The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC, the second interaction surface consists of strand C, strand F, and loop FG, and the third interaction surface consists of strand A and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-s time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, ϳ10 M. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nM.

SUMMARY Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that were implicated in the pathology of diabetes, chronic inflammation, Alzheimer’s disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). Nε-carboxy-methyl-lysine (CML) and Nε-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL) modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells. We show that RNAs are an important component of protein quinary interactions. Protein quinary interactions are unique to the target protein and may affect physicochemical properties, protein activity, and interactions with drugs.