Protein Transduction

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Protein Transduction

• David Shiuan
• Department of Life Science
• Institute of Biotechnology
• National Dong Hwa University

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Protein Transduction
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.
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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.

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In vivo protein transduction delivery of a
biologically active protein into the mouse
Science 285 (1999) 15691572 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.

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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 37C and 4C 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
  ß-galactosidase, intracellularly

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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

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13 (2002) 52-56
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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

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A three-step model of membrane transduction by
cationic peptides TIB 31(2003)497
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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
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PNAS USA 98(2001)8786
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PNAS USA 98(2001)8786
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PNAS USA 101(2001)17867
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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 (???)of complex events and
  activities can be performed on the cargo

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Mechanisms of PTD-mediated transduction

• Peptide internalized at 4C? 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

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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 23C.
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Arginine-rich Peptides J. Biol. Chem.
276(2001) 5836
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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

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Confocal microscopy of noncovalent protein
internalization via AID peptides in animal cells
(L) and plant cells BBRC 346(2006)758
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Cytoxicity of AIDs BBRC 346(2006)758
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Regulated portals of entry into the cellNature
422 (2003) 37-44
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What can we do to PTD?

• Transport capabilities
• Transport mechanisms
• Delivery vehicles