Donald Engelman

pH-dependent transmembrane peptide insertion: mechanism and uses in tumor targeting and drug delivery

 

Laboratories of Oleg A. Andreev, Donald M. Engelman (speaking),

and Yana K. Reshetnyak

University of Rhode Island and Yale

 

The discovery that the C helix of Bacteriorhodopsin exhibits spontaneous, pH-dependent insertion to form a helix across lipid bilayers has led to the use of related peptides, pHLIPs (pH (Low) Insertion Peptides), to study the insertion process, to selectively image acidic tissues in vivo and to translocate cargo molecules across the plasma membranes of living cells.

a pHLIP has three kinds of states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an alpha-helix. At physiological pH, the equilibrium is toward water and the membrane surface, which explains its lower affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. Using a thermodynamic analysis, we have defined energies and inferred lipid involvement in these states. We have used a family of molecules to begin a definition of the parameters of molecules that can be translocated. We have also explored variations of the pHLIP sequence.

Because pHLIP is unfolded on the surface of a bilayer and folding is pH-triggered, we are able to apply kinetic analysis to begin to understand the molecular events that accompany the insertion and folding of a peptide entering a bilayer. When the pH is dropped, it is found that a helix forms rapidly on the surface of a bilayer, followed by a slow insertion across it in several kinetically distinct steps. The exit pathway when the pH is jumped is more rapid, and includes partial unfolding of the helix while still in the bilayer.

Translocation is selective for low pH, and various types of cargo molecules attached by disulfides can be released by reduction in the cytoplasm, including peptide nucleic acids, a polar cyclic peptide (phalloidin), and organic compounds. Because a high extracellular acidity is characteristic of a variety of pathological conditions (such as tumors, infarcts, stroke-afflicted tissue, atherosclerotic lesions, sites of inflammation or infection, or damaged tissue resulting from trauma), the pH (low) insertion peptide may prove a useful tool for selective delivery of agents for drug therapy, diagnostic imaging, genetic control, or cell regulation. Finally, application of the technology to image tumors in mice will be discussed.

 

 

Selected Publications ( see www.yale.edu/engelman/publications.html )

Reshetnyak, Y. K., Andreev, O. A., Lehnert, U. and Engelman, D. M. "Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix" PNAS 103, 6460-6465 (2006)

Andreev, O. A., Dupuy, A. D., Segala, M., Sandugu, S., Serra, D. A., Chichester, C. O., Engelman, D. M. and Reshetnyak, Y. K. "Mechanism and uses of a peptide that targets tumors and other acidic tissues in vivo" PNAS 104, 7893-7898 (2007)

Reshetnyak, Y. K., Segala, M., Andreev, O. A. and Engelman, D. M. "A monomeric membrane peptide that lives in three worlds: in solution, attached to and inserted across lipid bilayers" Biophys. J. 93,2363-72 (2007)

Reshetnyak YK, Andreev OA, Segala M, Markin VS, Engelman DM "Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane "

Proc Natl Acad Sci USA 105,15340-5 (2008).

Segala J, Engelman DM, Reshetnyak YK, Andreev OA "Accurate analysis of tumor margins using a fluorescent pH Low Insertion Peptide (pHLIP)"

Int. J. Mol. Sci 10, 3478-3487(2009)

Thévenin D, An M, Engelman DM"pHLIP-Mediated Translocation of Membrane-Impermeable Molecules into Cells" Chem. Biol. 16, 754-762(2009).

Andreev OA, Karabadzhak AG, Weerakkody D, Andreev GO, Engelman DM, Reshetnyak YK "pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path." Proc Natl Acad Sci USA (2010) 107: 4081-4086.

Musical-Siwek M, Karabadzhak A, Andreev OA, Reshetnyak YK, Engelman DM "Tuning the insertion properties of pHLIP " Biochim. Biophys. Acta (2010) 1798:1041-6.

An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM. “pH-(low)-insertion-peptide (pHLIP) translocation of membrane-impermeable phalloidin toxin inhibits cancer cell proliferation” Proc Natl Acad Sci U S A. 2010 107:20246-50.