CURRENT MODELS FOR THE STRUCTURE OF BIOLOGICAL MEMBRANES

II. Problems in the characterization of biological membranes A. Structural characteristics B. Functional characteristics III. The model

Targeting Diseased Tissues by pHLIP Insertion at Low Cell Surface pH

The discovery of the pH Low Insertion Peptides (pHLIPs®) provides an opportunity to develop imaging and drug delivery agents targeting extracellular acidity. Extracellular acidity is associated with many pathological states, such as those in cancer, ischemic stroke, neurotrauma, infection, lacerations, and others. The metabolism of cells in injured or diseased tissues often results in the acidification of the extracellular environment, so acidosis might be useful as a general marker for the imaging and treatment of diseased states if an effective targeting method can be developed. The molecular mechanism of a pHLIP peptide is based on pH-dependent membrane-associated folding. pHLIPs, being moderately hydrophobic peptides, have high affinities for cellular membranes at normal pH, but fold and insert across membranes at low pH, allowing them to sense pH at the surfaces of cells in diseased tissues, where it is the lowest. Here we discuss the main principles of pHLIP interactions with membrane lipid bilayers at neutral and low pHs, the possibility of tuning the folding and insertion pH by peptide sequence variation, and potential applications of pHLIPs for imaging, therapy and image-guided interventions

pH-sensitive membrane peptides (pHLIPs) as a novel class of delivery agents

Here we review a novel class of delivery vehicles based on pH-sensitive, moderately polar membrane peptides, which we call pH (Low) Insertion Peptides (pHLIPs), that target cells located in the acidic environment found in many diseased tissues, including tumours. Acidity targeting by pHLIPs is achieved as a result of helix formation and transmembrane insertion. In contrast to the earlier technologies based on cell-penetrating peptides, pHLIPs act as monomeric membrane-inserting peptides that translocate one terminus across a membrane into the cytoplasm, while the other terminus remains in the extracellular space, locating the peptide in the membrane lipid bilayer. Therefore pHLIP has a dual delivery capability: it can tether cargo molecules or nanoparticles to the surfaces of cells in diseased tissues and/or it can move a cell-impermeable cargo molecule across the membrane into the cytoplasm. The source of energy for moving polar molecules attached to pHLIP through the hydrophobic layer of a membrane bilayer is the membrane-associated folding of the polypeptide. A drop in pH leads to the protonation of negatively charged residues (Asp or Glu), which enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion. The process is accompanied by the release of energy that can be utilized to move cell-impermeable cargo across a membrane. That the mechanism is now understood, and that targeting of tumours in mice has been shown, suggest a number of future applications of the pHLIP technology in the diagnosis and treatment of disease

Targeting Acidic Diseased Tissues by pH-Triggered Membrane-Associated Peptide Folding

The advantages of targeted therapy have motivated many efforts to find distinguishing features between the molecular cell surface landscapes of diseased and normal cells. Typically, the features have been proteins, lipids or carbohydrates, but other approaches are emerging. In this discussion, we examine the use of cell surface acidity as a feature that can be exploited by using pH-sensitive peptide folding to target agents to diseased cell surfaces or cytoplasms

Specificity in transmembrane helix-helix interactions defines a hierarchy of stability for sequence variants,

The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alaninescanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several ''hot spots'' for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design. G enome sequencing efforts reveal that approximately 20% of ORFs in complex organisms may encode proteins containing at least one helical transmembrane segment (1). Despite these numbers, as well as the fact that membrane proteins carry out many essential cell functions, our understanding of the sequence-structure-function relationships for this class of proteins lags far behind that of soluble proteins. These realities underscore the importance of biophysical and structural work aimed toward understanding chemical principles of helical membrane protein structural stability. Because the phospholipid bilayer places structural constraints on a helical membrane protein, the folding of a polypeptide sequence into a helical membrane protein can be considered, experimentally and theoretically, in separable thermodynamic steps (2, 3). The usefulness of this framework arises from the fact that individual energetic processes can be independently studied. The principal features of a polypeptide sequence that will give rise to the formation of an independently stable transmembrane ␣-helix are generally known (3). This information has been used extensively in computational search algorithms with reasonable accuracy rates to identify potential helical transmembrane proteins (reviewed in ref. 3). Once this is accomplished, however, the helical membrane protein folding problem then becomes focused on understanding and predicting the side-to-side associations in which these preformed transmembrane ␣-helices will participate. It is this final thermodynamic step in helical membrane protein folding that we investigate in this study. In a continuing effort to understand the structural and energetic principles of the side-to-side interactions of transmembrane ␣-helices, we have quantitatively examined the sequence dependence of the glycophorin A transmembrane helix dimerization. The propensity of the glycophorin A transmembrane domain to dimerize in a sequence-specific manner has been a paradigm for study of transmembrane helix-helix association in hydrophobic environments (4-9). An additional advantage for detailed thermodynamic analysis of the GpA transmembrane segment (TMS) dimerization is the fact that a solution NMR structure has been solved (10). Together with considerations of principles of stability of helices in membranes, the NMR structure provides a three-dimensional model for interpretation of potential structural consequences due to mutation. Understanding the chemical principles driving the selfassociation of the glycophorin transmembrane ␣-helix is of particular interest because both the NMR structure and the exquisite sequence dependence determined by SDS͞PAGE suggest that a detailed geometry of van der Waals interactions specify and stabilize the dimer (4, 8, 10). Only one residue with a polar side-chain, Thr 87 , is found at the dimer interface. The solution NMR structure of the glycophorin A transmembrane dimer in dodecylphosphocholine micelles reveals no interhelical hydrogen bond at this position (10), although the recent solid state NMR data from Smith and coworkers (11) hints that Thr 87 might participate in an intermonomer hydrogen bond in lipid bilayers. Nevertheless, the energetic stabilization from such a hydrogen bond is uncertain. Recent studies on the introduction of polar side chains into model transmembrane peptides find that residues containing two polar side-chain atoms (such as asparagine) have a much greater tendency to drive transmembrane helix association than residues containing only one polar sidechain atom (threonine or serine; refs. 12 and 13). It has been proposed that side-chain rotamer entropy is not expected to play a large role in the self-association of the glycophorin A transmembrane ␣-helix (10). The interacting surface of the glycophorin A TMS contains only three residues with some rotamer freedom in an ␣-helix (Leu Abbreviations: TMS, transmembrane segment; C8E5, pentaoxylethylene-octylether. † To whom reprint requests should be addressed

Tuning the insertion properties of pHLIP

The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pKapp of membrane insertion

Measuring Tumor Aggressiveness and Targeting Metastatic Lesions with Fluorescent pHLIP

Purpose: Malignant cancer foci develop acidic extracellular environments. Mild acidic conditions trigger insertion and folding of the pH (low) insertion peptide (pHLIPTM) across a cellular membrane, enabling targeting of such lesions. Procedures: We employed optical imaging to follow targeting by fluorescent pHLIP given i.v. in mice. For visualization, Cy5.5 and Alexa750 were covalently attached to the N terminus of pHLIP, which stays outside of a cell membrane after transmembrane insertion. Results: We demonstrate that pHLIP targets: (a) tumors of different origins established by subcutaneous injection of cancer cells, (b) spontaneous prostate tumors in TRAMP mice and (c) metastatic lesions in lung pHLIP accumulation in tumors correlates with tumor aggressiveness. Within a tumor, it stains extracellular spaces and cellular membranes. Conclusions: Tissue acidity can be detected by pHLIP peptide insertion and used to diagnose primary tumors, metastatic lesions, and lipid bodies in necrotic tissues. The ability of pHLIP to differentially bind metastatic and non-metastatic tumors may provide a new approach for evaluating cancer prognosis