Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains with high affinity and selectivity. This study utilizes this cutting edge technology to study the activation mechanism of oncogenic Latent Membrane Protein 1 (LMP-1) of Epstein-Barr virus (EBV). EBV is a human tumor virus associated with a number of malignancies and lymphoproliferative syndromes. EBV's ability to infect and immortalize B lymphocytes underlies its contribution to human disease. EBV's transforming activity depends on the expression and activity of LMP-1, the viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 functions as a constitutively active Tumor Necrosis Factor Receptor (TNFR) whose activity requires the function of its hydrophobic transmembrane domain. LMP-1 most resembles the TNFR CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1's activity is constitutive and ligand-independent. Constitutive homo-oligomerization and lipid raft association, activities of LMP-1's transmembrane domain, play a key role in activation of downstream signaling. This proposal focuses on LMP-1 as a model membrane protein target for the design of peptide inhibitors because of LMP-1's essential role in EBV-dependent B cell transformation, LMP-1's contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and EBV's dependence on LMP-1's hydrophobic transmembrane domain for activity. This study aims to develop an innovative approach to target LMP-1's transmembrane domain, using CHAMP-designed anti-peptide antagonists as probes to study the contribution of oligomerization and raft association to LMP-1 activation, with the goal of inhibiting downstream signaling. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes, will reveal the mechanism of LMP-1's constitutive activation of signaling, and will be applicable to the future development of novel therapeutics targeting diseases dependent on critical transmembrane proteins. Specifically, this proposal addresses the following Aims: 1) Can anti-TMD-1 peptides probes be developed that have high affinity and specificity for LMP-1's TMD-1? 2) Do identified peptides bind specifically and with affinity to LMP-1's TMD-1 in vitro? and 3) Can peptides that target TMD-1 (identified in Aims 1 and 2) interfere with LMP-1 homo-oligomerization, raft association, and constitutive signaling in intact cells?
. Many poorly characterized human cancers develop, in part, due to the activity of oncogenic transmembrane proteins which, because of the inability to target protein domains within the membrane bilayer, are inaccessible to molecular probes to study function and are poor targets for current drug treatment methods. This study examines a novel class of rationally engineered peptides capable of interacting with membrane-embedded protein domains for use in the study of oncogenic transmembrane proteins, and potentially for the future development of new therapeutic approaches to the prevention and treatment of such cancers. The Latent Membrane Protein-1 (LMP-1), an oncoprotein expressed by the human tumor virus Epstein-Barr virus (EBV), will be used as a model membrane protein target for the design of such peptide inhibitors, because of its contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and because of EBV's dependence on LMP-1's hydrophobic transmembrane domain for transformation.
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