Artificial Small Transmembrane Proteins as Novel Agents to Modulate Cell Behavior and Study Protein-protein Interactions

Based on our analysis of the BPV E5 protein, we devised a genetic approach to construct and identify small proteins that can alter cell behavior. We used a PCR-based method to construct complex retrovirus libraries in which various portions of the E5 protein are replaced with random sequences of hydrophobic amino acids to generate artificial transmembrane proteins we designate traptamers, for transmembrane aptamers. After expression in cells, isolates that induce cell transformation were selected on the basis of their ability to induce focus formation in rodent fibroblasts. We found that a surprisingly large fraction of random transmembrane domains with diverse sequences can induce cell transformation by interacting with the transmembrane domain of the PDGF ß receptor. Some of these proteins display altered specificity compared to the wild-type E5 protein in terms of their ability to recognize PDGF ß receptor transmembrane domains containing amino acid substitutions, and several of them can be converted by single amino acid substitutions from transmembrane activators into proteins that inhibit the action of PDGF.  In some cases, these proteins retain no amino acid sequences from the E5 protein or any other natural protein, yet can still bind and activate the PDGF receptor and cause tumors in animals (Fig. 3), and some of them have drastically reduced chemical complexity compared to naturally occurring proteins.

We have extended this approach to identify traptamers that activate the erythropoietin receptor and induce erythroid differentiation in primary human hematopoietic cells, or down-regulate the HIV co-receptor CCR5 and inhibit HIV infection in human T cells (Fig. 4).  Several of these proteins utilize different mechanisms to modulate the same target, recognize the target in different ways, or induce differential signaling. These findings provide insight into protein-protein interactions, oncogenesis, and possibly protein evolution and the origin of life, and they have important implications for artificial protein engineering.  Because 30% of all cell proteins are integral membrane proteins, it is possible that this approach can be used to modulate many potential targets. We are currently focusing on determining the mechanism of traptamer-mediated CCR5 down-regulation, determining the basis for altered signaling by different traptamers that activate the same target, and exploring the implications of proteins with minimal chemical complexity in terms of their specificity and signaling profiles.

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