The goal of my research is to understand the mechanisms by how proteins transmit signals from biological membranes. Our approach uses biophysical and biochemical methods such as NMR spectroscopy, circular dichroism, and isothermal titration calorimetry, and GST-pull down assays. With these tools, we characterize protein:protein interactions and ligand binding pockets as well as identify and quantify differences in small molecules that allow a cell to proliferate and to communicate with neighboring cells.
Role of the EH Domain in Endocytosis and Cellular Proliferation
The movement of molecules to and from the surface of a cell is fundamental to many biological processes including glucose uptake, fertilization, cholesterol metabolism, and cellular signaling. These vesicle-mediated sorting and cellular remodeling mechanisms are mediated in part by interactions between proteins containing EH domains and proteins containing asparagine-proline-phenylalanine (NPF) sequences. The human genome encodes 17 EH domains contained in 11 proteins, and about twenty proteins contain NPF motifs that are available for interaction. Although all the EH domain-containing proteins are involved in vesicle trafficking, the factors that govern selectivity of NPF-containing partners are largely unknown. The gap in knowledge regarding the most likely EH domain/NPF partners is a problem as it prevents us from understanding the molecular interactions that guide the movement of proteins within a cell. For example, the EHD1 protein functions in the recycling of vesicles and thus plays an important role in cellular physiology by directing proteins such as the β1-integrins back to the cell surface as well as controlling the level of the GLUT4-glucose transporter present on the cell surface. Likewise, the Eps15 protein is an accessory factor for the endocytosis of many proteins, such as the LDL receptor and the β1-integrins. Therefore, EH domain containing proteins affect communication of cells with the extracellular matrix, and the homeostasis of glucose and cholesterol within the cell.
Various complexes with EH domains are being characterized to understand specificity at the domain/NPF interface (Figure 1). Our data suggest new protein-protein partners that are being verified using co-localization, GST-pulldown, and coimmunoprecipitation experiments. In addition, the data have provided a basis for the design of EHD1-specific inhibitors that are being developed and characterized in collaboration with Dr. Joshua Kritzer in the Department of Chemistry (School of Arts and Sciences) and Dr. Addy Alt-Holland in the Department of Endodontics (School of Dental Medicine)
Figure 1. A model of the EHD1 EH domain bound to an inhibitory peptide. The model predicts that the domain uses positively charged residues (in blue) to contact negatively charged residues at the C-terminus of the NPF motif (Henry et al 2010 )
Another example is the EH domain-containing X-linked Reps2 (aka POB1) protein is part of a protein complex that directly interacts with a GTPase activating protein, RalBP1. Via RalBP1, Reps2 inhibits growth factor signaling and alters signaling molecules and drug efflux. Despite the importance of Reps2 in building the networks responsible for protein trafficking and signaling, we do not know how these proteins assemble. Such a lack of knowledge is a problem because without it we cannot understand key features of cellular function that, in the long term, may be manipulated for therapeutic purposes.
Characterization of small molecules in controlling cell fate
A second area of interest in the lab lies in using solution NMR methods to quantify chemical fingerprints of specific cellular processes, or metabolomics. The metabolome is the set of all metabolites in a tissue, or cell line, growth media, or other biofluid. NMR does not require separation prior to measurement, the sample can be recovered for further analyses, and the method is highly reproducible. NMR is also the basis for magnetic resonance spectroscopy, a procedure that can be added to MRI procedures that provides a non-invasive (without biopsy) biochemical readout of a tissue or organ.
The levels of many metabolites change in response to the changing needs of a cell. For example, many tumors show changes called the Warburg effect, and are characterized by higher glycolytic flux with increased biosynthesis of amino acids, lipids, and nucleic acids to provide for the increased proliferation demands of a tumor. In addition changes occur in the way that cells communicate with each other by the secretion of different metabolites. These changes in metabolites will also change in response to chemotherapy, often before structural changes in tissue are observed. Thus, metabolite levels may provide a valuable biomarker for treatment efficacy. Tumors are heterogeneous. Novel metabolic pathways include the production of 2-hydroxyglutarate in many glioblastomas and the dependence on asparagine exhibited in acute lymphoblastic leukemia (ALL). It is hypothesized the unique metabolic addictions will exist for many types of cancer, and more broadly, many different types of disease. We are currently investigating novel metabolic pathways in cancers of the skin, in collaboration with Dr. Alt-Holland, and in breast cancer, in collaboration with Dr. Amy Yee, Department of Biochemistry.
Figure 2. An example of a 1H NMR spectrum illustrating the complexity of molecules present and secreted into growth medium. The ppm scale indicates the resonance frequency that identifies a molecule; concentration is proportional to the height of the peak.