A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Kise, Yoshiaki; Lee, Sang Won; Park, Sang Gyu; Fukai, Shuya; Sengoku, Toru; Ishii, Ryohei; Yokoyama, Shigeyuki; Kim, Sung Hoon; Nureki, Osamu

doi:10.1038/nsmb722

Cited by 69 publications

(87 citation statements)

References 35 publications

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“…8 Notably, human TrpRS manifests its anti-angiogenic activity only when the WHEP domain is removed. Full length (FL) TrpRS cannot inhibit angiogenesis, 5,6,10 due to the steric hindrance of the WHEP domain that blocks the access of Trp2 and Trp4 of VE-cadherin to the active site pocket. 8 …”

Section: Introductionmentioning

confidence: 99%

“…16 Interestingly, both bovine and human TrpRS were shown to bind zinc to enhance their aminoacylation activities. 17,18,19 However, no zinc was found in the crystal structures of TrpRS, 4,10,20,21,22,23 suggesting that the zinc-bound conformation may be different. Interestingly, Wakasugi has identified a single amino acid substitution in TrpRS (H130R) that eliminates zinc-induced stimulation and renders constitutively high enzymatic active, 24 suggesting H130R TrpRS may mimic a zinc-bound conformation.…”

Section: Introductionmentioning

confidence: 99%

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An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

Zhou

et al. 2017

RNA Biology

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Tryptophanyl-tRNA synthetase (TrpRS) in vertebrates contains a N-terminal extension in front of the catalytic core. Proteolytic removal of the N-terminal 93 amino acids gives rise to T2-TrpRS, which has potent anti-angiogenic activity mediated through its extracellular interaction with VE-cadherin. Zinc has been shown to have anti-angiogenic effects and can bind to human TrpRS. However, the connection between zinc and the anti-angiogenic function of TrpRS has not been explored. Here we report that zinc binding can induce structural relaxation in human TrpRS to facilitate the proteolytic generation of a T2-TrpRS-like fragment. The zinc-binding site is likely to be contained within T2-TrpRS, and the zinc-bound conformation of T2-TrpRS is mimicked by mutation H130R. We determined the crystal structure of H130R T2-TrpRS at 2.8 Å resolution, which reveals drastically different conformation from that of wild-type (WT) T2-TrpRS. The conformational change creates larger binding surfaces for VE-cadherin as suggested by molecular dynamic simulations. Surface plasmon resonance analysis indicates more than 50-fold increase in binding affinity of H130R T2-TrpRS for VE-cadherin, compared to WT T2-TrpRS. The enhanced interaction is also confirmed by a cell-based binding analysis. These results suggest that zinc plays an important role in activating TrpRS for angiogenesis regulation.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

Zhou

et al. 2017

RNA Biology

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“…Tryptophanyl-tRNA synthetase (TrpRS) is a prime example of a synthetase procytokine. Through alternative splicing or natural proteolysis, fragments known as mini-TrpRS (alternative splicing), or the closely similar T2-TrpRS (natural proteolysis), are potent anti-angiogenic factors that, after exocytosis, bind to cell surface VE-cadherin and trigger arrest of neo-angiogenesis through the Akt signaling pathway (5)(6)(7)(8)(9)(10)(11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

Tumor endothelial cell tube formation model for determining anti-angiogenic activity of a tRNA synthetase cytokine

Zhou

Kiosses

Liu

et al. 2008

Methods

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In addition to their key role in protein biosynthesis, aminoacyl-tRNA synthetases have other biological functions that appeared during their long evolutionary development. In mammalian cells, specific members of this family of enzymes are also procytokines that, upon conversion, are active cytokines in pathways for angiogenesis, and thereby connect translation to control of blood vessel development. Here we describe an in vitro assay for tube formation by tumor endothelial cells on a matrigel substrate. In contrast to normal endothelial cells, tumor endothelial cells have strong angiogenic capabilities and the ability to form vessel-like tubes on a solid substrate. In particular, we found that a SV40-immortalized mouse lymphoid endothelial cell line was robust in this assay and yielded data that could be quantified with high precision. Consequently, this specific tube formation model provides an opportunity to discover and analyze potent agents that specifically affect angiogenesis. It has proven effective for studying the angiogenic functions of tRNA synthetase cytokines.

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“…In normal cells, human TrpRS exists as two forms. The major form is the full-length protein, and the other is a truncated TrpRS (mini-TrpRS) in which most of the extra NH 2 -terminal domain is deleted because of alternative splicing of the pre-mRNA (Kise et al 2004;Jorgensen et al 2000), with Met-48 being deduced as the NH 2 -terminal residue of mini-TrpRS (Kise et al 2004). PMN elastase digestion of recombinant full-length TrpRS produced two fragments designated T 1 -TrpRS and T 2 -TrpRS, respectively.…”

Section: Introductionmentioning

confidence: 99%

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture

Zeng¹,

Chen²,

Zeng³

et al. 2008

Cytotechnology

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Aim We studied the role of mini-TyrRS and mini-TrpRS in angiogenesis by using small interfering RNA-mediated mini-TyrRS/mini-TrpRS knockout in hypoxic culture of human umbilical vein endothelial cells. Methods SiRNA was used as the main method to inhibited the gene function. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction and western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and Matrigel-induced capillary tube formation in hypoxic culture. Cell proliferation was determined by crystal violet staining. Results The results showed that levels of the miniTyrRS/mini-TrpRS gene and protein in mock transfection group and negative control group were higher, but noticeably decreased in experimental group. However, no significant difference was detected between mock transfection group and negative control group, but there was a statistically significant difference compared with experimental group. For miniTyrRS-siRNA group, the cell migration, tube formation and the rate of cell proliferation were respectively inhibited by (47.4, 56.3, 65.4, 73.7%), (60.5, 69.1, 75.9, 83.6%) and (40.4, 56.2, 61.2, 68.0%). For miniTrpRS-siRNA, were respectively increased by (18.0, 33.8, 45.1, 56.4%), (18.3, 31.2, 40.3, 45.7%) and (8.4, 26.4, 38.2, 46.6%). Conclusion These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in matrigel models in hypoxic culture, raising the possibility that mini-TyrRS stimulates a common downstream signaling event. Thus, naturally occurring fragments of two proteins involved in translation, TyrRS and TrpRS, have opposing activity on endothelial cell angiogenesis in the matrigel assays. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro-and anti-angiogenic stimuli.

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A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Cited by 69 publications

References 35 publications

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

Tumor endothelial cell tube formation model for determining anti-angiogenic activity of a tRNA synthetase cytokine

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture

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