Taehee Kang

Secreted human glycyl-tRNA synthetase implicated in defense against ERK-activated tumorigenesis

Although adaptive systems of immunity against tumor initiation and destruction are well investigated, less understood is the role, if any, of endogenous factors that have conventional functions. Here we show that glycyl-tRNA synthetase (GRS), an essential component of the translation apparatus, circulates in serum and can be secreted from macrophages in response to Fas ligand that is released from tumor cells. Through cadherin (CDH)6 (K-cadherin), GRS bound to different ERK-activated tumor cells, and released phosphatase 2A (PP2A) from CDH6. The activated PP2A then suppressed ERK signaling through dephosphorylation of ERK and induced apoptosis. These activities were inhibited by blocking GRS with a soluble fragment of CDH6. With in vivo administration of GRS, growth of tumors with a high level of CDH6 and ERK activation were strongly suppressed. Our results implicate a conventional cytoplasmic enzyme in translation as an intrinsic component of the defense against ERK-activated tumor formation.

Dual role of methionyl-tRNA synthetase in the regulation of translation and tumor suppressor activity of aminoacyl-tRNA synthetase-interacting multifunctional protein-3

Mammalian methionyl-tRNA synthetase (MRS) plays an essential role in initiating translation by transferring Met to initiator tRNA (tRNAiMet). MRS also provides a cytosolic anchoring site for aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3)/p18, a potent tumor suppressor that is translocated to the nucleus for DNA repair upon DNA damage. However, the mechanism by which this enzyme mediates these two seemingly unrelated functions is unknown. Here we demonstrate that AIMP3 is released from MRS by UV irradiation-induced stress. Dissociation was induced by phosphorylation of MRS at Ser662 by general control nonrepressed-2 (GCN2) following UV irradiation. Substitution of Ser662 to Asp (S662D) induced a conformational change in MRS and significantly reduced its interaction with AIMP3. This mutant possessed significantly reduced MRS catalytic activity because of loss of tRNAMet binding, resulting in down-regulation of global translation. According to the Met incorporation assay using stable HeLa cells expressing MRS S662A or eukaryotic initiation factor-2 subunit-α (eIF2α) S51A, inactivation of GCN2-induced phosphorylation at eIF2α or MRS augmented the role of the other, suggesting a cross-talk between MRS and eIF2α for efficient translational inhibition. This work reveals a unique mode of regulation of global translation as mediated by aminoacyl-tRNA synthetase, specifically MRS, which we herein identified as a previously unidentified GCN2 substrate. In addition, our research suggests a dual role for MRS: (i) as a coregulator with eIF2α for GCN2-mediated translational inhibition; and (ii) as a coupler of translational inhibition and DNA repair following DNA damage by releasing bound tumor suppressor AIMP3 for its nuclear translocation.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress

ABSTRACT Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNAMet, leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

AIMP3/p18 controls translational initiation by mediating the delivery of charged initiator tRNA to initiation complex.

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNA(i)(Met)) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNA(i)(Met)in vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNA(i)(Met). In addition, AIMP3 discriminates Met-tRNA(i)(Met) from Met-charged elongator tRNA based on filter-binding assay. Pull-down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNA(i)(Met) for the delivery of Met-tRNA(i)(Met) to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNA(i)(Met) bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNA(i)(Met) transfer from MRS to eIF2 complex for the accurate and efficient translation initiation.

2-[2-Substituted-3-(3,4-dichlorobenzylamino)propylamino]-1H-quinolin-4-ones as Staphylococcus aureus methionyl-tRNA synthetase inhibitors.

New analogues of 2-[2-substituted-3-(3,4-dichlorobenzylamino)propylamino]quinolin-4-ones, 26a, 26b, 31a-e, 34, 35, 38 and 40, have been synthesized and evaluated against Staphylococcus aureus methionyl-tRNA synthetase. All of the synthesized compounds were less active than the reference compound 2. The compounds were also screened against various strains of S. aureus and Enterococci for their antibacterial activities. Among the compounds, 26b, 31c and 31e displayed significant inhibitory properties against various strains of Enterococci compared to compound 2.

Caspase-8 controls the secretion of inflammatory lysyl-tRNA synthetase in exosomes from cancer cells

Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-&agr; and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.

Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function

Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.