts.

12

We then examined whether macropinocytosis, during which rearrangement of the

13

actin cytoskeleton takes place, and macropinosomes are generated by actin-guided

14

membrane ruffling

15

by A549 cells. For this, we incubated A549 cells in the presence of 5-(N-ethyl-N-isopropyl)

16

amiloride (EIPA), a Na+,H+-exchanger inhibitor, which specifically blocks macropinocytosis.

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Fig. 4A (top panel) shows that the cellular uptake of TCTP-PTD was significantly lower in

18

the presence of EIPA, implying participation of macropinocytosis. We then examined

19

whether actin rearrangement occurs during TCTP-PTD penetration, by treating the cells

20

with various doses of cytochalasin D, which causes depolymerization of F-actin and

21

disruption of actin cytoskeleton. We found that the ability of TCTP-PTD to penetrate into

22

cells was also reduced by cytochalasin D (Fig. 4A, middle panel). Both EIPA and

23

cytochalasin D consistently reduced the ability of TCTP-PTD to penetrate into the cells by

26, 27

, contributes to the cellular uptake of TAMRA-labeled TCTP-PTD

12


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about 60% at 50 µM of inhibitors (Fig. 4A, top and middle panel). Confocal fluorescence

2

microscopic studies (Fig. 4B) and representative dot plots of flow cytometry (Fig. 3S in

3

Supporting Information), also confirmed that EIPA and cytochalasin D similarly decreased

4

the ability of FITC-TCTP-PTD to penetrate.

5

Since microtubules are also known to mediate the processes of endocytic pathways 28,

6

we next tested the possible role of microtubules in TCTP-PTD’s uptake in A549 cells.

7

Following the incubation of the cells with TAMRA-TCTP-PTD or FITC-TCTP-PTD,

8

cellular uptake of TCTP-PTD was evaluated by flow cytometry and confocal fluorescence

9

microscopy in the presence and absence of nocodazole, a microtubule depolymerizer and

10

inhibitor for the vesicular trafficking through the endosomal route. As shown in Fig. 4A

11

(bottom panel), TCTP-PTD uptake was decreased more than 30% in the presence of

12

nocodazole when compared to untreated controls. Inhibition of FITC-TCTP-PTD uptake by

13

nocodazole treatment was also confirmed by confocal fluorescence microscopy (Fig. 4B)

14

and in flow cytometry dot plot study (Fig. S2 in Supporting Information). This suggests that

15

endocytic pathway of the TCTP-PTD penetration is microtubules-dependent process.

16

To sum up, the above results suggest that TCTP-PTD penetration into A549 cells is

17

principally mediated by the lipid-raft domain and macropinocytosis, but not by CME,

18

through the process via dynamin- and microtubule-dependent endocytic processes.

19 20

3.3. Localization of TCTP-PTD in lipid-rafts/caveolae fractions of A549 cells. Having

21

found that TCTP-PTD penetration is sensitive to cholesterol depletion and may therefore is

22

mediated via lipid rafts, we investigated the role of caveolae in TCTP-PTD penetration.

23

Because nystatin, an inhibitor of caveolar endocytosis disrupts lipid-raft/caveolae structure 13



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and function, we examined the effect of 50 µg/ml nystatin on the penetration of TAMRA-

2

labeled TCTP-PTD into A549 cells. We found that nystatin inhibited TCTP-PTD penetration,

3

confirming that TCTP-PTD enters A549 cells through caveolae-mediated pathway (Fig. 5A,

4

left panel).

5

We then examined whether the mechanism for internalization of TCTP-PTD into cells

6

depends on the cell type. We found that TCTP-PTD uptake by HeLa cells was less influenced

7

by nystatin than that by A549 cells (Fig. 5A). This raised the question whether the

8

mechanism for penetration of TCTP-PTD into cells depends on the cellular components of

9

the cell type’s endocytic machinery. Since caveolin is expressed in epithelial cells in varying

10

degrees depending on the cell type, we compared the expression level of caveolin-1 in A549

11

cells with that in HeLa cells. Fig. 5B shows that the expression of caveolin-1, determined by

12

western blotting, is higher in A549 cells than in HeLa cells. It is likely that TCTP-PTD

13

penetrates better via caveolae-mediated endocytosis in A549 cells than in HeLa cells since

14

TCTP-PTD penetration is inhibited more significantly by caveolae inhibitors in A549 cells,

15

than in HeLa cells (Fig. 5A) in which the expression of caveolin-1 is more abundant (Fig.

16

5B). In addition, cellular transduction of FITC-TCTP-PTD in HepG2 cells in which the

17

expression of caveolin-1 was not found

18

(Fig. S4B in Supporting Information), than that of TAMRA-TCTP-PTD uptake into A549

19

cells (Fig. 5A). These findings suggest that TCTP-PTD uptake is associated with the

20

expression of caveolin-1 and that penetration pathway of TCTP-PTD is dependent on the

21

cellular expression of endocytic machinery that varies with cell type.

29

(Fig. S4A in Supporting Infomation), was lower

22

We then, tried to determine where in the cell, TCTP-PTD localizes after penetrating the

23

cell membrane through lipid-raft/caveolae, using anti-caveolin-1 antibody. We purified Lipid14


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1

rafts/caveolae using iso-osmolar contrast agent, iodixanol (OptiPrep) after treating A549 cells

2

with GFP and TCTP-PTD-GFP. TCTP-PTD-GFP, appeared in the 30 to 40% of iodixanol

3

gradients, which are lipid-raft/caveolae fractions. As expected, GFP, which cannot penetrate

4

into cells without PTD, was not found in lipid-raft/caveolae (Fig. 5C), suggesting that TCTP-

5

PTD is specifically localized in lipid-raft/caveolae fraction during the endocytic route.

6 7

3.4. Effect of chloroquine and localization of TCTP-PTD in A549 cells. Previous studies

8

reported that uptake of TAMRA-labeled TCTP-PTD is not influenced by the pretreatment

9

with the endolysomotrophic agent, chloroquine (an inhibitor that disrupts endosomes by

10

perturbing the acidification), and that TCTP-PTD does not localize in lysosomes and nucleus,

11

in HeLa cells 8. In order to determine whether endosomal acidification is a prerequisite for

12

TCTP-PTD transport, cellular penetration of TCTP-PTD into A549 cells following pre-

13

treatment with chloroquine, was examined. As shown in Fig. 6A, cellular transport of TCTP-

14

PTD-GFP was not significantly altered by the presence of 50 µM chloroquine. In accordance

15

with this finding, FITC-TCTP-PTD internalization was not affected by chloroquine in flow

16

cytometry analysis (Fig. S2 in Supporting Information).

17

Next, we tried to determine the subcellular location of TCTP-PTD once it penetrates

18

the A549 cells. Extracts of A549 cells were prepared after treating them with TCTP-PTD-

19

GFP for various times, and subjecting them to subcellular fractionation using the commercial

20

subcellular fractionation kit and then to western-blotting using specific antibodies against

21

marker proteins of each subcellular fraction. We found TCTP-PTD-GFP localized in cytosol,

22

membranes, membrane organelles, and cytoskeletal matrix, but not in the nucleus (Fig. 6B).

23

These results indicate that intracellular pathway of TCTP-PTD-GFP following penetration 15



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Page 16 of 37

1

includes cytoskeletal matrix (tubulin and actin), membrane/membrane organelles and

2

cytoplasm in A549 cells (Fig. 6B).

3

We aimed to identify the organelles penetrated by TCTP-PTD and also to determine

4

how it is released into cytoplasm after entry. Since a connection between caveolin-mediated

5

endocytosis and Golgi or ER through retrograde pathway (reviewed in

6

some PTDs, this possibility was tested by tracing the TCTP-PTD contents in the ER fraction

7

of A549 cells. We purified ER fractions after treatment the A549 cells with TCTP-PTD-GFP,

8

and confirmed its purity by the positive signals of calnexin, an ER marker protein, using anti-

9

calnexin-specific antibody. TCTP-PTD-GFP was found both in whole cell lysate and in the

10

ER fractions, implying that TCTP-PTD-GFP may pass through the ER and be released into

11

cytosol (Fig. 6C). Thus, following the penetration into A549 cells, the intra-cellular pathway

12

and destination of TCTP-PTD-GFP seems to include cytoplasm and cytoskeleton, through ER.

16


30

) was reported in

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1

4. DISCUSSION

2

Like the cationic PTDs, TCTP-PTD may use several distinct mechanisms in their cellular

3

translocation, depending on the cell type and cargo. Based on this study we propose the

4

scenario for the penetration of TCTP-PTD into A549 cells. The cargos, TCTP-PTD

5

conjugated with TAMRA, FITC or GFP, seem to be internalized into A549 cells by

6

cholesterol-dependent lipid raft/caveolae-mediated endocytosis aided by dynamin (Fig. 3 and

7

5). Such caveolae-mediated endocytosis is known to promote vesiculation by dynamin and

8

actin polymerization31. In addition to caveolar endocytosis, macropinocytosis that dependents

9

on the action of dynamin-mediated budding of vesicles (Fig. 3) and ruffling of plasma

10

membrane driven by actin rearrangement also appears to play a partial role in the

11

translocation of TAMRA-labeled TCTP-PTD (Fig. 4). In using more than one pathway for

12

cellular uptake, TCTP-PTD seems to act like several other PTDs.

13

After the uptake, TCTP-PTDs reside in vesicles enclosed by caveolae-mediated

14

endocytosis and macropinocytosis. The actual endocytic process leading to the formation of

15

the vesicle may determine the fate of endocytosed TCTP-PTD-cargo. After pinching off from

16

the cell surface, by the caveolae-driven pathway, the cargo finds itself in neutral pH

17

caveosome

18

(reviewed in

19

fate of internalized GFP-tagged TCTP-PTD revealed, that, following the localization at the

20

caveolin-1-containing lipid-raft (Fig. 5C), it moves mainly to cytoplasm, and cytoskeletal

21

matrix, but not to nucleus (Fig. 6B). Since the TCTP-PTD-GFP was found in ER fraction

22

(Fig. 6C), it appears that TCTP-PTD adopts the same retrograde transport that is shown to

23

occur in the cytosolic transfer of caveosome-laden PTDs

32

, subsequently directed to ER or Golgi apparatus along the retrograde route

30

). Fractionation and western blotting studies that tracked down the route and

30

. Because nocodazole, which

17



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1

disrupts microtubules, inhibits cargo delivery from caveosome to ER33, the observed

2

reduction of uptake caused by nocodazole supports the hypothesis that ER takes part in the

3

cellular translocation of TCTP-PTD. Admittedly, this hypothesis on the sequential traffic of

4

PTDs through caveosome-ER-cytosol needs confirmation using the specific inhibitors that

5

perturb the sequence. An example of such inhibitors is brefeldin A that inhibits transport from

6

the Golgi to the ER. But we cannot rule out the possibility that PTDs per se are capable of

7

escaping from endosomes before acidification since the biological activity of cargos seems to

8

remain intact. Translocation of PTDs across the endosomal membrane by the mechanisms

9

including conformational change, promotion of the membrane leakage, or modulation of the 34

10

pH of vesicle

11

facilitate their escape from vesicles in the presence of pH gradient 35. It was also suggested

12

that high concentrations of CPP-cargo in the intravesicular conditions promote the membrane

13

leakage (Reviewed in 34). However, the mechanism on how PTDs can exit from endosomal

14

compartment yet remains elusive.

are also possible. For example, hydrophobicity of PTDs has been shown to

15

In addition to caveolar endocytosis, TCTP-PTD can be transferred to macropinosomes

16

through macropinocytosis followed by endosomal pathway, depending on the microtubules. It

17

is commonly accepted that fate of macropinosome varies according to the cell types

18

(reviewed in 30). Because TCTP-PTD was not found localized in the lysosomes in HeLa cells

19

8

20

localization in cytosol (Fig. 6B), it is possible that TCTP-PTD-cargo may exit endosomal

21

compartment by still unknown mechanisms.

and its uptake did not depend on endosomal acidification in A549 cells (Fig. 6C) for its

22

We speculate that sensitivity to specific inhibitors may be related to the differences in the

23

composition of cellular membranes in cells. The present study shows that penetration of 18


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1

TCTP-PTD into A549 cells occurs by lipid raft-dependent endocytosis and macropinocytosis,

2

also seen in HeLa cells 8. However, differences in sensitivity to nystatin among cell lines (Fig.

3

5A), suggest that differences in the endocytic machineries in various cell lines, such as the

4

amounts of caveolin-1, lipid and other protein concentrations of membrane, and the rates of

5

endocytosis (Fig. 5B) may contribute to the cell-type specific mode of PTD entry.

6

Further studies are clearly needed to precisely track down the defined pathway and the

7

players of caveolae-mediated endocytosis by TCTP-PTD. Electron microscopic studies to

8

monitor the movement of caveolae, might help track down the penetration route of TCTP-

9

PTD. Use of model membranes to study lipid-peptide interaction, and computational

10

prediction of PTD-membrane interactions using molecular modeling, may also help dissect

11

the mechanism. In this regard we are investigating the mechanisms underlying cytosolic

12

release and the cell-to-cell translocation of TCTP-PTD.

13

TCTP-PTD may be considered to be a better transducer than other polycationic

14

transducers that are subject to the possible risk for nuclear accumulation. In addition, since

15

TCTP-PTD does not appear to entrap to the lysosome, lysosomal degradation of PTD can be

16

avoided, which might contribute to robust cytosolic delivery of cargos. For cargo delivery,

17

the nature of TCTP-PTD internalization makes it a more useful vehicle for siRNA delivery

18

than cationic PTDs that, in general, are trapped inside of endosomes and accumulate in the

19

nucleus 36. We are currently attempting to investigate such potential advantage of TCTP-PTD

20

for the delivery of therapeutic siRNA aimed at silencing target genes both in vitro and in vivo.

21

To sum up our results and suggestions, TCTP-PTD moves through lipid raft/caveolae 37

22

by a clathrin-independent, dynamin- and cholesterol-dependent process

23

macropinocytosis. TCTP-PTD in caveolae escapes the endosomes after entry through the ER, 19


and by


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1

and localizes in cytoplasm in association with the cytoskeleton moieties such as actin and

2

tubulin. The picture provided here may not only help in the understanding of physiological

3

implications of TCTP transport, but also has the potential to help development of TCTP-

4

PTD-aided approaches for safer and more effective delivery of bioactive cargos.

20


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1

5. CONCLUSION

2

In this study directed at elucidating the internalization mechanism of TCTP-PTD into A549

3

cells by biochemical methods, we showed that TCTP-PTD is internalized by means of

4

caveolae/lipid raft-dependent endocytosis and clathrin-independnent route, through a process

5

that is dependent of dynamin and actin/microtubule polymerization. Following entrapment

6

into caveolin-1-laden vesicles, carrier vesicles thought to move to caveosomes from which

7

the TCTP-PTD is transported into ER and then released into cytoplasm.

8

21



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1

■ Supporting Information

2 3

Four supplemental figures are provided in Supporting Information. This material is available

4

free of charge via the Internet at http://pubs.acs.org.

5 6

22


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1


■ AUTHOR INFORMATION

2 3

Corresponding Author

4

Address: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans

5

University, Seodaemoon-Gu, Ewhayeodae-gil 52, Seoul 120-750, Korea. Telephone: +82-2-

6

3277-3024. Fax: +82-2-3277-2851. E-mail: [email protected]

7 8

Notes

9

The authors declare no competing financial interest.

23



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1

■ ACKNOWLEDGMENTS

2 3

This study was supported by a grant of the Korea Health Technology R&D Project, Ministry

4

of Health & Welfare [A111417 (HI11C1371)] and National Research Foundation of Korea

5

(NRF) grant funded by the Ministry of Science, ICT & Future Planning (MSIP)

6

[2012R1A1A2042142] [2012M3A9A8053272].

7

24


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1

■ ABBREVIATIONS:

2

CME, clathrin-mediated endocytosis; CPZ, chlorpromazine; EIPA, 5-(N-ethyl-N-isopropyl)

3

amiloride; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; GFP, green

4

fluorescent protein; MβCD, methyl-β-cyclodextrin; PTD, protein transduction domain; TCTP,

5

translationally controlled tumor protein

6

25



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8. (20) Vocero-Akbani, A.; Lissy, N. A.; Dowdy, S. F. Transduction of full-length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation-induced cell death. Methods Enzymol. 2000, 322, 508-21. (21) Macdonald, J. L.; Pike, L. J. A simplified method for the preparation of detergent-free lipid rafts. J. Lipid Res. 2005, 46 (5), 1061-7. (22) Wang, Y. N.; Lee, H. H.; Lee, H. J.; Du, Y.; Yamaguchi, H.; Hung, M. C. Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB2. J. Biol. Chem. 2012, 287 (20), 16869-79. (23) Zhang, X.; Wang, F. Intracellular transduction and potential of Tat PTD and its analogs: from basic drug delivery mechanism to application. Expert Opin. Drug Deliv. 2012, 9 (4), 457-72. (24) Chao, T. Y.; Raines, R. T. Mechanism of ribonuclease A endocytosis: analogies to cellpenetrating peptides. Biochemistry 2011, 50 (39), 8374-82. (25) Reeves, V. L.; Thomas, C. M.; Smart, E. J. Lipid rafts, caveolae and GPI-linked proteins. Adv. Exp. Med. Biol. 2012, 729, 3-13. (26) Jones, A. T. Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides. J. Cell. Mol. Med. 2007, 11 (4), 670-84. (27) Kerr, M. C.; Teasdale, R. D. Defining macropinocytosis. Traffic 2009, 10 (4), 364-71. (28) Soldati, T.; Schliwa, M. Powering membrane traffic in endocytosis and recycling. Nat. Rev. Mol. Cell Biol. 2006, 7 (12), 897-908. (29) Cokakli, M.; Erdal, E.; Nart, D.; Yilmaz, F.; Sagol, O.; Kilic, M.; Karademir, S.; Atabey, N. Differential expression of Caveolin-1 in hepatocellular carcinoma: correlation with differentiation state, motility and invasion. BMC Cancer 2009, 9, 65. (30) Patel, L. N.; Zaro, J. L.; Shen, W. C. Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm. Res. 2007, 24 (11), 1977-92. (31) Pelkmans, L.; Puntener, D.; Helenius, A. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 2002, 296 (5567), 535-9. (32) Pfeffer, S. R. Caveolae on the move. Nat. Cell Biol. 2001, 3 (5), E108-10. (33) Pelkmans, L.; Kartenbeck, J.; Helenius, A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat. Cell Biol. 2001, 3 (5), 473-83. (34) Raagel, H.; Saalik, P.; Pooga, M. Peptide-mediated protein delivery-which pathways are penetrable? Biochim. Biophys. Acta 2010, 1798 (12), 2240-8. (35) Magzoub, M.; Pramanik, A.; Graslund, A. Modeling the endosomal escape of cellpenetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers. Biochemistry 2005, 44 (45), 14890-7. (36) Dominska, M.; Dykxhoorn, D. M. Breaking down the barriers: siRNA delivery and endosome escape. J. Cell Sci. 2010, 123 (Pt 8), 1183-9. (37) Nabi, I. R.; Le, P. U. Caveolae/raft-dependent endocytosis. J. Cell Biol. 2003, 161 (4), 673-7.

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1

Figure legends

2 3

Figure 1. Internalization of TCTP-PTD fusion proteins and peptides in A549 cells. (A)

4

Expression vector construct for TCTP-PTD-GFP fusion protein (TCTP-PTD-GFP).

5

Coomassie staining of purified TCTP-PTD-GFP proteins. After bacterial expression of GFP-

6

tagged TCTP-PTD fusion protein, the protein was purified using Ni2+-charged columns and

7

confirmed by Coomassie staining in SDS-PAGE. (B) After treatment of TCTP-PTD-GFP

8

protein with A549 cells, cellular uptake of TCTP-PTD-GFP was assayed by western blotting

9

with anti-GFP and -GAPDH-specific antibodies. (C) Cellular uptake of Rhodamine-labeled

10

TCTP-PTD and (D) FITC-labeled TCTP-PTD by A549 cells was performed by flow

11

cytometry and confocal fluorescence microscopy, respectively. Data means the average ± S.D.

12

The data from three independent experiments from FACS analysis were averaged. Error bars

13

indicate S.D.

14 15

Figure 2. Uptake of TCTP-PTD into A549 cells is not mediated by clathrin-mediated

16

endocytosis. Ten to 50 µM chlorpromazine was pretreated in A549 cells for 30 min, and the

17

FITC- or TAMRA-TCTP-PTD was incubated for 2 h in the presence of the CPZ.

18

Internalization of TAMRA-labeled TCTP-PTD was analyzed using (A) flow cytometry and

19

(B) confocal fluorescence microscopy. Bar graphs represent the relative percentage of

20

internalized TCTP-PTD compared to that of TCTP-PTD-treated control. Data were presented

21

as the means ± S.D. of three independent experiments.

22 23

Figure 3. TCTP-PTD enters the A549 cells using lipid-raft/caveolae- and dynamin-dependent

28


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1

endocytosis. (A) MβCD (5-10 mM) and dynasore (40-160 µM) were pretreated in A549 cells

2

for 30 min and then 10 µM TAMRA-labeled TCTP-PTD was added. Internalization of

3

TAMRA-TCTP-PTD was analyzed by flow cytometry. Histogram displays the results using

4

the optimum concentration of these inhibitors. Bar graphs represent the percentage of

5

internalized TCTP-PTD with respect to a control without inhibitor treatment. Data were

6

presented as the means ± S.D. of three independent experiments. A549 cells were treated with

7

optimum concentration of the inhibitors (5 mM MβCD, 80 µM dynasore) for 30 min, added

8

with 10 µM FITC-, TAMRA-labeled TCTP-PTD or TCTP-PTD-GFP for 2 h at 37 °C, and

9

then analyzed by (B) confocal fluorescence microscopy and by (C) western-blotting using

10

anti-GFP, and -GAPDH-specific antibodies. Nuclei of cells were stained with DAPI. Band

11

intensities of western blots were quantified using the ImagJ software after normalization with

12

that of GAPDH.

13 14

Figure 4. TCTP-PTD penetrates the cells using, in part, macropinocytosis. A549 cells were

15

pretreated with 10-50 µM concentrations of EIPA, cytochalasin D (Cyt D), and nocodazole

16

for 30 min and then incubated 10 µM FITC- or TAMRA-labeled TCTP-PTD for 2 h at 37 °C.

17

Internalization of TAMRA-TCTP-PTD was analyzed using (A) flow cytometry and (B)

18

confocal fluorescence microscopy. Bar graphs represent the percentage of internalized TCTP-

19

PTD with respect to a control without inhibitor treatment. Histogram displays the result from

20

the treatment of optimum concentration of inhibitors. Data were presented as the means ±

21

S.D. of three independent experiments. Nuclei of cells were stained with DAPI.

22 23

Figure 5. Subcellular localization of TCTP-PTD in lipid-rafts/caveolae fractions. (A) HeLa 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

and A549 cells were pretreated with 50 µg/ml nystatin (a lipid-rafts/caveolae inhibitor) for 30

2

min, followed by treatment with TAMRA-TCTP-PTD (10 µM) for 2 h, and then analyzed by

3

flow cytometry. (B) Differential expression of caveolae in HeLa and A549 cells, as assessed

4

by immunoblotting using the antibody detecting the caveolae-specific marker protein,

5

caveolin-1. (C) Localization of TCTP-PTD in the lipid-rafts/caveolae fractions. A549 cells

6

were incubated with GFP and TCTP-PTD-GFP proteins, and internalized TCTP-PTD-GFP

7

proteins were graded by OptiPrep gradient fractionations using iodixanol gradient

8

purification. Isolated lipid-rafts/caveolae fractions were subjected to immunoblotting using

9

anti-caveolin-1 and anti-GFP antibodies, respectively.

10 11

Figure 6. Effect of chloroquine and localization of TCTP-PTD in cytoplasm, cytoskeleton

12

and ER. (A) Ten µM TCTP-PTD-GFP treated in A549 cells with or without 50 µM

13

chloroquine (an endolysomotrophic agent), and analyzed by confocal fluorescence

14

microscopy. (B) Following the treatment of A549 cells with TCTP-PTD-GFP or GFP, cells

15

were fractionated into cytoplasm, organelle membrane, nucleus, and cytoskeleton fractions

16

using a ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem). Purity and

17

specificity of each fractions were validated using specific antibodies against tubulin (a marker

18

for cytoplasm), cytochrome C (a marker for organelle membranes), actin (a marker for

19

cytoskeletal matrix), and PARP (a marker for nucleus). (C) TCTP-PTD-GFP or GFP proteins

20

were incubated with A549 cells and the ER fractions were isolated from cells. Purity of total

21

cell lysates and the purified ER fractions was immunoblotted with specific antibodies

22

detecting the calnexin (a marker for ER fraction), PARP (a marker for nucleus) and

23

cytochrome C (a marker for mitochondria). 30


Page 30 of 37

Page 31 of 37

A

Fig 1. Kim HY et al

B (kDa) GFP

T7---ATG-6His--TCTP(1-10)-HA---GFP

TCTP-PTD-GFP

43

TCTP-PTD-GFP

34

(kDa) GFP TCTP-PTD-GFP 43

TCTP-PTD -GFP Ampr

43

34

GAPDH

128

120 Untreated Rhodamine Rhodamine TCTP-PTD

Counts

80

40

FITC-TCTP-PTD

D

C

FITC

DAPI

Merge

20X 20X

Z-stack Rhodamine -TCTP-PTD

100

Rhodamine

0

Untreated

internalization (% of Rhodamine-TCTP-PTD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


101

102 FL2

103

104

20X


150

128 Untreated TAMRA-TCTP-PTD + 50 μM CPZ

100

50

Counts

A

Page 32 of 37 Fig 2. Kim HY et al

0

0

TAMRA-TCTP-PTD CPZ (μM)

-

+ -

FITC-TCTP-PTD

B

+ 10

+ 20

FITC

+ 50

100

101

102

FL-2

DAPI

+ CPZ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

internalization (% of control)



Merge

40X

40X

103

104

Page 33 of 37

+ 5

+ 7.5

+ 10


+ -

Untreated TAMRA-TCTP-PTD + 5 mM MβCD

0 100

101

102

103

FITC

DAPI

Merge

20X

+ MβCD

Counts

128

FITC-TCTP-PTD

B


A

Fig 3. Kim HY et al

20X

+ Dynasore

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 TAMRA-TCTP-PTD 18 MβCD (mM) 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 TAMRA-TCTP-PTD 36 Dynasore (μM) 37 38 39 40 41 42


104

FL-2

20X

C Untreated TAMRA-TCTP-PTD + 80 µM Dynasore

Counts

128

0

+ -

+ 40

+ + 80 160

100

MβCD Dynasore TCTP-PTD-GFP GFP

+

+ -

+ + -

+ + TCTP-PTDGFP

101

102

103

104

FL-2


1.00

0.78

0.44

GAPDH

B




0 + 10

+ 20

+ 50

FL-2

+ 10

+ 20

+ 50


+ -

Untreated TAMRA-TCTP-PTD + 50 µM Cyt D

0

+ 20

+ 50

Merge

40X

40X

Untreated TAMRA-TCTP-PTD + 20 µM Nocodazole

0 + 10

DAPI

40X

FL-2

128

+ -

FITC

+ Cyt D

Counts

128

+ Nocodazole

+ -

FITC-TCTP-PTD

Untreated TAMRA-TCTP-PTD + 20 µM EIPA

+ EIPA

Counts

128


1 2 3 4 5 6 7 8 9 10 TAMRA-TCTP-PTD 11 EIPA (μM) 12 13 14 15 16 17 18 19 20 21 22 23 24 25TAMRA-TCTP-PTD 26 Cyt D (μM) 27 28 29 30 31 32 33 34 35 36 37 38 39 40 TAMRA-TCTP-PTD 41Nocodazole (μM) 42

Counts

A

ACS Paragon Plus Environment FL-2

40X

Page 35 of 37

A

Fig 5. Kim HY et al

A549 128

Untreated TAMRA-TCTP-PTD + Nystatin

Untreated TAMRA-TCTP-PTD + Nystatin

Counts

128

HeLa

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


0

0

100

101

102

103

100

104

101

102

103

104

FL-2

FL-2

B

C HeLa

OptiPrep gradient fractions 26% 40% 11 12 13 14 15 16 17 18 19

A549 Caveolin-1

Caveolin-1 TCTP-PTD-GFP

Tubulin


GFP


A

B DAPI

Merge

TCTP-PTD-GFP

GFP

Cytoplasm TCTP-PTD-GFP

GFP 0.5 2 4

Membrane/Organelle

GFP

TCTP-PTD-GFP

0.5 2 4 (h)

40X

TCTP-PTD-GFP Tubulin

+ CQ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Cytochrome C 40X

C

Total

Calnexin

ER

Nucleus GFP

TCTP-PTD-GFP

 TCTP-PTD-GFP

0.5 2 4

Cytoskeleton GFP

TCTP-PTD-GFP

0.5 2 4 (h) TCTP-PTD-GFP PARP Actin

 Calnexin

Calnexin

 PARP

 Cytochrome C  Caveolin


Page 37 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


98x89mm (300 x 300 DPI)


Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma

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