Laboratory of Structural Biology, Sao Carlos Institute of Physics, University of São Paulo, São Carlos, SP, Brazil.
Laboratory of Cell Cycle and Center of Toxins, Immune Response and Cell Signaling-CeTICS, Butantan Institute, São Paulo, SP, Brazil.
PLoS Negl Trop Dis. 2020 Oct 5;14(10):e0008091. doi: 10.1371/journal.pntd.0008091. eCollection 2020 Oct.
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-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-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.
真核生物的挖掘超门已被用作模型来研究细胞分子过程的进化。引人注目的是，锥体虫科（T. brucei、T. cruzi 和 L. major）的人类寄生虫保留了负责硒代半胱氨酸生物合成和掺入硒蛋白的复杂机制（SELENOK/SelK、SELENOT/SelT 和 SELENOTryp/SelTryp），尽管这些蛋白质在实验室控制条件下似乎对寄生虫的生存力不是必需的。硒代磷酸合成酶 (SEPHS/SPS) 在硒代谢中起着不可或缺的作用，负责催化硒代磷酸的形成，硒代磷酸是硒代半胱氨酸合成的生物硒供体。我们解决了 L. major 硒代磷酸合成酶的晶体结构，并证实其二聚体组织在整个生命领域中具有功能重要性。我们还证明了它与硒代半胱氨酸裂解酶（SCLY）的相互作用，并表明它不存在于其他涉及硒代半胱氨酸途径的稳定组装中，即磷酸丝氨酸-tRNASec 激酶（PSTK）-Sec-tRNASec 合酶（SEPSECS）复合物和 tRNASec 特异性延伸因子（eEFSec）复合物。在 Procyclic T. brucei 细胞中敲除硒代磷酸合成酶后，用二硫苏糖醇 (DTT) 或衣霉素处理会导致生长缺陷。另一方面，只有 DTT 在表达硒代磷酸合成酶-RNAi 的血液 T. brucei 中表现出负面影响。此外，硒蛋白 T（SELENOT）对于两种形式的寄生虫都是可有可无的。总之，我们的数据表明 T. brucei 硒代磷酸合成酶在调节寄生虫内质网应激反应中起作用。
