The pH-low insertion peptide (pHLIP) is a leading peptide technology to target the extracellular acidosis that characterizes solid tumors. The pHLIP binds to lipid membranes, and responds to acidification by undergoing a coupled folding/membrane insertion process. In the final transmembrane state, the C terminus of pHLIP gets exposed to the cytoplasm of the target cell, providing a means to translocate membrane-impermeable drug cargoes across the plasma membrane of cancer cells. There exists a need to develop improved pHLIP variants to target tumors with greater efficiency. Characterization of such variants typically relies on determining the pK parameter, the pH midpoint of peptide insertion into the lipid bilayer. Here we report that the value of the pK can be strongly dependent on the method used for its determination. Membrane insertion of pHLIP involves at least four intermediate states, which are believed to be linked to the staggered titration of key acidic residues. We propose that some spectroscopic methods are influenced more heavily by specific membrane folding intermediates, and as a result yield different pK values. To address this potential problem, we have devised an assay to independently monitor the environment of the two termini of pHLIP. This approach provides insights into the conformation pHLIP adopts immediately before the establishment of the transmembrane configuration. Additionally, our data indicate that the membrane translocation of the C terminus of pHLIP, the folding step more directly relevant to drug delivery, occurs at more acidic pH values than previously considered. Consequently, such a pK difference could have substantial ramifications for assessing the translocation of drug cargoes conjugated to pHLIP.
Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism.
The pH-low insertion peptide (pHLIP) is used for targeted delivery of drug cargoes to acidic tissues such as tumors. The extracellular acidosis found in solid tumors triggers pHLIP to transition from a membrane-adsorbed state to fold into a transmembrane a-helix. Different factors influence the acidity required for pHLIP to insert into lipid membranes. One of them is the lipid headgroup composition, which defines the electrostatic profile of the membrane. However, the molecular interactions that drive the adsorption of pHLIP to the bilayer surface are poorly understood. In this study, we combine biophysical experiments and all-atom molecular dynamics simulations to understand the role played by electrostatics in the interaction between pHLIP and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. We observed that the solution ionic strength affects the structure of pHLIP at the membrane surface as well as the acidity needed for different steps in the membrane insertion process. In particular, our simulations revealed that an increase in ionic strength affected both pHLIP and the bilayer; the coordination of sodium ions with the C-terminus of pHLIP led to localized changes in helicity, whereas the coordination of sodium ions with the phosphate moiety of the phosphocholine headgroups had a condensing effect on our model bilayer. These results are relevant to our understanding of environmental influences on the ability of pHLIP to adsorb to the cell membrane and are useful in our fundamental understanding of the absorption of pH-responsive peptides and cell-penetrating peptides.
The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces antioncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using styrene maleic acid lipid particles to measure dimerization in membranes. We observed that the signaling lipid PIP 2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TMs are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP 2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions and suggest that PIP 2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.
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