Book of Abstracts: Albany 2007
June 19-23 2007
Molecular Mechanism of Molecules Translocation and Targeting Acidic Tissue In Vivo by pHLIP ? pH (Low) Insertion Peptide
Many pathological conditions, including tumors, inflammation, infarction, stroke, infections, and others, create a local acidic environment. We found a way of selective targeting of tissue with elevated level of acidity and translocation of molecules through the membrane in cytoplasm of cells with low extracellular pH. Membrane peptide pHLIP [pH (Low) Insertion Peptide] derived from the bacteriorhodopsin C helix has unique properties: it is soluble in aqueous solution and weakly interacts with cell membrane at normal pH; however, it inserts in membrane and forms a transmembrane helix at mild acidic pH (<7.0). The insertion is unidirectional: C-terminus goes inside cell and the N-terminus stays outside. A variety of cargo molecules attached to the C-terminus of pHLIP via cleavable S-S bond could be translocated and released in cytoplasm in the environment of low extracellular pH. Among the cargo molecules translocated by pHLIP are cyclic peptide phalloidin (mushroom toxin, which stabilizes actin cytoskeleton), 12-mer PNA (peptide nucleic acids), and fluorescent dyes. Our data indicate that the pathway of peptide entry into the membrane and the translocation of molecules into cells is not mediated by endocytosis, by interactions with cell receptors or by formation of pores in cell membranes: it is the formation of a helix across the lipid bilayer, triggered by the increase of the peptide hydrophobicity due to the protonation of Asp residues induced by low pH. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an α-helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The replacement of two key Asp residues located in the transmembrane part of pHLIP by Lys or Asn led to the loss of pH-sensitive insertion into membranes of liposomes, red blood cells, and cancer cells in vivo, as well as to the loss of specific accumulation in tumors. pHLIP technology introduces a new concept to detect, target, and treat acidic diseased tissue by employing the selective insertion and folding of membrane peptides.