Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family ( T . brucei , T . cruzi and L . major ) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L . major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNA Sec kinase (PSTK)-Sec-tRNA Sec synthase (SEPSECS) complex and the tRNA Sec -specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T . brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T . brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T . brucei selenophosphate synthetase in the regulation of the parasite’s ER stress response.
Early branching eukaryotes have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasite of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life.We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable complexes involved in the selenocysteine pathway, namely the phosphoseryl-tRNA Sec kinase (PSTK)-Sec-tRNA Sec synthase (SEPSECS) and the tRNA Sec -specific elongation factor (eEFSec)-ribosome. Endoplasmic reticulum stress with ditiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Although selenoprotein T (SELENOT) was dispensable for both forms of the parasite, SELENOT-RNAi procyclic T. brucei cells were sensitive to DTT. Together, our data suggest a role for the T. brucei selenophosphate synthetase in regulation of the parasite's ER stress response. SynopsisSelenium is both a toxic compound and a micronutrient. As a micronutrient, it participates in the synthesis of specific proteins, selenoproteins, as the amino acid selenocysteine. The synthesis of selenocysteine is present in organisms ranging from bacteria to humans. The protozoa parasites of the Trypanosomatidae family, that cause major tropical diseases, conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins. However, this pathway has been considered dispensable for the protozoa cells. This has intrigued us, and lead to question that if maintained in the cell it should be under selective pressure and therefore be necessary.Also, since the intermediate products of selenocysteine synthesis are toxic to the cell, it has been proposed that these compounds need to be sequestered from the cytoplasm. Therefore, extensive and dynamic protein-protein interactions must happen to deliver those intermediates along the pathway. In this study we have investigated the molecular and structural interactions of different proteins involved in selenocystein synthesis and describe its involvement in the endoplasmic reticulum protection to oxidative stress. Our results also show how the interaction of different proteins leads to the protection of the c...
RESUMO -Pigmentos são substâncias de caráter histórico sendo utilizados desde os primórdios da humanidade. Atualmente, possuem grande aplicabilidade industrial passando pelos ramos alimentício, farmacêutico, têxtil, entre outros; sendo a produção sintética destes a mais comum. Devido aos correntes riscos apresentados por corantes sintéticos, o desenvolvimento de rotas alternativas à obtenção de pigmentos, em especial aquelas que envolvam fontes naturais, são imperativas e tem apresentado grande receptividade. Sendo assim, o presente projeto visa avaliar o processo de extração de pigmento produzido pelo fungo Talaromyces complexo minioluteus isolado no Cerrado da região de São Carlos, obtendo um composto mais puro a ser utilizado para a identificação estrutural do mesmo. Mais especificamente, avaliar-se-ão as condições dos processos para a extração de pigmento utilizando duas fases líquidas, estabelecendo parâmetros para esta operação unitária no processamento "downstream". Além disso, com vista a otimizar os processos de identificação, testar-se-á técnicas de purificação do pigmento visando à obtenção de um grau de pureza, que será utilizado em outros trabalhos para a sua caracterização utilizando técnicas de RMN, espectrometria de massas e de infravermelho. INTRODUÇÃOCorantes e pigmentos são substâncias que permeiam as atividades humanas desde os seus primórdios, sendo as pinturas rupestres (uma das manifestações artísticas mais antigas da humanidade) evidências claras de suas remotas origens (Ebinuma, 2013;Ferreira, 2014). Atualmente, corantes e pigmentos possuem papel de destaque no meio industrial sendo utilizados nas indústrias têxtil, de alimentos, cosmética, farmacêutica etc., com vista a tornar seus produtos mais atraentes aos olhos do consumidor ou ainda conferir a estes funcionalidades ou propriedades especiais. É alta a gama de corantes existentes, sendo suas propriedades organolépticas advindas de diferentes estruturas moleculares. Por exemplo, a quantidade de instaurações da molécula é a principal responsável pela intensidade da cor manifestada.Entretanto, quando os corantes são utilizados na indústria alimentícia ou para fins farmacêuticos, há uma preocupação quanto a seus efeitos na saúde humana. Há estudos de certos compostos, presentes, sobretudo nos corantes sintéticos (os mais utilizados atualmente), que podem ser prejudiciais à saúde, com potenciais carcinogênicos, mutagênicos
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