No description available.

No description available.

Sunkieji metalai yra vieni didžiausių ekologinių nuodų. Kadangi apsinuodijimų sunkiaisiais metalais dažnis tebėra didelis, prevencinės strategijos bei veiksmingo gydymo poreikis išlieka aktualūs. Savo eksperimentais mes siekėme įvertinti apsauginį cinko jonų poveikį baltymų biosintezės sistemai bei svarbiausiems jos komponentams (tRNR ir aminoacil-tRNR-sintetazėms) esant slopinančiam kadmio jonų poveikiui. Eksperimentai atlikti su baltosiomis laboratorinėmis pelėmis. Cinko apsauginio poveikio įvertinimui, baltymų biosintezės intensyvumas pelių kepenyse vertintas po 0,5 LD50 CdCl2 (1,6 mg Cd2+ vienam kg kūno masės) ir/arba 0,3 LD50 ZnSO4 (3,1 mg Zn2+ vienam kg kūno masės) tirpalų sušvirkštimo į laboratorinių pelių pilvo ertmę. Baltymų biosintezės intensyvumas pelių organuose nustatytas praėjus 2, 8 ir 24 val. po metalų sušvirkštimo, pagal radioaktyviai žymėto [14C]-leucino įjungimą į naujai susintetintus peptidus ir baltymus. tRNRLeu ir leucyl-tRNR sintetazių aktyvumas nustatytas vykstant reakcijai su [14C]-leucinu. Gauti rezultatai parodė, jog 2 val. po CdCl2 sušvirkštimo, cinko jonai apsaugojo baltymus sintezuojančią sistemą nuo toksinio kadmio poveikio. Praėjus 8 val. po šių abiejų metalų sušvirkštimo, cinko jonai iš dalies normalizavo baltymų biosintezę, tačiau praėjus 24 val., baltymų biosintezės intensyvumas išliko tokio paties aktyvumo, kaip ir kadmiu paveiktose pelėse. Vadinasi, praėjus ilgesniam laikui (24 val.), cinko jonai neapsaugo kepenų transliacijos... [toliau žr. visą tekstą] / The aim of this study was to evaluate protective effects of zinc ions on the total protein synthesis in mouse liver and key components of liver translation machinery (tRNR ir aminoacil-tRNR synthetases) in the present of toxic cadmium ions effects. Experiments were done on white mice using intraperitoneal injections of 0,5 LD50 CdCl2 solution (1,6 mg Cd2+ per 1 kg of body mass) and/or 0,3 LD50 ZnSO4 (3,1 mg Zn2+ per 1 kg of body mass). Protein synthesis was evaluated by incorporation of 14C-labelled leucine into newly synthesized peptides and proteins after 2, 8 and 24 hours of intoxication. Activities of tRNALeu and leucyl-tRNA synthetase were measured by an aminoacylation reaction using 14C-labelled leucine. The data showed that at the 2nd h after CdCl2 injection, Zn2+ abolished deleterious effect of Cd2+ on the protein synthesis in the liver. Although pronounced activation of the protein synthesis was observed after 8 h of intoxication with either Cd2+ or Zn2+, this effect was lower in the presence of both ions. At the 24th h the protein synthesis was as active as in the liver of Cd-treated mice. Thus, Zn2+ can counteract Cd-induced inhibition of protein synthesis in mice liver only at the early stage of Cd2+ intoxication (at the 2nd h). Zn2+ abolished deleterious effect of Cd2+ on the activity of leucyl-tRNA synthetase within 24 h of mice intoxication with CdCl2. In vitro conditions, Zn2+ increased the acceptor activity of leucyl-tRNA synthetase only in low (1... [to full text]

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum (ER) stress response and in angiogenesis. Using Western blotting, immunofluorescence microscopy and RT-qPCR we were able to investigate changes in TARS protein and transcript levels. We initially hypothesized that TARS was secreted by exosomal release, and so we treated a human ovarian cancer cell line (CaOV-3) with monensin, an ionophore that increases exosome production, and VEGF to observe changes in intracellular and extracellular TARS protein. Monensin treatment consistently increased extracellular and intracellular TARS protein, however CD63, an exosome marker protein, levels were unaffected by monensin treatment. VEGF had no effect on intracellular TARS. We therefore hypothesized that the TARS response was a result of ER stress. The unfolded protein response (UPR) is a series of signaling pathways that are activated upon ER stress. When CaOV-3 cells were treated with increasing concentrations of monensin, intracellular levels of TARS and p-eIF2α, a downstream UPR target, increased accordingly. Monensin increased intracellular TARS protein and transcript levels in CaOV-3 cells. Monensin also increased DNAJB9, an ER chaperone protein, transcript levels, further confirming ER stress. Interestingly, monensin increased VEGF transcript levels about 6-fold. Borrelidin, a natural TARS inhibitor, also increased VEGF transcript levels, and caused an increase in p-eIF2α protein. Although the mechanism of TARS secretion remains unresolved, these data indicate that intracellular TARS expression increases in response to ER stress by monensin. Given TARS and VEGF transcript expression increased accordingly, it is possible that intracellular TARS may have pro-angiogenic function. Future directions may include investigating TARS interactions with translational control machinery.

No description available.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

Nos organismos eucariotos, aproximadamente 50% das proteínas traduzidas no citoplasma são transportadas para as organelas, onde irão desempenhar suas funções. Com isso, surgiu um intricado sistema de transporte intracelular de proteínas. Nas plantas, a presença de uma segunda organela endossimbionte, o plastídio, tornou este sistema mais complexo e gerou demanda adicional por transporte. Ainda, grande maioria das proteínas mitocondriais e plastidiais são codificadas por genes nucleares e importadas do citosol. O dogma uma proteína-uma localização foi associado ao conceito de um gene-uma proteína na biologia celular. Entretanto, proteínas individuais podem ter mais de uma função, e mais recentemente, proteínas codificadas por um único gene foram identificadas em mais de um compartimento subcelular, o que deu origem ao conceito de duplo direcionamento (DD). Um exemplo bem estudado de DD vem das proteínas da família das aminoacil-tRNA sintetases (aaRS), que participam da síntese protéica ao acoplar o aminoácido ao seu tRNA cognato. Dentre as aaRSs, a glutamil-tRNA sintetase citosólica (GluRS), através de sua extensão N-terminal, parece estar envolvida com outras funções além da tradução. Em Arabidopsis thaliana, há dois genes nucleares que codificam a GluRS, um para uma proteína de duplo direcionamento (DD) e outro para uma proteína citosólica. Resultados recentes em nosso laboratório mostraram que a GluRS citosólica pode estar relacionada ao controle da localização subcelular de proteínas organelares em Arabidopsis. Para verificar um eventual papel desta proteína na localização subcelular de outras proteínas, foram realizados ensaios de duplo-híbrido em levedura, os quais mostraram interação entre a GluRS e a glutamina sintetase (GS) de Arabidopsis thaliana, proteína de DD para mitocôndrias e cloroplastos Esta interação foi confirmada in planta, sendo a sequência da GluRS responsável pela interação localizada na região N-terminal, do resíduo 207 ao 316. Análises filogenéticas apontam que esta região encontra-se ausente nas bactérias e que originou-se provavelmente em Archea, entre 2,6 e 1,8 bilhões de anos. Além disso, observa-se que esta sequência é conservada em fungos, musgos e plantas vaculares, tendo originado-se em Arabidopsis há cerca de 2 bilhões de anos. / In eukaryotic organisms, about 50% of cytoplasmic translated proteins are transported to the organelles, where they can play their roles. Thus, a complex system for intracellular transport was established. In plants, the presence of a second endosymbiont organelle, the plastid, turned this system still more intricated and required an additional transport mechanism. Besides, most of organellar proteins are coded by nuclear genes and imported from the cytosol. The one protein-one localization was associated to the idea of one gene-one protein, which has long been established in molecular biology. However, individual proteins can show more than one function, and recently, proteins coded by one single gene were identified in more than one subcellular compartment, which has originated the concept of dual targeting. One of the most studied example of dual targeted proteins is the aminoacyl-tRNA synthetase (aaRS) family, which are related to protein synthesis by attaching the correct amino acid onto the cognate tRNA molecule. Among the aaRSs, cytosolic glutamyl-tRNA synthetase (GluRS), through its N-terminal extension, seems to be involved in other cellular role beyond translation. In Arabidopsis thaliana, there are two genes encoding GluRS, one for a dual-targeted protein and other for a cytosolic protein. Recent results in our laboratory showed that GluRS interacts with proteins destinated to other organelles, which suggest that this protein might have a role in interfering on protein localization in Arabidopsis. In order to gain some information on the role of this protein in subcellular localization, yeast two-hybrid assays were performed. These studies showed the interaction between GluRS and glutamine synthetase (GS), a mitochondrial and chloroplastic dual-targeted protein. This interaction was confirmed in planta. In addition, the GluRS sequence associated to protein interaction was localized at its N-terminal portion, between the residues 207 316. Phylogenetic analysis revealed that this region is absent in bacteria and it probably arose from Archea between 2.6 and 1.8 billion years ago. Also, this sequence is conserved in fungi, moss and all the green plants investigated. Finally, datation analysis showed that this sequence arose in Arabidopsis between 2 and 1.7 billion years ago.

A regulação da localização de proteínas é um dos aspectos fundamentais na biologia celular vegetal. Os cloroplastos importam mais de 90% de suas proteínas do citosol, portanto, é importante caracterizar os fatores citosólicos que podem estar envolvidos no direcionamento de proteínas para as organelas. Um ensaio de duplohíbrido em leveduras com as proteínas cloroplastidiais HMPPK/TMPPase (TH1) e Glutamina Sintetase (GS) II usados como iscas revelou que a forma citosólica da glutamil-tRNA sintetase - GluRS (At5g26710) de Arabidopsis thaliana interagiu com ambas as proteínas. Estudos de Complementação da Fluorescência Bimolecular (BiFC) confirmaram tais interações in planta. Estudos com deleções na região Nterminal da GluRS mostraram que esta região é responsável pelas interações com HMPPK/TMPPase e GSII. Além disso, seis resíduos de aminoácidos parecem ser cruciais para a interação entre as proteínas. Curiosamente, foi mostrado que a GluRS está envolvida na localização de proteínas em leveduras. A fim de obter mais informações sobre o envolvimento da GluRS ns localização de proteínas nos cloroplastos, foram produzidos plantas de tabaco transgênicas expressando uma proteína quimérica, feita pela fusão do gene codificador da HMPPK/TMPPase, TH1- GFP, e GSII-GFP e posteriormente usados em ensaios de agroinfiltração com RNA de interferência (RNAi) para GluRS. Análises em microscópio confocal mostraram que TH1-GFP e GSII-GFP acumulam no citosol em vez de serem direcionados aos cloroplastos. Neste trabalho, mostramos pela primeira vez que a GluRS está envolvida na localização de proteínas cloroplastidiais em plantas e esse mecanismo é também conservado em Saccharomyces cerevisiae. / Regulation of protein localization is one of the key aspects in plant cell biology. Chloroplasts import more than 90% of their proteins from the cytosol, therefore, it is important to identify and characterize cytosolic factors that might be involved in protein delivery to the organelar envelope. A yeast two-hybrid screen with a chloroplastlocalized HMPPK/TMPPase protein and glutamine synthetase (GS), used as baits, revealed that the cytosolic form of the glutamyl-tRNA synthetase (GluRS) (At5g26710) from Arabidopsis thaliana interacted with both proteins. Bimolecular Fluorescence Complementation (BiFC) studies confirmed such interactions in planta. Deletion studies of GluRS showed that the N-terminal region of the protein is responsible for proteinprotein interactions (PPI) with TH1 and GS. In addition, six amino acid residues appeared to be crucial for PPI. Interestingly, GluRS has been also shown to be involved in regulating protein localization in yeast. In order to gain more information about the involvement of GluRS on protein localization in chloroplasts, we produced transgenic tobacco plants expressing a chimeric protein made by the fusion of TH1- GFP and GSIIGFP and agroinfiltrated with a RNA interference (RNAi) construct against GluRS. Confocal analysis showed that TH1-GFP and GSII-GFP accumulated in the cytosol instead of being targeted to chloroplasts. Here, we show for the same time that GluRS is involved in protein localization in plants and this mechanism is also conserved in Saccharomyces cerevisiae.

A síntese de selenocisteína e sua incorporação co-traducional em selenoproteínas como resposta a um códon UGA em fase requerem uma complexa maquinaria molecular. Em eucariotos, foram identificados componentes que participam da reação de formação de selenocisteína: Seril-tRNA sintetase (SerRS), O-fosfoseril-tRNA quinase (PSTK), SECIS Binding Protein 2 SBP2, um fator de elongação específico para Sec (EFSec), selenofosfato sintetase 1 (SPS1) e selenofosfato sintetase 2 (SPS2), SEPSECS, proteína ligante de RNA SECp43, proteína ribossomal L30, um tRNA de inserção de selenocisteína (tRNASec, SELC) e uma sequência específica no RNA mensageiro (elemento SECIS). O primeiro passo da incorporação de selenocisteína em proteínas é realizado pela SerRS, que aminoacila o tRNA com serina através da ativação da serina por Mg+2 e ATP, levando a formação de um intermediário ligado a enzima (Ser-AMP). Posteriormente, ocorre a mudança do radical Ser do intermediário Ser-AMP para o tRNASec, e subsequentemente, a conversão enzimática de Ser-tRNASec para Sec-tRNASec. Através de análises in sílico nosso grupo identificou componentes da maquinaria de inserção de selenocisteína em espécies de Kinetoplastida. Foram identificados homólogos de tRNASec e as enzimas TbSerRS, TbSPS2, TbPSTK, TbSepSecS e TbEFSec. Nosso principal alvo é o estudo estrutural da SerRS de Trypanosoma brucei nativa e em complexo com o tRNASec e com as isoformas do tRNASer. Uma nova metodologia no processo de purificação desta enzima foi desenvolvida e, através das técnicas de cromatografia de exclusão molecular, espalhamento de luz dinâmico e ultracentrifugação analítica conseguimos determinar o estado oligomérico da TbSerRS. O resultado de dímeros em solução corroborou com dados reportados na literatura, além de verificarmos por meio de estudos de cinética enzimática que a enzima encontra-se ativa sob as condições utilizadas. A técnica de ultracentrifugação analítica de sedimentação em equilíbrio também nos permitiu verificar a formação do complexo SerRS-tRNA, mas não nos possibilitou definir a estequiometria deste complexo. Estudos estruturais da enzima nativa e em interação com os tRNAs SELC e com as isoformas do tRNASer, L-serina, um análogo não hidrolisável de AMP, MgCl2, e com porções menores dos tRNAs foram realizados por meio da cristalografia por difração de raios X. Através dessa técnica, dezessete conjunto de dados foram coletados, processados e estão em fase de refinamento. Algumas análises estruturais possibilitaram confirmar a presença de duas moléculas de glicerol em cada monômero na região do sítio ativo para a estrutura da TbSerRS nativa e uma molécula de dAMP para o complexo TbSerRS-dAMP. / The synthesis of selenocysteine and its co-translational incorporation in selenoproteins in response to a UGA codon in frame require complex molecular machinery. In eukaryotes, components that participate in the reaction of selenocysteine formation were identified: SeryltRNA synthetase (SerRS), O-phosphoseryl-tRNA kinase (PSTK), SECIS Binding Protein 2 - SBP2, a selenocysteine-specific elongation factor (EFSec), selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2), SEPSECS, SECp43 RNA binding protein, ribosomal protein L30, selenocysteine tRNA (tRNASec, SELC), and a specific sequence in the messenger RNA (SECIS element). The first step for selenocysteine incorporating is performed by SerRS that aminoacylates the tRNA with serine through serine activation by Mg2+ and ATP leading to the formation of an intermediate linked to the enzyme (Ser-AMP). Subsequently, the change of the Ser radical to tRNASec takes place followed by the enzymatic conversion of Ser-tRNASec to Sec-tRNASec. Through in silico analysis our group has identified components of the selenocysteine insertion machinery in species of Kinetoplastida. Homologues of tRNASec and the enzymes TbSerRS, TbSPS2, TbPSTK, TbSepSecS and TbEFSec were identified. Our main target is the structural study of the native SerRS from Trypanosoma brucei and SerRS in complex with the tRNASec and the tRNASer isoforms. A new methodology in the purification process of this enzyme has been developed, and through molecular exclusion chromatography, dynamic light scattering and analytical ultracentrifugation techniques we were able to determine the oligomeric state of TbSerRS. The result of dimers in solution corroborated with the data reported in the literature. Moreover, we were able to verify through studies of enzyme kinetics that the enzyme is active. The sedimentation equilibrium analytical ultracentrifugation technique also demonstrated the formation of the SerRS-tRNA complex, however, it did not allow the definition of the complex stoichiometry. Structural studies of the native enzyme and its interaction with SELC, tRNASer isoforms, L-serine, a non-hydrolyzable AMP analog, MgCl2, and smaller portions of tRNAs were performed by X-ray diffraction crystallography. Through this technique, seventeen data sets were collected, processed, and are being submitted to refinement processes. Initial structural analysis allowed the confirmation of the presence of two glycerol molecules in each monomer in the active site region in the native structure of TbSerRS and one dAMP molecule in the TbSerRS-dAMP complex.

Malaria is a treatable communicable disease yet remains a common cause of death and disease especially among pregnant women and children. Most of malaria's worldwide burden disproportionately lies in Southeast Asia and Sub-Saharan Africa. Western medicine's 100+ year history of combating Plasmodium falciparum has taught us that the global population of malaria parasites has a unique and dangerous ability to rapidly evolve and spread drug resistance. Recently it was documented that resistance to the first-line antimalarial artemisinin may be developing in Southeast Asia.