The goals of the proposed research are to determine the structural basis for a phosphorylation-directed crosstalk between the eukaryotic translation and transcription machineries. By releasing potent pro-inflammatory mediators, mast cells are essential effectors in the elicitation of allergic responses. Activation of a specific transcriptio factor - MITF - is a critical step for the production of these mediators. Transcription of MITF target genes depends on a co-activator, lysyl-tRNA synthetase (LysRS), an essential component of the cytoplasmic protein synthesis machinery. Phosphorylation of Ser207 of LysRS (to give LysRSp207) directs release of this enzyme from the cytoplasmic multi-tRNA synthetase complex (MSC). LysRSp207 then translocates to the nucleus where it catalyzes the synthesis of a critical signaling molecule, diadenosine tetraphosphate (Ap4A). In the nucleus, nascent Ap4A disrupts the interaction of MITF with the HINT1 suppressor of MITF's function. With the disruption of the MITF-HINT1 complex, LysRSp207 binds to and activates MITF to promote transcription of target genes. In mast cells, a S207D phosphor-mimetic LysRS mutant (LysRSS207D) induces the production of Ap4A and can recapitulate the activity of LysRSp207, which promotes transcription of MITF target genes. This pathway is the first example of phosphorylation-directed crosstalk between the translation and transcription machineries. Our central goal is to understand the structural basis for the axis of the MSC-LysRS-MITF/HINT1 pathway. Our preliminary work showed that the S207D phosphor-mimetic mutation triggers a dynamic structural opening that completely switches off the translational function of LysRS. Thus, phosphorylation acts as a functional switch.
Our aim i s to determine the mechanism by which phosphorylation of Ser207 leads to dissociation of LysRS from the MSC scaffold protein p38 and therefore from the MSC. We will also attempt to understand how LysRS generates Ap4A and dissociates the suppressor HINT1 from MITF. Lastly, we will investigate the structural basis for formation of the LysRS-MITF complex. These investigations can result in the first structural understanding of the crosstalk between eukaryotic translation and transcription machineries. In addition, the insights gained by these studies could open up new therapeutic approaches to disrupting MITF function for treating allergic disorders, a common medical condition that afflicts more than one in five people in the United States.
The goals of the proposed research are to determine the structural basis for the axis of the MSC-LysRS-MITF/HINT1 pathway in controlling mast cell activation. Combining X-ray crystallography, HDX-MS and various functional analyses, the proposed studies will result in the first structural understanding of the phosphorylation-directed crosstalk between eukaryotic translation and transcription machineries. In addition, the insights gained by these studies could open up new therapeutic approaches to disrupting MITF function for treating allergic disorders, a common medical condition that afflicts more than one in five people in the United States.
|Fang, Pengfei; Han, Hongyan; Wang, Jing et al. (2015) Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor. Chem Biol 22:734-44|
|Schaub, Franz X; Reza, Md Shamim; Flaveny, Colin A et al. (2015) Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis. Cancer Res 75:5023-33|
|Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je et al. (2015) Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains. J Biol Chem 290:29313-28|
|Mirando, Adam C; Fang, Pengfei; Williams, Tamara F et al. (2015) Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor. Sci Rep 5:13160|
|Slade, Daniel J; Fang, Pengfei; Dreyton, Christina J et al. (2015) Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design. ACS Chem Biol 10:1043-53|
|Fang, Pengfei; Yu, Xue; Jeong, Seung Jae et al. (2015) Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase. Nat Commun 6:6402|
|Fang, Pengfei; Guo, Min (2015) The Nature's Clever Trick for Making Cyclic Dinucleotide. Structure 23:801-2|
|Fang, Pengfei; Guo, Min (2015) Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification. Life (Basel) 5:1703-25|
|Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon et al. (2014) Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction. Nat Chem Biol 10:29-34|
|Lo, Wing-Sze; Gardiner, Elisabeth; Xu, Zhiwen et al. (2014) Human tRNA synthetase catalytic nulls with diverse functions. Science 345:328-32|
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