Summary Selective neuronal loss is a hallmark of neurodegenerative diseases, which counter-intuitively are often caused by mutations in widely-expressed genes1. Charcot-Marie-Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies2,3. A subtype of the diseases—CMT2D—is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-tRNA synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons leading to deficits in distal motor function4. How mutations in GlyRS (GlyRSCMT2D) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRSCMT2D acquires a neomorphic binding activity that directly antagonizes an essential signaling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRSCMT2D to bind the Neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRSCMT2D that antagonizes the VEGF/Nrp1 interaction, and indicate the VEGF/Nrp1 signaling axis is an actionable target for treating CMT2D.

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.A s ancient proteins that arose as part of the development of the genetic code, aminoacyl-tRNA synthetases (AARSs) are essential components of the translation apparatus. The 20 enzymes, 1 for each amino acid, catalyze the attachment of each amino acid to its cognate tRNA in the cytoplasm, where the charged tRNAs are then used for ribosomal protein synthesis (1). Surprisingly, ex-translational functions have been discovered for many tRNA synthetases, including gene regulation in Escherichia coli, RNA splicing in mitochondria of Neurospora crassa (2), and a diverse variety of functions in vertebrates that include among others regulation of inflammatory responses and of angiogenesis (3). These expanded functions are associated with the accretive additions of specialized motifs and domainssuch as internal short sequence motifs and appended GST, leucine zipper, and helix-turn-helix domains (4). The specialized motif and domain additions facilitate new protein-protein interactions that confer novel functions. Some of the many disease connections to AARSs, and to proteins that are part of the multi-tRNA synthetase complex in mammalian cells, are thought to result from disruptions to, or alterations of, their ex-translational functions (5, 6). Indeed, there are dominant CharcotMarie-Tooth disease-causing mutations in tyrosyl-and glycyltRNA synthetases that do not disrupt aminoacylation activity (7,8).Also surprising for essential components of the translation apparatus was the observation that specific fragments (produced by alternative splicing or natural proteolysis) of tyrosyl-and tryptophanyl-tRNA synthetases (YRS and WRS) bind to and signal through extracellular receptors, including chemokine receptor 1 and 2 (CXCR1 and -2) on polymorphonuclear leukocyte (PMN) cells (YRS) (9) and vascular endothelial (VE)-cadherin on endothelial cells (WRS) (10). These two synthetases are secreted from mammalian cells under specific conditions that potentiate their ex-translational functions (11, 12). Collectively, these observations raised th...

The question of how dispersed mutations in one protein engender the same gain-of-function phenotype is of great interest. Here we focus on mutations in glycyl-tRNA synthetase (GlyRS) that cause an axonal form of Charcot-Marie-Tooth (CMT) diseases, the most common hereditary peripheral neuropathies. Because the disease phenotype is dominant, and not correlated with defects in the role of GlyRS in protein synthesis, the mutant proteins are considered to be neomorphs that gain new functions from altered protein structure. Given that previous crystal structures showed little conformational difference between dimeric wild-type and CMT-causing mutant GlyRSs, the mutant proteins were investigated in solution by hydrogen-deuterium exchange (monitored by mass spectrometry) and small-angle X-ray scattering to uncover structural changes that could be suppressed by crystal packing interactions. Significantly, each of five spatially dispersed mutations induced the same conformational opening of a consensus area that is mostly buried in the wild-type protein. The identified neomorphic surface is thus a candidate for making CMT-associated pathological interactions, and a target for disease correction. Additional result showed that a helix-turn-helix WHEP domain that was appended to GlyRS in metazoans can regulate the neomorphic structural change, and that the gain of function of the CMT mutants might be due to the loss of function of the WHEP domain as a regulator.Overall, the results demonstrate how spatially dispersed and seemingly unrelated mutations can perpetrate the same localized effect on a protein.aminoacyl tRNA synthetase | hereditary motor and sensory neuropathy | dimer formation

Fidelity of translation, which is predominately dictated by the accuracy of aminoacyl-tRNA synthetases in pairing amino acids with correct tRNAs, is of central importance in biology. Yet, deliberate modifications of translational fidelity can be beneficial. Here we found human and not E. coli AlaRS has an intrinsic capacity for mis-pairing alanine onto non-alanyl-tRNAs including tRNACys. Consistently, a cysteine-to-alanine substitution was found in a reporter protein expressed in human cells. All human AlaRS-mischarged tRNAs have a G4:U69 base pair in the acceptor stem. The base pair is required for the mischarging. By solving the crystal structure of human AlaRS and comparing it to that of E. coli AlaRS, we identified a key sequence divergence between eukaryotes and bacteria that influences mischarging. Thus, the expanded tRNA specificity of AlaRS appears to be an evolutionary gain-of-function to provide posttranscriptional alanine substitutions in eukaryotic proteins for potential regulations.

Although great success has been achieved in asymmetric fluoroalkylation reactions via nucleophilic or electrophilic processes, the development of asymmetric radical versions of this type of reactions remains a formidable challenge because of the involvement of highly reactive radical species. Here we report a catalytic asymmetric radical aminoperfluoroalkylation and aminodifluoromethylation of alkenes with commercially available fluoroalkylsulfonyl chlorides as the radical sources, providing a versatile platform to access four types of enantioenriched α-tertiary pyrrolidines bearing β-perfluorobutanyl, trifluoromethyl, difluoroacetyl and even difluoromethyl groups in excellent yields and with excellent enantioselectivity. The key to success is not only the introduction of the CuBr/chiral phosphoric acid dual-catalytic system but also the use of silver carbonate to suppress strong background and side hydroamination reactions caused by a stoichiometric amount of the in situ generated HCl. Broad substrate scope, excellent functional group tolerance and versatile functionalization of the products make this approach very practical and attractive.