LysRS Serves as a Key Signaling Molecule in the Immune Response by Regulating Gene Expression
ABSTRACT Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.
FEBS Letters 11/2014; 588(23):4478-4486. DOI:10.1016/j.febslet.2014.10.019 · 3.34 Impact Factor
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ABSTRACT: Background: One of the activities of histidine triad nucleotide-binding protein 1 (Hint1) under in vitro conditions is the conversion of nucleoside 5'-O-phosphorothioate (NMPS) to its 5'-O-phosphate (NMP), which is accompanied by the release of hydrogen sulfide. Methods: Non-hydrolyzable derivatives of AMPS and dCMPS, each containing the residue able to form a covalent bond in nucleic acid-protein complexes via photocrosslinking (at 308 nm), were applied at the complexing experiments with recombinant and cellular Hint1. The cellular lysates prepared after RNAi-mediated knockdown of Hint1 were incubated with AMPS and the level of desulfuration was measured. Results: Recombinant Hint1 and Hint1 present in the cellular lysate of A549 cells, formed complexes with the used substrate analogs. Computer modeling experiments, in which the ligand was docked at the binding pocket, confirmed that direct interactions between Hint1 and the screened analogs are possible. Using RNAi technology, we demonstrated lowered levels of AMPS substrate desulfuration in reactions that employed the cell lysates with a reduced Hint1 level. Conclusions: The enzymatic conversion of AMPS to AMP occurred with the participation of cellular Hint1, the protein, which is present in all organisms. General significance: The intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5'-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H2S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H2S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.Biochimica et Biophysica Acta (BBA) - General Subjects 09/2014; 1840(12). DOI:10.1016/j.bbagen.2014.08.016 · 3.83 Impact Factor
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ABSTRACT: Aim. Analyze the interaction between Lysyl-tRNA synthetase (LysRS) and HIV-1 GagPol to know whether a particular N-terminal sequence of mitochondrial LysRS triggers a specific recognition with GagPol. Methods. Yeast two-hybrid analysis, immunoprecipitation. Results. We have shown that LysRS associates with the Pol domain of GagPol. Conclusions. A model of the assembly of the LysRS:tRNA3Lys:GagPol packaging complex is proposed.Biopolymers and Cell 09/2011; 27(5):381-2. DOI:10.7124/bc.000128