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Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.

The pH-low insertion peptide (pHLIP) targets acidic diseases such as cancer. The acidity of the environment causes key aspartic acids in pHLIP to become protonated, causing the peptide to insert into membranes. Here we investigate how the negative charge of the membrane influences how pHLIP enters and exits the lipid bilayer. We found that electrostatic repulsion affected differently the membrane entry and exit of pHLIP for negatively charged membranes. As a consequence, a large hysteresis was observed. We propose this is not a consequence of structural changes but results from local changes in the environment of aspartic acids, shifting their pK values.

The interferon alpha receptor is composed of two subunits: IFNaR1 and IFNaR2. Interferon alpha binding to the receptor induces phosphorylation of tyrosine 466 on IFNaR1, which in turn binds the SH2 domain of the latent transcription factor Stat2 to initiate signaling. Stat2 also binds to IFNaR2 in a constitutive, phosphorylation-independent manner. To explore the function of the Stat2-IFNaR2 interaction and its possible relationship to the SH2-dependent docking of Stat2 to phosphorylated IFNaR1, the affinity of Stat2 for each of the receptor subunits was determined. Recombinant proteins corresponding to the cytoplasmic domains of the receptor subunits and the central core region of Stat2 were partially purified and used in affinity precipitation experiments to demonstrate that Stat2 binds more avidly to IFNaR2 than to phosphorylated IFNaR1. Surface plasmon resonance based biosensor analysis confirmed this finding; Stat2 bound IFNaR2 (K(d) = 45 nM) approximately 6-fold stronger than it bound tyrosine 466-phosphorylated IFNaR1 (K(d) = 245 nM). Affinity precipitation experiments involving all three proteins (Stat2, phosphorylated IFNaR1, and IFNaR2) indicated that the Stat2-receptor interactions are independent of one another. The relevance of these data to possible models of interferon alpha signal transduction is discussed.