Cell Signaling Through the Eyes of an Adapter Protein
Mount Sinai Hospital ♦ University of Toronto ♦ Published in Nature Biotechnology, June 26, 2011
A novel approach to analyzing the protein machinery in cells utilizes affinity purification (AP) to identify protein-protein interactions, and a unique form of mass spectrometry, known as selected reaction monitoring (SLR), to identify novel proteins and quantify the kinetics behind the formation and disappearance of protein networks. To link the cell’s external environment with intracellular signaling, researchers focused on a transmembrane adapter protein, GRB2, which is at the core of an extraordinarily diverse group of protein interactions that are essential for cellular function.
GRB2 is a player in many tyrosine kinase receptor (RTK) pathways, so researchers hypothesized that by capturing it with affinity purification they would also capture a number of proteins involved in RTK signaling. To test their hypothesis they engineered a HEK239T cell line to express a FLAG-tagged version of GRB2 that would allow for purification of the adapter protein and any associated proteins. In order to broadly identify proteins in the RTK network that associate with GRB2, researchers treated 293 cells with the phosphatase inhibitor pervanadate and then purified GRB2 out of cell lysates with liquid chromatography.
Analysis of protein clusters allowed for the construction of a protein network with GRB2 at its hub. Further investigation allowed researchers to determine how this network fluctuates temporally in response to stimulation. By exposing 293 cells to epidermal growth factor for different periods of time, researchers used AP-SLR to create a dynamic version of the protein network illustrating changes in protein involvement over time.
GABA Exerts Dual Effects on Diabetic Condition
St. Michael’s Hospital ♦ University of Toronto ♦ Published in PNAS, June 27, 2011
Diabetes is an autoimmune disease characterized by the progressive loss of insulin-producing β-cells in the pancreas. The research community is investigating endogenous growth factors as a means to encourage the proliferation of β-cells in vivo. This approach does not remedy the underlying problem, however, as the body’s immune cells will continue to attack β-cells, exacerbating the disease. New findings provide evidence that suggests the central nervous system neurotransmitter GABA can play a dual role in treating the diabetic condition as it stimulates the proliferation of β-cells while suppressing their immune-mediated destruction.
Contrary to its function in the brain, GABA causes membrane depolarization, calcium-influx, and the activation of Pl3-K/Akt-dependent growth and survival pathways in the pancreas. Researchers found that GABA therapy can increase β-cell mass in vivo, and can actually reverse the disease in severely diabetic mice. Interestingly, GABA also seems to suppress the cytokine storms that cause systemic inflammation and pancreatic infiltration by T lymphocytes and macrophages.