Identification of Novel Signaling Pathways in NF2
Abstract
Neurofibromatosis type 2 is a genetic disorder characterized by bilateral schwannomas derived from Schwann cells of the eighth cranial nerve. Because conventional chemotherapy is not an effective treatment for these insidious, slow-growing tumors, other therapies must be devised. Unfortunately, the molecular basis of NF2 is poorly understood. Therefore, to develop new, effective therapies it is critical to understand exactly how NF2 progresses at the molecular level. Neurofibromatosis type 2 is caused by a mutation in the tumor suppressor gene NF2, which results in the loss of a protein called merlin. Although it is unclear exactly how loss of merlin leads to schwannoma formation, it is now understood that unlike normal Schwann cells, schwannoma cells no longer stop growing when they come in close contact with one another. This abnormality, called loss of contact inhibition, is thought to be critical in the development of many kinds of tumors, but it is unclear how loss of merlin causes it. What is clear is that merlin functions by binding to specific proteins within the cell, although there is no clear consensus as to which of these proteins is critical for tumor suppression. Critical questions about merlin function remain to be answered. What are the proteins that merlin interacts with to prevent schwannoma formation? What does merlin do to these proteins when it binds them and what happens to these proteins when merlin is gone that causes tumors to form? Since conventional experimental techniques have not led to answers to these questions, we decided to explore unconventional technologies. We have adopted a new method, proximity biotinylation, which represents a powerful new way to investigate merlin function. Proximity biotinylation is a novel technique that allows us to identify all the proteins that merlin interacts within a living Schwann cell. This is achieved by making a hybrid protein consisting of merlin bound to a modified bacterial enzyme called BirA. BirA normally functions to attach a small molecule, biotin, to a specific protein in bacteria. We have modified BirA such that it will attach biotin to any protein that is in close proximity. When expressed in Schwann cells, the hybrid protein we made, merlin-BirAR118G, attaches biotin to proteins that merlin is bound to; therefore, any proteins that have biotin are likely to interact with merlin. These biotin-containing proteins are readily purified and identified by a process call Streptavidin affinity chromatography and MALDI mass spec, resulting in a list of proteins that merlin interacts with. It would be a significant understatement to say that this worked well. Proximity biotinylation has proven itself as vastly superior to conventional methods that identify protein interactions. We identified 53 proteins as merlin proximal and likely interaction partners for merlin. The majority of these proteins are known to be components of cell junctions, multi-protein complexes that control interactions between adjacent cells or cells and surrounding tissue. Cell junctions are now seen a complexes structures that connect mechanical forces with downstream cell signaling machinery. We hypothesize that the loss of merlin destabilizes or impairs assembly of these structures, causing abnormal, cancer-causing signals. To test this hypothesis, we will again use proximity biotinylation, in conjunction a much better method to attach BirA to proteins in cell junctions, to look for changes in key cell junction components in the presence or absence of merlin. We expect that the experiments described in this proposal will help us understand exactly what merlin does in cells. Once this is known in detail, we will have a much better idea what the consequences of the loss of merlin are and a greater appreciation of the types of drugs, or combinations of drugs, that may be used to treat this disease.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 07, 2017
- Source ID
- W81XWH1710152
Entities
People
- Robert F Hennigan
Organizations
- United States Army