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Pathway for polyarginine entry into mammalian cells.
Stephen M Fuchs1, Ronald T Raines
1Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Biochemistry
|March 3, 2004
Summary
Protein transduction domains (PTDs) like nonaarginine enter cells via endocytosis after binding to cell surface heparan sulfate. This mechanism facilitates molecule delivery into mammalian cells.
Area of Science:
- Cell biology
- Biochemistry
- Molecular biology
Background:
- Protein transduction domains (PTDs) are cationic peptides that facilitate cellular molecule delivery.
- Nonaarginine (R(9)) is a highly effective PTD, but its internalization pathway requires elucidation.
Purpose of the Study:
- To determine the cellular uptake mechanism of nonaarginine (R(9)).
- To identify the role of heparan sulfate (HS) in PTD internalization.
- To understand the complete pathway for PTD-mediated delivery.
Main Methods:
- Live-cell imaging of R(9) uptake.
- Colocalization studies with vesicular markers.
- Assays using cells deficient in heparan sulfate.
- Liposome leakage experiments.
Main Results:
- R(9) is localized in endocytic vesicles in live cells, not the cytosol or nucleolus.
- HS-deficient cells do not internalize R(9), indicating HS binding is essential.
- R(9) binds to heparin with high affinity (Kd = 109 nM).
- R(9) causes liposome leakage at high peptide:lipid ratios.
Conclusions:
- PTD internalization occurs via endocytosis, initiated by binding to cell surface HS.
- Release from endocytic vesicles is facilitated by HS degradation and vesicle leakage.
- This pathway enables efficient PTD-mediated delivery of molecules into mammalian cells.