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Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial trna^(Lys)

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Science China(Life Sciences) 2020年第8期63卷 1227-1239页

作者： Yong Wang Jing-Bo Zhou Qi-Yu Zeng Siqi Wu Mei-Qin Xue Pengfei Fang En-Duo Wang Xiao-Long Zhou State Key Laboratory of Molecular Biology CAS Center for Excellence in Molecular Cell ScienceShanghai Institute of Biochemistry and Cell BiologyChinese Academy of SciencesUniversity of Chinese Academy of SciencesShanghai 200031China School of Life Science and Technology ShanghaiTech UniversityShanghai 201210China State Key Laboratory of Bioorganic and Natural Products Chemistry Center for Excellence in Molecular SynthesisShanghai Institute of Organic ChemistryChinese Academy of SciencesShanghai 200032China

aminoacyl-trna synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-trnas for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes both cytosolic and mitochondrial lysyl-trna synthetases(LysRSs).Previously,two mutations(c.1129 G>A and c.517 T>C)were identified that led to hearing impairment;however,the underlying biochemical mechanism is unclear.In the present study,we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol,but affect the cytosolic LysRS level,its tertiary structure,and cytosolic trna aminoacylation in vitro.As for mitochondrial translation,the two mutations have little effect on the steady-state level,mitochondrial targeting,and trna binding affinity of mitochondrial LysRS.However,they exhibit striking differences in charging mitochondrial trnaLys,with the c.517T>C mutant being completely deficient in vitro and in vivo.We constructed two yeast genetic models,which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels.Overall,our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.

关键词： aminoacyl-trna synthetase trna aminoacylation protein synthesis

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Dual Targeting to Mitochondria and Chloroplasts： Characterization of Thr-trna synthetase Targeting Peptide

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Molecular Plant 2009年第6期2卷 1298-1309页

作者： Anna-Karin Berglund Erika Spanning Henrik Biverstahl Gianluca Maddalo Christian Tellgren-Roth Lena Maler Elzbieta Glaser Department of Biochemistry and Biophysics Arrhenius Laboratories for Natural Science Stockholm University SF-10691 Stockholm Sweden Department of Genetics and Pathology Rudbecklaboratoriet Uppsala University SE-751 85 Uppsala Sweden

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-trna synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

关键词： Dual targeting mitochondria chloroplast targeting peptide aminoacyl-trna synthetase

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The Role of Anionic Protein Residues on the Salt Dependence of the Binding of aminoacyl-trna synthetases to trna:A Poisson-Boltzmann Analysis

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Communications in Computational Physics 2008年第5期3卷 1051-1070页

作者： Johan H.Bredenberg Alexander H.Boschitsch Marcia O.Fenley Institute of Molecular Biophysics Florida State UniversityTallahasseeFL 32306USA. Continuum-Dynamics Inc. 34 Lexington AvenueEwingNJ 08618USA. Department of Physics Florida State UniversityTallahasseeFL 32306USA.

Long-range electrostatic interactions in proteins/peptides associating to nucleic acids are reflected in the salt-dependence of the binding process.According to the oligocationic binding model,which is based on counterion condensation theory,only the cationic residues of peptides/proteins near the binding interface are assumed to affect the salt dependence in the association of peptides and proteins to nucleic acids.This model has been used to interpret and predict the binding of oligocationic chains-such as oligoarginines/lysines-to nucleic acids,and does an excellent job in these kinds of systems.This simple relationship,which is used to compare or count the number of ionic interactions in protein-nucleic acid complexes,does not hold when acidic residues,i.***.glutamate and aspartate,are incorporated in the protein matrix.Here,we report a combined molecular mechanics(by means of energy-minimization of the structure under the influence of an empirical energy function)and Poisson-Boltzmann(PB)study on the salt-dependence in binding to trna of two important enzymes that are involved in the seminal step of peptide formation in the ribosome:Glutamine synthetase(GluRS)and Glutaminyl synthetase(GlnRS)bound to their cognate trna.These two proteins are anionic and contain a significant number of acidic residues distributed over the entire protein.Some of these residues are located in the binding interface to trna.We computed the salt-dependence in association,SKpred,of these enzyme-trna complexes using both the linear and nonlinear solution to the PoissonBoltzmann Equation(PBE).Our findings demonstrate that the SKpred obtained with the nonlinear PBE is in good agreement with the experimental SKobs,while use of the linear PBE resulted in the SKpred being anomalous.We conclude that electrostatic interactions between the binding partners in these systems are less favorable by means of charge-charge repulsion between negatively charged protein residues and phosphateoxygens in the trna backbone but also play a significant role

关键词： Enzyme electrostatics molecular mechanics Poisson-Boltzmann equation oligocation salt-dependence aminoacyl-trna synthetase polyelectrolyte counterion condensation theory

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Does transfer ribonucleic acid act as a ribozyme?

Does transfer ribonucleic acid act as a ribozyme?

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第九届全国酶学学术讨论会暨邹承鲁诞辰85周年纪念会

作者： Wang-Yi Liu Institute of Biochemistry and Cell Biology,the Chinese Academy of Scineces,320 Yue-yung Road,Shanghai 200031

In modem cell,protein biosynthesis starts from aminoacyl-trna formation that results from two successive reactions in which three substrates:amino-acid,ATP and trna react to yield three products:aminoacyl-trna,AMP and... 详细信息

关键词： aminoacyl-trna synthetase ribozyme trna

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