Methionyl-tRNA Synthetase-induced Misacylation as a Defense Mechanism against Oxidative Stresses

Abstract

Aminoacyl-tRNA synthetases (ARS) are essential enzymes for protein synthesis, linking codons to their corresponding amino acids. Among them, methionyl-tRNA synthetase (MRS) is in charge of transferring methionine (Met) to methionyl-tRNA (tRNAMet). Aminoacylation mediated by ARS is tightly controlled under normal condition assuring translational fidelity. Accumulation of reactive oxygen species (ROS), produced as byproducts of diverse cellular activities, induces various cellular damages including unfolded protein responses (UPR). Cells are equipped with multiple mechanisms to cope with oxidative stresses and usage of thiol group in Met as a ROS scavenger is supposed to be a way of cell protection. Recent study suggested that MRS can misacylate non-methionyl tRNAs with Met at a high frequency under oxidative stresses; however, it was unclear why and how the translational fidelity was modulated by MRS. To elucidate the mechanism of misacylation by MRS under oxidative stresses, post translational modification of MRS was examined. Phosphorylation of MRS was identified and it was dependent on extracellular signal-regulated protein kinase (Erk1/2) which plays a role in maintaining cell proliferation and growth in response to ROS stresses. MRS was dually phosphorylated by Erk1/2 and the phosphorylation sites were identified as serine 209 and 825 (S209 and S825). To verify the effect of dual phosphorylation on aminoacylation activity of MRS, phosphorylation-mimicking MRS mutants were generated and subjected to tRNA binding and aminoacylation assays with tRNAMet and tRNALys. Interestingly, the double mutant whose serine was substituted to aspartate at 209 and 825 showed increased Met-acylation activity to tRNALys while its acylation to tRNAMet was decreased. These results suggested that dual phosphorylation at S209 and S825 compelled MRS to charge non-methionyl tRNAs such as tRNALys more preferentially than tRNAMet. Increased misincorporation of Met into protein being translated was also observed in vivo. However UPR was not induced under the experimental condition used in this study and the cell viability was reduced when the phosphorylation of MRS was blocked. This study suggested that dual phosphorylation of MRS under oxidative stresses modulated translational fidelity by inducing mis-methionylation to non-cognate tRNAs and subsequent misincorporation of Met into growing polypeptides would protect cells from ROS attacks.

활성산소는 일반적인 세포 운동의 부산물로 체내에서 생성되며, 지속적으로 축적될 경우 세포에 산화적 스트레스를 유도하여 다양한 질병의 원인이 되므로 체내 활성산소의 농도 균형을 맞춰주는 것이 중요하다. 이러한 조절을 위해 황 함유 아미노산인 methionine이 활성산소 제거자로써 사용되는데, 최근 세포에 활성산소를 유도하였을 경우 단백질 합성에서 사용되는 전체 methionine의 10%가 misacylation에 의해 non-methionyl-tRNA에 붙어서 단백질 전사에 사용됨이 밝혀졌다. 이와 같은 mistranslation은 새로이 합성되는 단백질의 methionine 잔기 비율을 높임으로써, 세포가 methionine을 통해 활성산소를 낮추게 도와준다. 본 논문에서는 활성산소에 의한 methionine의 misacylation 기작을 밝히기 위해, methionine을 tRNA에 붙여주는 효소인 methionyl-tRNA synthetase (MRS)에 의한 mis-methionylation을 알아보았다. MRS는 특정 아미노산을 상보적인 tRNA에 붙여주는 효소인 Aminoacyl-tRNA synthetases (ARSs) 중 하나로써, 체내 단백질 합성에 관여한다. MRS가 활성산소에 의해 methionyl- tRNA가 아닌 non-methionyl tRNA에 methionine을 붙여주는 것을 확인하기 위해 세포에 활성산소를 유도하였고, 이 때에 MRS의 post translational modification (PTM)을 발견하였다. 이러한 PTM은 산화적 스트레스에 의해 활성화되는 Extracellular signal-regulated protein kinase (Erk1/2)에 의해 일어나며, Erk 1/2 저해제에 의해 methionine의 misacylation이 감소하는 것이 확인되었다. Erk1/2에 의해 인산화가 일어난 MRS는 methionyl-tRNA에 대한 aminoacylation 능력이 감소하였고, lysyl-tRNA에 대한 aminoacylation 능력이 증가하였다. 하지만 활성산소를 유도 후 세포의 Unfolded protein response (UPR)을 측정한 결과 UPR은 관찰되지 않았으며, MRS의 인산화가 저해되었을 경우 세포의 생존 능력이 저해됨이 보여졌다. 이러한 결과는 활성산소에 의한 단백질의 mis-methionylation이 세포 손상을 유발하지 않는다는 것을 보여준다. 활성산소에 의한 MRS의 인산화는 non-methionyl-tRNA에 대한 MRS의 methionylation 비율을 높임으로써, 세포가 활성산소 저해자인 methionine을 통해 체내 활성산소를 줄이는 데에 기여할 것으로 기대한다.