Structural and functional investigation of mammalian tyrosyl-tRNA synthetase

Kornelyuk, A. I.

doi:10.7124/bc.0004df

Cited by 20 publications

(18 citation statements)

References 34 publications

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“…It can be the cause the development of type 2 diabetes, impaired reproductive system functions, cardiovascular, and is accompanied by complications such as chronic kidney disease. Kidneys, like the cardiovascular system, are the main target organs, which sensitive to obesity, because some of the first take on the metabolism correction function during increasing excess of adipose tissue in the body [2].…”

Section: Out the Catalytic Function Of Binding The Amino Acid To Trnamentioning

confidence: 99%

“…Аміноацил-тРНК синтетаза (АРСаза) є одним із основних ферментів білкового синтезу. На дорибосомному етапі трансляції у високоспецифічній енергозалежній реакції синтетаза каталізує активацію амінокислоти та приєднання її до гомологічної транспортної РНК, здійснюючи таким чином першу стадію декодування інформації про структуру білка, закладенну в нуклеотидній послідовності ДНК та РНК [1,2].…”

unclassified

“…Тирозил-тРНК синтетаза Bos taurus складається із двох структурних модулів, N-кінцевого каталітичного (відповідає укороченій формі фермента міні BtTyrRS, 342 а.з., 39 кДа) та С-кінцевого цитокіноподібного (166 а.з., 20 кДа) [2]. У повнорозмірній BtTyrRS (528 а.з., 59.2 кДа) N-кінцевий модуль здійснює каталітичну функцію ковалентного зв'язування відповідної амінокислоти із тРНК, тоді як С-модуль корегує та стабілізує розміщення тРНК в активному центрі фермента [1,2]. Після розщеплення тирозил-тРНК синтетази еластазою на міні BtTyrRS та С-модуль останні виявляють цитокінові властивості [3][4][5].…”

unclassified

“…У фізіологічних умовах тирозил-тРНК синтетаза Bos taurus являє собою гомодимер α2 типу, мономером якого є повнорозмірний фермент. При виділенні BtTyrRS із печінки бика було показано, що поряд із основною фо-рмою виділяється також і функціонально активна протеолітично модифікована форма тирозил-тРНК синтетази з молекулярною вагою 39 кДа, яка має повну ферментативну активність в експериментах in vitro [2,[6][7].…”

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Isolation and characterization of the mutant N-terminal catalytical module of the B. taurus Tyrosyl-tRNA synthetase with the replacement of Trp87 and Trp283 by alanine

et al. 2019

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Aminoacyl-tRNA synthetase is one of the major enzymes of protein synthesis. The mammalian tyrosyl-tRNA synthetase consists of two structural units, the N-terminal catalytic (mini TyrRS) and the C-terminal cytokine-like modules. In a full length TyrRS, the N-terminal module carries out the catalytic function of binding the amino acid to tRNA, while the C-module adjusts and stabilizes the placement of tRNA in the active center of the enzyme. After cleavage of tyrosyl-tRNA synthetase with elastase on the mini TyrRS and C-module, the latter exhibit cytokine properties. The aim of the work was to optimize the expression of cloned cDNA miniTyrRS Bos taurus in plasmid pET30a-39KYRS in which the tryptophan codons at position 87 and 283 are replaced with alanine codons using the site-directed mutagenesis, and to obtain the mutant one-tryptophan protein of the mini BtTyrRS for further study on using methods of fluorescence spectroscopy of conformational changes of the enzyme at the stage of tyrosyladenylate formation and in interaction with the acceptor end of tRNATyr, as well as determination of the effect of tryptophan residus in positions 87 and 283 in its structure on the structurally dynamic and functional properties of the enzyme. It was found that the replacement of two tryptophan codons into the alanine codons in the cDNA of the mini TyrRS cloned in the expressing plasmid pET30a-39KYRSW40 does not affect the synthesis and solubility of the mutant form of the enzyme in the strain E.coli BL21 (DE3) pLysE. The amount of soluble form of the recombinant mutant mini BtTyrRS in the cytoplasm of bacterial cells, when expressed in E. coli BL21 (DE3) pLysE strain, is significantly enhanced by incubation of bacterial culture at a temperature 25 ° C compared to a culture incubation at 37° C. The yield of the obtained purified protein of the mutant mini BtTyrRS is 2.5 mg per average from 100 ml of culture medium, which is sufficient for further structural and functional studies of the mutant form of the enzyme. The compact structure of the recombinant protein is shown by fluorescence spectroscopy.

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Section: Out the Catalytic Function Of Binding The Amino Acid To Trnamentioning

confidence: 99%

unclassified

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Isolation and characterization of the mutant N-terminal catalytical module of the B. taurus Tyrosyl-tRNA synthetase with the replacement of Trp87 and Trp283 by alanine

et al. 2019

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“…(14) PEST sequences enriched by proline, serine, glutamic acid, and threonin usually are natural substrates for cleavage by many intracellular proteinases. (24) Consequently, pathological processes that lead to the release of proteinases could trigger proteolytic cleavage of TyrRS at the PEST-like sequence.…”

Section: Production and Characterization Of Recombinant Tyrrs Mini Tmentioning

confidence: 99%

Monoclonal Antibodies Against Tyrosyl-tRNA Synthetase and Its Isolated Cytokine-Like Domain

Kondratiuk

Khoruzenko

Cherednyk

et al. 2013

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH 2 -terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXCchemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N-and C-terminal domains of TyrRS. Recombinant TyrRS and its N-and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization.

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Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme–substrate complexes

2014

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Tyrosyl-tRNA synthetase from Mycobacterium tuberculosis (MtTyrRS) is an enzyme that belongs to class I of aminoacyl-tRNA synthetases, which catalyze the attachment of L-tyrosine to its cognate tRNATyr in the preribosomal step of protein synthesis. MtTyrRS is incapable of cross-recognition and aminoacylation of human cytoplasmic tRNATyr, so this enzyme may be a promising target for development of novel selective inhibitors as putative antituberculosis drugs. As a class I aminoacyl-tRNA synthetase, MtTyrRS contains the HIGH-like and KFGKS catalytic motifs that catalyze amino acid activation with ATP. In this study, the conformational mobility of MtTyrRS catalytic KFGKS loop was analyzed by 100-ns all-atoms molecular dynamics simulations of the free enzyme and its complexes with different substrates: tyrosine, ATP, and the tyrosyl-adenylate intermediate. It was shown that in the closed state of the active site, the KFGKS loop, readily adopts different stable conformations depending on the type of bound substrate. Molecular dynamics simulations revealed that the closed state of the loop is stabilized by dynamic formation of two antiparallel β-sheets at flanking ends which hold the KFGKS fragment inside the active center. Prevention of β-sheet formation by introducing point mutations in the loop sequence results in a rapid (<20 ns) transition of the loop from its functional "closed" M-like structure to an inactive "open" O-like structure, i.e. rapid diffusion of the catalytic loop outside the active site. The flexibility and rapid dynamics of the wild-type aaRS catalytic loop structure are crucial for formation of protein-substrate interactions and subsequently for overall enzyme functional activity.

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Structural and functional investigation of mammalian tyrosyl-tRNA synthetase

Cited by 20 publications

References 34 publications

Isolation and characterization of the mutant N-terminal catalytical module of the B. taurus Tyrosyl-tRNA synthetase with the replacement of Trp87 and Trp283 by alanine

Isolation and characterization of the mutant N-terminal catalytical module of the B. taurus Tyrosyl-tRNA synthetase with the replacement of Trp87 and Trp283 by alanine

Monoclonal Antibodies Against Tyrosyl-tRNA Synthetase and Its Isolated Cytokine-Like Domain

Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme–substrate complexes

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