Thiemann, O. H. | bioRxiv Science

bioRxiv2025

Extracellular glutamine is essential in cell division and flagellar function in bloodstream forms of Trypanosoma brucei

Trypanosoma brucei is the etiologic agent of African trypanosomiasis, also known as sleeping sickness. In the mammalian host, T. brucei is present as replicative and non-replicative bloodstream forms (BSF) referred to as long slender and stumpy, respectively. In the insect vector, the developmental forms are procyclic (PCF), epimastigote, and metacyclic trypomastigote. It is well established that BSF use glucose as the main energy source, and PCF can use glucose but, in its absence, uses amino acids, mainly proline. Due to these facts, the metabolism of amino acids has been well studied in PCF, but less attention has been given to it in BSF. In the present work, we unveil the participation of glutamine (Gln), the most abundant amino acid in the human blood, in BSF proliferation. According to the data presented herein, in the absence of extracellular Gln, cell cycle progress, as well as kinetoplast and flagella distribution and function among daughter cells, are widely compromised, yielding polyploid multiflagellated cells with reduced motility. In addition, the Gln-poor medium resulted in cells with a massive alteration in the proteome glutamylation. These alterations were reverted when Gln was added to the culture media. An analysis of the glutamylproteome of cells incubated in Gln-poor medium, followed by a Gene Ontology (GO) analysis, suggested a relationship between glutamylation and biological processes such as gene expression and regulation, cell cycle, and bioenergetics metabolism, in addition to cytoskeleton dynamics in BSF of T. brucei.

cell biology↗

bioRxiv2020

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase

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-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) and the tRNASec-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 parasites 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 cell against the toxic effects of seleium compounds during selenocysteine synthesis.

molecular biology↗