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細胞穿透胜肽之分子運送細胞穿透胜肽之分子運送Molecular Delivery of Cell-Molecular Delivery of Cell-

Penetrating PeptidesPenetrating Peptides

李漢榮李漢榮

國立東華大學國立東華大學自然資源與環境學系自然資源與環境學系

Speaker: Dr. Han-Jung LeeSpeaker: Dr. Han-Jung LeeDepartment of Natural Resources and Department of Natural Resources and

Environmental StudiesEnvironmental StudiesNational Dong Hwa UniversityNational Dong Hwa University

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Plasma Plasma MembraneMembrane

Extracellular Intracellular

IntroductionIntroduction

Schwarze et al., Science 285: 1569-1572, 1999

-Gal

PTD

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Transactivator (Tat) protein of the human immunodeficiency virus type 1 (HIV-1) was able to enter into cells and activate HIV-LTR.

Green and Loewenstein, Cell 55: 1179-1188, 1988

Frankel and Pabo, Cell 55: 1189-1193, 1988

Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

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DNA

RNA

protein

Tat

Plasma Plasma MembraneMembrane

O2

H2O

Extracellular Intracellular

Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

Functional domains of HIV-1 Tat :

Gaynor, Curr. Top. Microbiol. Immunol. 193: 51-77, 1995

Vives et al., J. Biol. Chem. 272: 16010-16017, 1997

acidiccysteine-rich core basic augmenting

2 11 22 37 47 57 721

CxxCxCxxCCxxCxxC

YGRKKRRQRRREPVDPRLEPW

86

Basic amino acid-rich domain

Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

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CPPs, also called protein transduction domains (PTDs), are a group of short, highly basic peptides which can penetrate into cells either alone or associated with cargoes.

TatYGRKKRRQRRR

(CPP)

Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

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ResultsResults

Chang et al., Plant Cell Physiol. 46: 482-488, 2005

8Covalent protein transduction into onion root tip cells

Tat-GFP (400x)

R9-GFP (400x)

Tat-GFP (200x)

R9-GFP (200x)

GFP

(200x)

9Covalent protein transduction in plant cells

R9-RFP

R9-RFP

RFP(onion epidermal cells)

(onion epidermalcells)

(tomato root cells)

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protein

covalent protein transduction

(CPT)

noncovalent protein transduction

(NPT)

Covalent and Noncovalent Protein Covalent and Noncovalent Protein TransductionsTransductions

Plasma MembranePlasma Membrane

protein

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GFP

RFP

R9/GFP

R9/RFP

Noncovalent protein transduction in onion root cells

Chang et al., New Phytol. 174: 46-56, 2007

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Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

CPPs are able to deliver proteins into cells in a covalent protein transduction (CPT) and noncovalent protein transduction (NPT).

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Synthetic nona-arginine (SR9)Synthetic nona-arginine (SR9) N-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CN-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-C

Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

14Chen et al., FEBS Lett. 581: 1891-1897, 2007

SR9 DNA

SR9/DNA SR9/DNA

SR9/DNADNA

(mung bean)

(mung bean)

(mung bean) (mung bean)

(soybean) (soybean)

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GC-EGFP cellsdsRNA

SR9/ dsRNA

Wang et al., J. Biochem. Biophy. Methods 70: 579-586, 2007

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Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

CPPs can deliver proteins, DNAs and RNAs into cells in a noncovalent manner.

17Hu et al., Peptides 30: 1669-1678, 2009

PBS

GFP RFP

RFP

R9-GFP R9-GFP

R9-GFP/RFP R9-GFP/RFP

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Cell-Penetrating PeptidesCell-Penetrating Peptides(CPPs)

CPPs are able to deliver proteins into cells in a covalent protein transduction (CPT), noncovalent protein transduction (NPT) and covalent and noncovalent protein transductions (CNPT).

19Liu et al., Biomaterials 32: 3520-3537, 2011

Lyso Tracker

Hoechst

(merge)

Quantum dots

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Summary

We demonstrate that CPPs can effectively deliver proteins, DNAs, RNAs and nanoparticles into live cells. 17

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Summary

However, the mechanism of protein transduction is unknown yet.

CPP Translocation CPP Translocation MechanismsMechanisms

Cargo CPP