The research interests of the Bishop laboratory are predominantly focused at the interface between organic chemistry and molecular biology. We use a combination of chemical and biochemical approaches to study two central biological processes: cellular signal transduction and protein synthesis. The two major ongoing projects of the lab are described below.
Project I: Target-specific control of protein tyrosine phosphatase activity
A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. The protein tyrosine phosphatases (PTPs) represent attractive protein targets, as small-molecule ligands that can specifically inhibit or activate individual PTPs would be valuable tools for dissecting protein-phosphorylation networks. However, the size of the PTP superfamily—roughly 100 are encoded in mammalian genomes—and the common architecture of PTP active sites impede the discovery of selective PTP ligands via conventional medicinal chemistry.
To avoid the specificity problems that are inherent to small-molecule ligand discovery, the Bishop lab has developed strategies for generating “ligand-sensitized” PTPs—engineered phosphatases whose activities are uniquely responsive to control by small molecules. Two distinct strategies—active-site engineering and WPD-loop targeting—have been employed to generate PTPs that possess novel sensitivity to applied small-molecule inhibitors and activators. In both approaches, functionally silent mutation(s) on a target PTP sensitize the enzyme to a small molecule that does not affect the activity of wild-type PTPs. A significant advantage of these engineered-sensitivity approaches to controlling PTP activity is that they can potentially yield general strategies for targeting multiple members of a large protein family—the amino-acid residues identified for sensitization are present across the protein family, eliminating the need to redesign a protein/ligand interface for each new PTP target. Once highly sensitizing mutations are discovered on model PTPs, primary-sequence alignments allow for the identification of the corresponding positions in other PTPs, enabling the design, expression, and analysis of an array of sensitized PTPs for target-specific inhibition and activation. The long-term objectives of this research are to generate allele-specific ligands for a substantial fraction of the PTP superfamily; to validate the potency and selectivity of target-specific PTP control in living cells; and to use the PTP inhibitors and activators in mammalian cell-signaling experiments toward the delineation of PTP functions in signaling cascades, as well as the validation of PTPs as therapeutic targets.
Project II: Biochemical characterization of the tRNA-dihydrouridine synthases
Transfer RNA (tRNA), which acts as an essential link between the cellular pool of amino acids and the decoding machinery of the ribosome, contains many modified RNA bases, in addition to the four canonical bases A, C, G, and U. Although the functions of some modified tRNA bases have been well characterized, the biological role of most of these chemically interesting nucleotides remains unclear. 5,6-Dihydrouridine is one of the most abundant modified bases in prokaryotic and eukaryotic tRNAs. This non-aromatic base is found almost exclusively at conserved positions in the D-loop and is formed post-transcriptionally by the reduction of uridines in tRNA transcripts. Despite its widespread occurrence, little is known about dihydrouridine’s biological roles and the enzyme family responsible for dihydrouridine formation, the tRNA-dihydrouridine synthases (DUSs).
Our goal is to gain a molecular level understanding of how DUSs act to reduce D-loop uridines to dihydrouridine. The relatively recent identification of the DUS family (2002) and the dearth of biochemical studies on these enzymes allow various lines of questioning to emerge: How do DUSs recognize and bind tRNA? DUSs contain no known RNA-binding domains. Thus, it is very likely that DUSs utilize a yet undiscovered RNA-binding motif. One of the primary objectives of our work is to develop an understanding of how DUSs recognize and bind tRNA. The identification of a novel DUS RNA-binding motif may also have broad implications outside the DUS family, as similar RNA-binding moieties are often found in otherwise unrelated protein families. What is the substrate specificity of the DUSs and how it is achieved? The E. coli genome encodes three members of the DUS family. From our initial data, it is clear that these enzymes have non-redundant tRNA-substrate specificities. However, the determination of the substrate specificities for the three E. coli DUSs has not been carried out. We are attempting to map the precise tRNA positions that are modified by each of the E. coli DUSs. In addition, the portions of the enzymes that give rise to this specificity will be delineated.