Eric First, PhD
Associate Professor of Biochemistry
Bachelor of Science, Biochemistry (1979) - University of Wisconsin, Madison, WI
Bachelor of Science, Chemistry (1980) - University of Wisconsin, Madison, WI
PhD, Biochemistry (1987) - University of California at San Diego, La Jolla, CA
Post-Doctoral Fellow, Biochemistry, Molecular Biology (1988) - Imperial College, London, England
Post-Doctoral Fellow, Biochemistry, Molecular Biology (1991) - Cambridge University, Cambridge, England
U.S. Patent granted October 3, 2017 - High throughput assay for monitoring AMP production and aminoacyl-tRNA synthetase activity
Figure 1 – Reaction scheme for the tyrosyl-tRNA synthetase assay. The production of AMP by tyrosyl-tRNA synthetase is monitored by coupling it to AMP deaminase and IMP dehydrogenase and following the increase in absorbance at 340 nm resulting from the reduction of NAD+. The Tyr-tRNATyr product is cleaved by M. tuberculosis cyclodityrosine synthase, releasing cyclodityrosine and regenerating the tRNATyr substrate. Tyr, AMP, IMP, XMP, and PPi represent L-tyrosine, adenosine 5’-monophosphate, inosine 5’-monophosphate, xanthine 5’-monophosphate, and inorganic pyrophosphate, respectively.
Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. This reaction occurs by a two-step mechanism in which the amino acid is first activated and then transferred to the 3’-end of the cognate tRNA. Misacylation of tRNA with the wrong amino acid occurs less than one time for every 10,000 rounds of catalysis that the enzyme performs. Our current research focuses on three areas related to aminoacyl-tRNA synthetases. First, we are developing methods to expand the genetic code to include D-amino acids. Current efforts are aimed at engineering orthogonal tyrosyl-tRNA synthetase variants that are specific for either D- or L-tyrosine. Specifically, we have introduced editing domains into tyrosyl-tRNA synthetase that selectively hydrolyze L-tyrosyl-tRNA but not D-tyrosyl-tRNA. Second, we are developing high-throughput assays and inhibitor screens for aminoacyl-tRNA synthetases and related enzymes. This research is based on a high-throughput tyrosyl-tRNA synthetase assay that we recently developed in which the tyrosyl-tRNA product is cleaved, regenerating the tRNA substrate. We have extended this assay to monitor the activities of cyclodityrosine synthase and D-tyrosyl-tRNA deacylase. We have also adapted this assay to monitor post-transfer editing by aminoacyl-tRNA synthetases. We are in the process of using this assay to identify inhibitors of post-transfer editing by the human phenylalanyl-tRNA synthetase. Third, we are investigating the role that the tyrosyl-tRNA synthetase plays in Charcot-Marie-Tooth disorder. Charcot-Marie-Tooth disorder (CMT) is the most common inherited peripheral neuropathy, affecting 150,000 individuals in the U.S. Patients experience degeneration of their peripheral nerve cells, leading to atrophy of the muscles controlling their hands, feet, forearms, and lower legs. Ultimately, this leads to loss of the ability to perform routine tasks such as holding a pencil or turning a door knob. Four different mutations in the gene encoding human tyrosyl-tRNA synthetase are responsible for a form of CMT known as Dominant Intermediate Charcot-Marie-Tooth disorder (DI-CMTC). Our research suggests that DI-CMTC may be due to a defect in the fidelity of tyrosyl-tRNA synthetase.
Figure 1 – Aminoacyl-tRNA synthetases play a central role in protein synthesis. Aminocyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. As the energy in the aminoacyl-tRNA linkage is used to drive peptide bond formation, the tRNA aminoacylation reaction provides 50% of the energy used in protein synthesis. Furthermore, the aminoacyl-tRNA synthetases are responsible for ensuring the fidelity of protein synthesis, as aminoacylation of tRNA with a noncognate amino acid results in misincorporation of the wrong amino acid during translation of the mRNA.
Figure 2 – Insertion of the phenylalanyl-tRNA synthetase editing domain into tyrosyl-tRNA synthetase alters the enantioselectivity of tyrosyl-tRNA synthetase. Tyrosyl-tRNA synthetase is able to aminoacylate tRNA with both L- and D-tyrosine. Insertion of the phenylalanyl-tRNA synthetase editing domain into tyrosyl-tRNA synthetase results in hydrolysis of the L-Tyr-tRNA product, but not D-Tyr-tRNA, altering the enantioselectivity of the enzyme.
Figure 3 – Insertion of the phenylalanyl-tRNA synthetase editing domain chimera into tyrosyl-tRNA synthetase. The structures of the Geobacillus sterarothermophilus tyrosyl-tRNA synthetase dimer (PDB 3TS1) and Pyrococcus horikoshii phenylalanyl-tRNA synthetase editing domain (residues 83-275 in the β-subunit; PDB 2CXI) are shown. The insertion sites for the editing domain (residues 161-162 in tyrosyl-tRNA synthetase) are shown as blue cartoons.
- First, E. A., and Richardson, C. J. (2017) Spectrophotometric assays for monitoring tRNA aminoacylation and aminoacyl-tRNA hydrolysis reactions. Methods 113, 3-12.
- Richardson CJ, First EA. Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain. Biochemistry. 2016 Mar 15;55(10):1541-53. [Editors’ Choice Award]
- Richardson CJ, First EA. Hyperactive Editing Domain Variants Switch the Stereospecificity of Tyrosyl-tRNA Synthetase. Biochemistry. 2016 May 3;55(17):2526-37.
- First, E. A. Analytical Biochemistry 483, 34-39 (2015). “A continuous spectrophotometric assay for monitoring AMP production.”
- Richardson, C. J. and First, E. A. Analytical Biochemistry 486, 86-95 (2015). “A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate.”
- Richardson, C. J. and First, E. A. Data in Brief 4, 253-256 (2015). “Expanding a tyrosyl-tRNA synthetase assay to other aminoacyl-tRNA synthetases.”
- Leiman, S. A., Richardson, C., Foulston, L., Elsholz, A. K., First, E. A., and Losick, R. Journal of Bacteriology 197, 1632-1639 (2015). “Identification and characterization of mutations conferring resistance to D-amino acids in Bacillus subtilis.”
- Froelich, C. A. and First, E. A., Biochemistry 50, 7132-7145 (2011). “Dominant Intermediate Charcot-Marie-Tooth disorder is not due to a catalytic defect in tyrosyl-tRNA synthetase.”
- Sharma, G. and First, E.A., Journal of Biological Chemistry 284, 4179-4190 (2009). “Thermodynamic analysis reveals a temperature-dependent change in the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase.”
- Sheoran, A., Sharma, G., and First, E.A., Journal of Biological Chemistry 283, 12960-12970 (2008). “Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Pre-steady state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine.”
- Sheoran, A. and First, E.A., Journal of Biological Chemistry 283, 12971-12980 (2008). “Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme.”
Complete List of my Published Work: BIBLIOGRAPHY
Several lines of evidence indicate that malignant cells are under higher levels of proteotoxic stress than nontransformed cells, suggesting that they are susceptible to drugs that increase protein misfolding and/or the ability of the cell to handle misfolded proteins. The goal of this research is to determine whether post-transfer editing by aminoacyl-tRNA synthetases represents a novel target for developing chemotherapy agents. This requires developing a high-throughput assay that can be used to screen for inhibitors of the human phenylalanyl-tRNA synthetase editing domain. This screen is based on a continuous spectrophotometric editing assay previously developed by the applicant. Once inhibitors have been identified, they will be tested to determine their efficacy with respect to inhibiting cell proliferation and migration in MDA-MB-231 triple negative breast cancer cells.