Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Nuclear translocation of an aminoacyl-tRNA synthetase may mediate a chronic "integrated stress response".

Various stress conditions are signaled through phosphorylation of translation initiation factor eukaryotic initiation factor 2α (eIF2α) to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. Here, we report that tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family that responds to diverse stress conditions through cytosol-nucleus translocation to activate stress-response genes, also inhibits global translation. However, it occurs at a later stage than eIF2α/ATF4 and mammalian target of rapamycin (mTOR) responses. Excluding TyrRS from the nucleus over-activates translation and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. We propose that TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and strategically located nuclear localization signal and integrate them by nucleus translocation to effect protective responses against chronic stress.

Mechanistic perspectives on anti-aminoacyl-tRNA synthetase syndrome.

Antisynthetase syndrome (ASSD) is an autoimmune disease characterized by circulating autoantibodies against one of eight aminoacyl-tRNA synthetases (aaRSs). Although these autoantibodies are believed to play critical roles in ASSD pathogenesis, the nature of their roles remains unclear. Here we describe ASSD pathogenesis and discuss ASSD-linked aaRSs - from the WHEP domain that may impart immunogenicity to the role of tRNA in eliciting the innate immune response and the secretion of aaRSs from cells. Through these explorations, we propose that ASSD pathogenesis involves the tissue-specific secretion of aaRSs and that extracellular tRNAs or tRNA fragments and their ability to engage Toll-like receptor signaling may be important disease factors.

Inflammatory platelet production stimulated by tyrosyl-tRNA synthetase mimicking viral infection.

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

Tissue Modification in Nonsurgical Facelift Options.

Nonsurgical facelifts are a term for a heterogeneous group of procedures used by physicians to improve facial rejuvenation without the use of operative techniques. Patients demand these services due to the reduced recovery time and generally lower risk. However, nonsurgical techniques, to be effective, must induce conformational change in the cells and tissues of the face. Therefore, these techniques are significant procedures that have associated risks. Understanding the tissue modifications and mechanisms of action of these techniques is vital to their safe and effective use. The purpose of this article is to provide a background of tissue modification in nonsurgical facelift options.

Extracellular tyrosyl-tRNA synthetase cleaved by plasma proteinases and stored in platelet α-granules: Potential role in monocyte activation.

BACKGROUND: Tyrosyl-tRNA synthetase (YRS) belongs to the family of enzymes that catalyzes the tRNA aminoacylation reaction for protein synthesis, and it has been recently shown to exert noncanonical functions. Although database results indicate extremely low levels of YRS mRNA in platelets, YRS protein is abundantly present. The source of YRS in platelets, as well as the physiological role of platelet-stored YRS, remains largely unknown. OBJECTIVES: To clarify how YRS accumulates in platelets and determine the potential role of platelet-stored YRS. METHODS: Recombinant YRS proteins with epitope tags were prepared and tested in vitro for proteolytic cleavage in human plasma. Fluorescent-labeled YRS was examined for uptake by platelets, as demonstrated by western blotting and confocal microscopy analysis. Using RAW-Dual reporter cells, Toll-like receptor and type I interferon activation pathways were analyzed after treatment with YRS. RESULTS: Full-length YRS was cleaved by both elastase and matrix metalloproteinases in the plasma. The cleaved, N-terminal YRS fragment corresponds to the endogenous YRS detected in platelet lysate by western blotting. Both full-length and cleaved forms of YRS were taken up by platelets in vitro and stored in the α-granules. The N-terminal YRS fragment generated by proteolytic cleavage had monocyte activation comparable to that of the constitutive-active mutant YRS (YRSY341A) previously reported. CONCLUSION: Platelets take up both full-length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N-terminal YRS fragment stored in α-granules may have potential to activate monocytes.

Tyrosyl-tRNA synthetase stimulates thrombopoietin-independent hematopoiesis accelerating recovery from thrombocytopenia.

New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.

A simple cost-effective method of microsurgical simulation training: the turkey wing model.

The rat femoral artery (RFA) anastomosis model has been the gold standard in microsurgical simulation training. While effective, live animal use requires animal use committee regulation and costly maintenance. Our institution's animal laboratory is remote to the hospital, limiting access by our busy surgical residents with their limited duty hours. We present an alternative convenient, cost-effective model. Ten frozen turkey wings were divided into distal and proximal segments. Vessel diameter, length, and anastomosis perfusion were assessed. Proximal brachial arteries ("humeral" segments) measured 8.85 ± 1.14 cm long with diameter 1.69 ± 0.27 mm. Distal brachial arteries ("forearm") measured 10.5 ± 2.06 cm long with diameter 1.25 ± 0.25 mm. An 8-lb box (~20 wings) cost $13.76. Separate use of the segments provides two training sessions with $0.35 per session effective cost. Our average cost for RFA microsurgical training sessions was $120 dollars for a single rat 2-hour session and $66 per rat if a maximum crate load of six rats was used. Besides significant cost, not all training programs are equipped to house, care for, and use rats in microsurgical training. We now use turkey wings for microvascular training. They are cheap, abundant, readily accessible for training, and consistent with tissue quality and vessel size approximating human systems.

Structural switch of lysyl-tRNA synthetase between translation and transcription.

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

Structural context for mobilization of a human tRNA synthetase from its cytoplasmic complex.

Human lysyl-tRNA synthetase is bound to the multi-tRNA synthetase complex (MSC) that maintains and regulates the aminoacylation and nuclear functions of LysRS. The p38 scaffold protein binds LysRS to the MSC and, only with the appropriate cue, mobilizes LysRS for redirection to the nucleus to interact with the microphthalmia associated transcription factor (MITF). In recent work, an (α(2))(2) LysRS tetramer crystallized to yield a high-resolution structure and raised the question of how LysRS is arranged (dimer or tetramer) in the MSC to interact with p38. To understand the structural organization of the LysRS-p38 complex that regulates LysRS mobilization, we investigated the complex by use of small angle X-ray scattering and hydrogen-deuterium exchange with mass spectrometry in solution. The structure revealed a surprising α(2)ß(1):ß(1)α(2) organization in which a dimeric p38 scaffold holds two LysRS α(2) dimers in a parallel configuration. Each of the N-terminal 48 residues of p38 binds one LysRS dimer and, in so doing, brings two copies of the LysRS dimer into the MSC. The results suggest that this unique geometry, which reconfigures the LysRS tetramer from α(2):α(2) to α(2)ß(1):ß(1)α(2), is designed to control both retention and mobilization of LysRS from the MSC.

Packaging HIV virion components through dynamic equilibria of a human tRNA synthetase.

Aminoacyl tRNA synthetases, components of the translation apparatus, have alternative functions outside of translation. The structural and mechanistic basis of these alternative functions is of great interest. As an example, reverse transcription of the HIV genome is primed by a human lysine-specific tRNA (tRNA(Lys3)) that is packaged (into the virion) by the HIV Gag protein with lysyl-tRNA synthetase (LysRS). Not understood is the structural basis for simultaneous packaging of tRNA(Lys3), LysRS, and Gag. Here, ab initio computational methods, together with our recent high-resolution 3-D structure of human LysRS, produced an energy-minimized model where Gag, tRNA(Lys), and LysRS form a ternary complex. Interestingly, the model requires normally homodimeric LysRS to dissociate into a monomer that bridges between Gag and tRNA(Lys3). Earlier experiments of others and new experiments presented here, which tested an engineered dissociated form of LysRS, were consistent with the ab initio "bridging monomer" model. The results support an emerging theme that alterative functions of tRNA synthetases may come, in part, from protein surfaces exposed by dynamic equilibria.

Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase.

Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an alpha-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered alpha-helix did not form a leucine zipper that interlocked with the same alpha-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization.

Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma.

Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

The C-Ala domain brings together editing and aminoacylation functions on one tRNA.

Protein synthesis involves the accurate attachment of amino acids to their matching transfer RNA (tRNA) molecules. Mistranslating the amino acids serine or glycine for alanine is prevented by the function of independent but collaborative aminoacylation and editing domains of alanyl-tRNA synthetases (AlaRSs). We show that the C-Ala domain plays a key role in AlaRS function. The C-Ala domain is universally tethered to the editing domain both in AlaRS and in many homologous free-standing editing proteins. Crystal structure and functional analyses showed that C-Ala forms an ancient single-stranded nucleic acid binding motif that promotes cooperative binding of both aminoacylation and editing domains to tRNA(Ala). In addition, C-Ala may have played an essential role in the evolution of AlaRSs by coupling aminoacylation to editing to prevent mistranslation.