Dept. of Cellular & Molecular Pharmacology
University of California, San Francisco
600 16th Street
Box 2280, GH-N512F
San Francisco, CA 94143-2280
2002 Searle Scholar
Electrophilic Capture of Protein Targets in Living CellsOur research follows two trajectories: a cell biology trajectory, in which we elucidate the signals that control actin assembly at specific membrane sites; and a chemistry trajectory, in which we synthesize small molecule tools to perturb and thereby illuminate cellular functions.
1. Endosome movement powered by actin assembly
Actin is an essential eukaryotic protein that couples ATP hydrolysis with self-assembly to form dynamic polymers that produce force. One of the outstanding questions in cell biology concerns the role played by the actin cytoskeleton in the movement of cellular membranes in such processes as endocytosis, exocytosis, and intracellular trafficking. We have developed a cell-free system that reconstitutes membrane-dependent actin assembly. Actin assembly in this system occurs specifically on the surface of endosomes and lysosomes and results in their physical movement at speeds of 10-20 microns per minute. Actin-dependent organelle movement is massively stimulated by phorbol myristate acetate, a tumor promoter that activates protein kinase C (PKC). Two of the proteins downstream of PKC are the GTP-binding protein, Cdc42 and the actin nucleation promoting factor, N-WASP. We are using biochemical approaches to understand how PKC, Cdc42, and N-WASP collaborate to drive actin-dependent organelle motility.
2. Cellular processes illuminated by small molecules
We are chemically synthesizing a variety of biologically active small molecules. In one project, our goal is to identify the protein target of an unusual cyclopeptide that was originally isolated from a marine sponge. At low nanomolar concentrations, this cyclopeptide blocks inflammatory cytokine signaling by an unknown mechanism. We will use synthetic variants of the natural cyclopeptide to figure out how it works.
We are also developing a chemical proteomics approach to identify and characterize proteins, including integral membrane proteins, directly from whole cell lysates and membrane fractions. Our approach relies on the ability of a small molecule containing an electrophilic functional group to form an irreversible, covalent bond with protein nucleophiles. Biotinylated "scaffolds" containing a diverse repertoire of electrophilic functionality (e.g. epoxide, chloroacetyl, acryloyl) are added to cell lysates, and specifically targeted proteins are purified and identified by mass spectrometry. We are currently exploring how changes in the scaffold and the electrophile influence protein targeting specificity.
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