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Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.

All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNALeu for protein synthesis and plays an important role in mTORC1 activation by sensing intracellular leucine concentrations. Here, we describe a protocol for the purification, reductive methylation, binding affinity measurement by microscale thermophoresis, T i value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed us to build the RagD binding domain, which was shown to be a dynamic region of LARS1 refractory to crystallization. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).

Leucyl-tRNA synthetase (LRS) is known to function as leucine sensor in the mammalian target of rapamycin complex 1 (mTORC1) pathway. However, the pathophysiological significance of its activity is not well understood. Here, we demonstrate that the leucine sensor function for mTORC1 activation of LRS can be decoupled from its catalytic activity. We identified compounds that inhibit the leucine-dependent mTORC1 pathway by specifically inhibiting the GTPase activating function of LRS, while not affecting the catalytic activity. For further analysis, we selected one compound, BC-LI-0186, which binds to the RagD interacting site of LRS, thereby inhibiting lysosomal localization of LRS and mTORC1 activity. It also effectively suppressed the activity of cancer-associated MTOR mutants and the growth of rapamycin-resistant cancer cells. These findings suggest new strategies for controlling tumor growth that avoid the resistance to existing mTOR inhibitors resulting from cancer-associated MTOR mutations.Leucyl-tRNA synthetase (LRS) is a leucine sensor of the mTORC1 pathway. Here, the authors identify inhibitors of the GTPase activating function of LRS, not affecting its catalytic activity, and demonstrate that the leucine sensor function of LRS can be a new target for mTORC1 inhibition.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Biomolecular and Physical Sciences, Science, Environment, Engineering and Technology, When cells are exposed to oxidative stress reactive oxygen species (ROS) are formed, causing severe damage in the cell. To maintain the redox homeostasis, antioxidant systems are switched on. Two of the major redox proteins include thioredoxin (Trx) and Methionine Sulfoxide Reductase (MSR). Trx interacts directly and indirectly with several transcription factors to modulate binding to gene promoters. In particular, Nrf2 is known to be an important transcription factor in redox regulation. To further define the mechanisms by which the Trx promoter is regulated by oxidative stress, the HEC1B endometrial cancer cell line and MDA-­‐MB-­‐231 breast cancer cell line were transfected with various Trx gene promoter constructs that drive expression of a luciferase reporter. When cells were transfected with constructs containing the Trx gene promoter region where the Antioxidant Responsive Element (ARE) was deleted, no induction was observed in response to tBHQ, diamide or H2O2 utilising luciferase reporter activity assays. Thus, the results confirmed the published data that the ARE is one of the most important elements in activating the Trx gene promoter during oxidative stress. Furthermore, it was established that luciferase reporter assays using the same cell line grown in media supplemented with different serum yielded significantly different results. Cells grown in medium supplemented with the serum containing the highest selenium level resulted in the lowest fold induction of the Trx gene promoter, while sera with the lowest level resulted in the highest folld inductions.