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 (TMDs) with high affinity and selectivity. Nonetheless, all previous work has been limited to single-pass TMDs. Among the membrane-associating proteins that account for approximately 25% to 30% of the human proteome, multi-spanning membrane proteins are particularly challenging to study due to the high degree of difficulty involved in preparing, characterizing, and analyzing these transmembrane proteins. With the proposed studies, we aim to develop a generally applicable method to study previously inaccessible multi-spanning membrane protein associations. Latent Membrane Protein 1 (LMP-1) was chosen as a model system to test this technology because of the essential role of its multi-spanning TMD in activation of signaling and its clinical relevance, particularly to lymphoid malignancies and lymphoproliferative syndromes associated to human Epstein-Barr virus (EBV). EBV's ability to infect and immortalize B lymphocytes depends on the expression and activity of LMP-1, the multi-spanning, viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 most resembles the Tumor Necrosis Factor Receptor (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 of LMP-1's TMD plays a key role in activation of downstream signaling. This study aims to develop an innovative approach to target LMP-1's TMD, using CHAMP-designed anti-TMD peptide antagonists as probes to study the contribution of oligomerization to LMP-1 activation, with the goal of inhibiting downstream signaling. To the CHAMP method, we will introduce a novel screening dimension as well as a next generation of algorithm. 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 multi-spanning transmembrane proteins. Specifically, this proposal addresses the following Aims: 1) Develop specific peptide probes targeting individual TMDs of LMP-1;and 2) Determine the role of TMD-mediated oligomerization in LMP-1 activation.

Public Health Relevance

Many poorly characterized human diseases develop, in part, due to the activity of integral membrane proteins. The inability to target transmembrane domains makes these proteins inaccessible to conventional molecular probes (e.g. antibodies) to study their functions and poor targets for drug treatment. This study examines a novel class of rationally engineered peptides capable of interacting with membrane-embedded protein domains for use in the study of multi-spanning transmembrane proteins, and potentially for the future development of new therapeutic approaches. Latent Membrane Protein-1 (LMP-1), an oncoprotein expressed by the human tumor virus Epstein-Barr virus (EBV), will be used as a model multi-spanning membrane protein target for the design of such peptide inhibitors because of its contribution to EBV-dependent lymphoma and lymphoproliferative syndromes as well as EBV's dependence on LMP-1's hydrophobic transmembrane domain for transformation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM103843-02
Application #
8721453
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fabian, Miles
Project Start
2013-09-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80303
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