This direction mainly focuses on elucidating the novel, expanded function of human amino-acyl tRNA synthetase (aaRS) in angiogenesis (formation of new blood vessels), for developing a new class of protein therapeutics for cancer treatment. The aaRS (one for each amino acid) are ancient, universal proteins that catalyze attachment (aminoacylation) of each amino acid to its cognate tRNA in the first step of protein synthesis (translation). Over evolution, aaRS acquired domains having no apparent connection to their aminoacylation reactions, but unusual functions had been accorded and regulated by these appended domains, angiogenesis is of the most interest. 

SerRS

Disruption of seryl-tRNA synthetase (SerRS) causes abnormal blood vessel formation and defective blood circulation in zebrafish, but the mechanism is unknown. Combining multiple approaches, including the first crystal structure of human SerRS, cellular biology, enzymatic analysis, hydrogen-deuterium exchange mass spectrometry and zebrafish animal experiments, we demonstrated that the essential role of SerRS in vascular development is dependent on a unique domain (named UNE-S) appeared in SerRS from fish to human, it is coincident with the appearance of closed circulatory system. A robust nuclear localization signal (NLS) embedded in the UNE-S domain directs SerRS to a different cellular compartment--the nucleus, but SerRS mutants with the NLS either deleted or sequestered in an alternative conformation are nuclear localization defective and form abnormal vasculature. This work is the first example for an essential role of an aaRS appended domain at the organism level (Nature Communications, 2012).

We then determined the crystal structure of human SerRS in complex with an aminoacylation reaction intermediate analog Ser-SA, binding of Ser-SA dramatically leverages the position of the tRNA binding domain of SerRS, a long range conformational and functional communication specific to higher eukaryotes were discovered (Structure, 2013).

Later, we found that the nuclear-localized SerRS and c-Myc are a pair of ‘Yin-Yang’ transcriptional regulator for proper development of a functional vasculature. First, SerRS blocks c-Myc from binding to the VEGFA promoter by direct head-to-head competition. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation (eLife, 2014). These work systematically illustrated the molecular mechanism of SerRS in angiogenesis.

TrpRS

Tryptophanyl-tRNA synthetases (TrpRS) catalyze the first step of protein synthesis (aminoacylation), providing amino acids for translation. NH2-terminally truncated variants mini-/T1-/T2-TrpRS are produced by alternative splicing or proteolytic digestion of the full-length TrpRS. These splice variants inhibit VEGF-induced endothelial cell proliferation and migration, also angiogenesis. The natural proteolytic fragment T2-TrpRS completely lost the aminoacylation activity, but it only contains anti-angiogenesis function. Since it inhibits new blood vessel formation, but with no disruption of existing blood vessels, T2-TrpRS has appeal for therapeutic applications to oncology. Currently, our group is focusing on the structural and functional characterization of T2-TrpRS&VE-cadherin interactions to discover the most robust form for cancer therapeutics, this innovation can lead to a candidate for human trials of malignant melanoma. This project is supported by NSFC (31400630, 2015-2017) and Zhejiang Provincial Natural Science Foundation (LY14C050002, 2014-2016).

It is revealed that T2-TrpRS binds tightly to N-terminal determinants of vascular endothelial cadherin (VE-cadherin), blocks cell-cell junctions needed for blood vessel formation, and inhibits activation of genes associated with angiogenesis (Nat Struct Mol Biol. 2010). Recently, we investigated the molecular mechanism of the interaction between T2-TrpRS and VE-Cadherin, through cell biology, biochemical and X-ray crystallographic approaches. We found that zinc induces conformational changes in human FL-/T2-TrpRS and facilitates proteolytic generation of T2-TrpRS. Interestingly, one H130R mutation not only reverses the species difference regulation of zinc on the aminoacylation activity of FL-TrpRS, it also mimics the zinc-bound conformation of T2-TrpRS. In addition, H130R mutation induces dramatic crystal structural changes in T2-TrpRS, dissociates the entire eukaryotic specific extension (ESE) away from the catalytic domain, thus decreases the space hindrance of the substrate binding pocket. Molecular dynamic simulation and surface plasma resonance studies of the complex further revealed that the opened substrate pocket provides larger contacting area for VE-Cadherin binding, and enhances the binding affinity of T2-TrpRS and VE-Cadherin. This study further investigated the molecular mechanism of the interaction between T2-TrpRS and VE-Cadherin, and explored a zinc insensitive T2-TrpRS mutant that binds to VE-Cadherin with enhanced affinity. Our work will benefit the development of an natural tumor angiogenesis inhibitor that targets VE-Cadherin (RNA biology. 2017).