Protein Transduction

David Shiuan

Department of Life Science Institute of Biotechnology

National Dong Hwa University

In general, the plasma membrane of eukaryotic cells is impermeable to the vast majority of peptides and proteins.

However, this dogma has recently ~1999 been shown to be untrue with the identification of several protein transduction domains (PTDs) that are capable of transducing cargo/protein across the plasma membrane, allowing the proteins to accumulate within the cell.

Protein Transduction

HIV Tat transactivator protein

Frankel Pabo: Intercellular uptake of the Tat protein from human immunodeficiency virus. Cell 55 (1988) 1189-1193.

Green Lowenstein: Autonomous functional domains of chemically synthesized HIV Tat tran-activator protein.

Cell 55 (1988) 1179-1188.

In vivo protein transduction: delivery of a biologically active protein into the mouse

Science 285 (1999) 1569–1572 S.R. Schwarze, A. Ho, A. Vocero-Akbani and S.F. Dowdy

This article demonstrated for the first time the ability to deliver via protein transduction peptides (2 kDa) and large proteins (120 kDa) into most, if not all, cells and tissues of a mouse.

Protein Transduction 2003 understandins/facts

Transduction across the membrane by PTDs occurs through an unidentified mechanism that is independent of receptors, transporters and endo-cytosis

Transduction occurs via a rapid process that at both 37°C and 4°C targets essentially 100% of cells in a concentration-dependent fashion

As recombinant fusion proteins or covalently cross-linked proteins, PTDs are capable of delivering biologically active proteins, such as β-galactosidase, intracellularly

Three most widely studied PTDs

1. The Drosophila homeotic transcription protein antennapedia (Antp).

2. The herpes simplex virus structural protein VP22 .

3. The human HIV-1 transcriptional activator Tat protein

13 (2002) 52-56

Bioconjugate Chem. 10 ( 1999) 186 -191

40 nm superparamagnetic iron nanoparticles were conjugated to Tat

PTD peptides and internalized into both hematopoietic and neural progenitor cells

A three-step model of membrane

transduction by cationic peptides TIB 31(2003)497

TAT Peptide on the Surface of Liposomes Affords Their Efficient Intracellular Delivery Even at Low Temperature and in the

Presence of Metabolic Inhibitors PNAS USA 98(2001)8786

PNAS USA 98(2001)8786

PNAS USA 98(2001)8786

PNAS USA 101(2001)17867

Mechanism of transduction across the cellular membrane is currently unknown

(1) PTD with acidic motifs on the membrane

(2) Internalization step is independent of

transporters, receptors and endocytosis

(3) a plethora (過多症 )of complex events and activities can be performed on the cargo

Mechanisms of PTD-mediated

transduction

Peptide internalized at 4°C it is unlikely that uptake required any cellular-mediated process or required physical arrangement

Internalization occurs through interaction between the Arg and charged members of the cell membrane

The cell-surface heparin sulfate (HS) proteoglycans are key mediators of peptide internalization in vivo

The membrane hydrophobic interior poses a significant barrier to the uptake of hydrophilic proteins

The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake PNAS 97(2000) 13003

FACS cellular uptake assay analogs Tat49-57 (Fl-ahx-RKKRRQRRR): Tat49-56 (Fl-ahx-RKKRRQRR), Tat49-55 (Fl-ahx-RKKRRQR), Tat50-57 (Fl-ahx-KKRRQRRR),Tat51-57 (Fl-ahx-KRRQRRR).

Jurkat cells were incubated with varying concentrations (12.5 µM shown) of peptides for 10 min at 23°C.

Arginine-rich Peptides

J. Biol. Chem. 276(2001) 5836

Arginine-rich intracellular delivery peptides noncovalently transport

protein into living cells BBRC 346(2006)758

FPs (GFP, RFP, tdTomato, mCherry, and mOrange)

AID peptides [HIV-Tat (Tat-PTD), R9, and R9Z]

Cells were treated for 10 min.

[1] different FPs

[2] FP + AID peptide mixtures

then, washed with PBS, and recorded by the confocal microscope system

Confocal microscopy of noncovalent protein internalization via AID peptides in animal cells (L) and

plant cells ® BBRC 346(2006)758

Cytoxicity of AIDs BBRC 346(2006)758

Regulated portals of entry into the cell

Nature 422 (2003) 37-44

What can we do to PTD?

Transport capabilities

Transport mechanisms

Delivery vehicles