Tyrosyl-transfer RNA Synthetase Research Articles

Low-Dose Trans-Resveratrol Ameliorates Diabetes-Induced Retinal Ganglion Cell Degeneration via TyrRS/c-Jun Pathway.

The purpose of this study was to investigate the protective effect of low-dose trans-resveratrol (trans-RSV) on diabetes-induced retinal ganglion cell (RGC) degeneration and its possible mechanism. A streptozotocin-induced diabetic mouse model was established and treated with or without trans-RSV intragastric administration (10 mg/kg body weight/day) for 12 weeks. Oscillatory potentials (Ops) of the dark-adapted electroretinogram (ERG) were recorded. The number of RGCs was detected by Tuj1 and TUNEL staining. The apoptosis markers in the retina were analyzed by Western blot. The cross sections of optic nerves were observed by transmission electron microscopy. In addition, mouse neuroblastoma N2a cells were injured by high-glucose (HG) treatment. Cell viability and apoptosis were measured with or without low-dose trans-RSV treatment. The intracellular localization of tyrosyl transfer-RNA synthetase (TyrRS) was observed in both mouse retinas and N2a cells. The effects of low-dose trans-RSV on the binding of TyrRS to the transcription factor c-Jun and the binding of c-Jun to pro-apoptotic genes were analyzed by co-IP and ChIP assays in HEK 293 cells. Trans-RSV relieved electrophysiological injury of retinas and inhibited RGC apoptosis in diabetic mice. It also protected N2a cells from HG-induced apoptosis. Additionally, it promoted TyrRS nuclear translocation in both diabetic mouse retinas and HG-treated N2a cells. Trans-RSV promoted TyrRS binding to c-Jun, inhibited the phosphorylation of Ser-63 of c-Jun, and downregulated pro-apoptotic gene transcription. Low-dose trans-RSV can ameliorate diabetes-induced RGC degeneration via the TyrRS/c-Jun pathway. It can promote TyrRS nuclear translocation and bind to c-Jun, downregulating c-Jun phosphorylation and downstream pro-apoptotic genes.

Resveratrol attenuates oxidative injury in human umbilical vein endothelial cells through regulating mitochondrial fusion via TyrRS-PARP1 pathway

Background/aimsOxidative stress-induced damage in endothelial cells is a crucial initiator of atherosclerosis (AS), which is highly related to excessive reactive oxygen species (ROS) and mitochondrial dynamics. Resveratrol (RSV) exerts beneficial effects against endothelial oxidative injury, while the underlying mechanisms have not been fully elucidated. Thus, we aimed to explore the role of mitochondria dynamics during the anti-oxidative activities of RSV in palmitic acid (PA)-stimulated human umbilical vein endothelial cells (HUVECs) and to verify whether tyrosyl transfer- RNA synthetase (TyrRS) and poly (ADP-ribose) polymerase 1 (PARP1) are targeted during this process.MethodsHUVECs were exposed to 200 μM of PA for 16 h before treated with 10 μM of RSV for 8 h. Cell viability was detected using Cell counting kit-8 (CCK-8) assay. The intracellular ROS level and mitochondria membrane potential (MMP) were measured using microplate reader and flow cytometry. The malondialdehyde and superoxide dismutase were measured using the microplate reader. The mitochondrial morphology and fusion process was observed under transmission electron microscopy and confocal microscopy. TyrRS and PARP1 were knocked down with the specific small interference RNAs (siRNA), and the protein expressions of TyrRS, PARP1, and mitochondrial fusion proteins (MFN1, MFN2, and OPA1) were measured by western blot.ResultsRSV treatment suppressed the PA-induced injuries in HUVECs, including the damage to cell viability, oxidative stress, and loss of MMP. Additionally, RSV improved the protein levels of MFN1, MFN2, and OPA1 as well as inhibited the PA-induced fragmentation of mitochondria. However, the effects of RSV on oxidative stress and mitochondrial fusion were abolished by the pretreatment of siRNAs of TyrRS and PARP1, indicating that these effects of RSV were dependent on the TyrRS-PARP1 pathway.ConclusionsRSV attenuated endothelial oxidative injury by regulating mitochondrial fusion via TyrRS-PARP1 signaling pathway.

PARP1 911

Although resveratrol is thought to provide many beneficial health effects, its cellular targets and mechanism of function are still under investigation. A new study found that resveratrol binds to tyrosyl transfer-RNA synthetase, resulting in the activation of the stress response effector PARP1.

Arrest, Migration, Survival and Proliferation of Leukocytes and Vascular Cells: The Many Faces of Chemokine Biology

This issue of Microcirculation explores the role of chemokines, a family of small, secreted peptides that regulate leukocyte traffic in health and disease. Leading experts review the diverse function of these mediators, with an emphasis on their roles in microvascular physiology and pathophysiology. The most amazing aspect of this field of research is the diversity of functions that have been attributed to chemokines. Chemokines are classified according to their amino acid sequence as CC (no intervening amino acid between first two cysteinyl residues), CXC (one intervening amino acid), CX3C (three intervening amino acids), and XC (only one cysteinyl residue). They are numbered as CCL1–27, CXCL1–16, CX3CL1, and XCL1 (10,11,15). They bind to at least 11 CC receptors (CCR1–11), 6 CXC receptors (CXCR1–6), CX3CR1, and XCR1 (10,11,15). Earlier notions that CXC chemokines attract granulocytes and CC receptors are responsible for mononuclear cell effects have been supplanted by more recent findings. Chemokines may have emerged early in evolution from proteolytic fragments of tyrosyl transfer RNA synthetase, an essential intracellular enzyme, even before a circulatory system developed (15,16). In complex mammalian organisms, chemokines not only regulate leukocyte development and chemotaxis, but also play important roles in their trafficking through the vascular and lymphatic systems. Some, but not all chemokines can be immobilized on the endothelial surface and can activate integrin adhesion receptors on rolling leukocytes to promote their arrest. Two reviews in this issue (1,8) focus on the function of arrest chemokines, presenting different perspectives on how chemokine receptor signaling may be integrated with other signals in phagocytes (8) and how chemokine receptor engagement in lymphocytes will promote local integrin avidity upregulation in a fraction of a second (1). One of the first chemokines discovered was CCL2 (monocyte chemoattractant protein-1), and it may

Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?

The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the α+β type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.

Disordered C-terminal Domain of Tyrosyl Transfer-RNA Synthetase: Evidence for a Folded State

The C-terminal domain (residues 320 to 419) of tyrosyl-tRNA synthetase from Bacillus stearothermophilus(Bst-TyrRS) is necessary for the binding of tRNA Tyrbut disordered in the crystal structure. Four different criteria showed that the isolated C-terminal domain of Bst-TyrRS was at least partially folded in solution. Its spectrum of circular dichroism was compatible with a high content of secondary structure elements (56% of its residues) and these structural elements disappeared in 7.5 M urea. It was unfolded by urea along a unique transition, around 6.0 M, according to the variations in the fluorescence of its tyrosine residues and in its electrophoretic mobility through transverse gradient gels. It was denatured by heat with a temperature of half-precipitation in 30 minutes that was equal to 67.9°C and close to the Bst-TyrRS one, 68.7°C. Its transitions of denaturation by urea or temperature were weakly cooperative. The C-terminal domains of the TyrRSs from Escherichia coli(Eco-TyrRS) and B. stearothermophiluscould be genetically exchanged without a significant loss of aminoacylation activity. A hybrid between the N-terminal domain of Bst-TyrRS and the C-terminal domain of Eco-TyrRS was precipitated by heat in 30 minutes following two transitions: 83% of the molecules were precipitated with a temperature of half-transition (51.6°C) close to the Eco-TyrRS one (48.6°C). The remainder was precipitated with a temperature of half-transition (65.5°C) close to the Bst-TyrRS one (67.2°C) or that of its N-terminal domain (68.0°C). These results showed that the C-terminal domain of Eco-TyrRS could undergo a transition from a soluble active conformation to an insoluble one. The denaturations of Bst-TyrRS and of its N-terminal domain by urea occurred with two successive transitions, around 4 M and 6 M, and thus according to a complex mechanism.

A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme.

Group I introns are highly structured RNAs which catalyse their own splicing by guanosine-initiated transesterification reactions. Their catalytic core is generally stabilized by RNA-RNA interactions within the core and with peripheral RNA structures. Additionally, some group I introns require proteins for efficient splicing in vivo. The Neurospora CYT-18 protein, the mitochondrial tyrosyl-transfer RNA synthetase (mt TyrRS), promotes splicing of the Neurospora mitochondrial large ribosomal RNA (LSU) and other group I introns by stabilizing the catalytically active structure of the intron core. We report here that CYT-18 functions similarly to a peripheral RNA structure, P5abc, that stabilizes the catalytic core of the Tetrahymena LSU intron. The CYT-18 protein and P5abc RNA bind to overlapping sites in the intron core, inducing similar conformational changes correlated with splicing activity. Our results show that a protein can play the role of an RNA structure in a catalytic RNA, a substitution postulated for the evolution of nuclear pre-messenger RNA introns from self-splicing introns.

Structure of a mutant of tyrosyl-tRNA synthetase with enhanced catalytic properties.

One surprising outcome of applying the techniques of protein engineering to the enzyme tyrosyl-transfer RNA synthetase was that the enzyme's activity could actually be increased by a specific sequence change. In particular, altering residue threonine 51 to a proline (mutant TP51) increased the enzyme's affinity for tyrosyl adenylate complexes. The non-additive effect of combining the TP51 mutation with a second sequence alteration (histidine 48 to a glycine) suggested that the effect of the TP51 change might be mediated by a structural change involving the peptide backbone. To address the question of the mechanism by which the TP51 change increases the activity of tyrosyl-tRNA synthetase we have determined the structure of the mutant enzyme. We find the change has a purely local effect on the structure of the enzyme, and conclude that the increased activity of the TP51 mutant probably results from the replacement of the polar threonine residue by a non-polar group: in the wild-type enzyme substrate binding is disfavoured by the displacement of solvent from the vicinity of threonine 51. This unfavourable effect is absent in the TP51 mutant.

Hydrogen bonding in enzymatic catalysis analysed by protein engineering

Two historic publications have suggested that enzymes can utilize the binding energy with their substrates to increase the rate of chemical catalysis—a concept termed strain1,2. Specifically, the structure of an enzyme was described as complementary to the structure of the transition state of its substrate rather than to that of the unreacted substrate, so that the substrate would tend to be distorted on binding to the enzyme. The current understanding of strain does not require such distortion. Instead, it is thought that there are groups on the enzyme which interact better with the transition state than with the unaltered substrate, an arrangement known as differential binding of substrates and transition states3. The improvement in binding energy caused by going from substrate to transition state lowers the activation energy of the reaction and so increases the catalytic rate. Here we report direct evidence in support of this notion. Rapid reaction kinetics on mutants of a tyrosyl-transfer RNA synthetase produced by protein engineering show that certain hydrogen-bonding side chains some distance from the reacting bonds of the substrate bind to ATP preferentially in the transition state of the reaction rather than in the unreacted state. In this way, the binding energies of the interactions are used primarily to lower the apparent activation energy of the chemical steps rather than to enhance binding.

Binding of tyrosine, adenosine triphosphate and analogues to crystalline tyrosyl transfer RNA synthetase

Crystalline complexes of tyrosyl tRNA synthetase were prepared with the following substrates and substrate analogues: ATP, AMP, α-β methylene ATP, tyrosine and tyrosinyl adenylate. Using 14C-labelled ligands, the binding constants for tyrosine and ATP to crystals were shown to be similar to those observed in solution. Two tyrosine molecules were found to bind to the symmetrical dimer in the crystalline enzyme, while only one tyrosine binds with high affinity in solution. Electron density difference maps show that tyrosine and the AMP derivatives all bind at the same site, in a cleft 10 Å deep at one side of the pleated sheet, tyrosine binding over 100 times more strongly. The phosphate groups of AMP and ATP are not unambiguously observed in the difference electron density maps. Tyrosinyl adenylate is clearly delineated in the electron density difference map, with the tyrosyl side-chain occupying the site previously observed. The adenosine group is in a wide cup-like depression outside the pocket, lying between the carboxyl-terminal continuations of strands 3 and 5 of the pleated sheet. The adenine ring is lying against an α-helix. The binding of tyrosinyl adenylate causes no detectable conformational changes of the enzyme.

The crystal structure of tyrosyl-transfer RNA synthetase at 2.7 Å resolution

Tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus has been crystallized as hexagonal plates, P3 121, a = b = 64.6 A ̊ , c = 238.8 A ̊ , with the dimeric molecule (molecular weight, 90,000) occupying two crystallographic asymmetric units (Reid et al., 1973). Three heavy-atom derivatives have been identified and X-ray diffraction measurements have been made to 2.7 Å resolution, using the oscillation method. The three heavy-atom derivatives were methyl mercury (two sites, half occupied, 3 Å apart), uranyl acetate (single fully occupied site) and chloroplatinite PtCl 4 2− (three sites of differing occupancy). The results were used to compute an electron density map at 2.7 Å resolution, which shows the monomer as a unit of about 60 Å × 60 Å × 40 Å. The maximum dimension of the dimer is about 130 Å. Most of the polypeptide chain has been traced uniquely. It includes five α-helices more than 12 Å long and several shorter helices. A six-stranded pleated-sheet structure lies in the centre of each subunit. The catalytic site of the enzyme is believed to be adjacent to the mercury-binding group.

Crystallization and preliminary X-ray diffraction studies on tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus

Conditions have been established for the crystallization of tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus at room temperature. The crystals are extremely well-ordered, exhibiting diffraction spots out to at least 2.7 Å, and can be grown to a convenient size for X-ray crystallographic analysis. The crystals are trigonal with a space group P3 121, the unit cell having dimensions of a = 64.4 A ̊ and c = 238 A ̊ ; the crystallographic asymmetric unit is probably one subunit of the dimeric (2 × 45,000, mol. wt) enzyme. The enzyme crystals are extremely stable and exhibit good resistance to radiation damage. This amino-acyl-tRNA synthetase appears to be amenable to complete structure determination by X-ray crystallography.