Selenoprotein T Protects Endothelial Cells against Lipopolysaccharide-Induced Activation and Apoptosis

Sepsis is an exaggerated immune response upon infection with lipopolysaccharide (LPS) as the main causative agent. LPS-induced activation and apoptosis of endothelial cells (EC) can lead to organ dysfunction and finally organ failure. We previously demonstrated that the first twenty amino acids of the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) are sufficient to inhibit EC apoptosis. To identify genes whose regulation by LPS is affected by this N-terminal APEX1 peptide, EC were transduced with an expression vector for the APEX1 peptide or an empty control vector and treated with LPS. Following RNA deep sequencing, genes upregulated in LPS-treated EC expressing the APEX1 peptide were identified bioinformatically. Selected candidates were validated by semi-quantitative real time PCR, a promising one was Selenoprotein T (SELENOT). For functional analyses, an expression vector for SELENOT was generated. To study the effect of SELENOT expression on LPS-induced EC activation and apoptosis, the SELENOT vector was transfected in EC. Immunostaining showed that SELENOT was expressed and localized in the ER. EC transfected with the SELENOT plasmid showed no activation and reduced apoptosis induced by LPS. SELENOT as well as APEX1(1-20) can protect EC against activation and apoptosis and could provide new therapeutic approaches in the treatment of sepsis.

Hepatic Proteomic Analysis of Selenoprotein T Knockout Mice by TMT: Implications for the Role of Selenoprotein T in Glucose and Lipid Metabolism

Selenoprotein T (SELENOT, SelT), a thioredoxin-like enzyme, exerts an essential oxidoreductase activity in the endoplasmic reticulum. However, its precise function remains unknown. To gain more understanding of SELENOT function, a conventional global Selenot knockout (KO) mouse model was constructed for the first time using the CRISPR/Cas9 technique. Deletion of SELENOT caused male sterility, reduced size/body weight, lower fed and/or fasting blood glucose levels and lower fasting serum insulin levels, and improved blood lipid profile. Tandem mass tag (TMT) proteomics analysis was conducted to explore the differentially expressed proteins (DEPs) in the liver of male mice, revealing 60 up-regulated and 94 down-regulated DEPs in KO mice. The proteomic results were validated by western blot of three selected DEPs. The elevated expression of Glycogen [starch] synthase, liver (Gys2) is consistent with the hypoglycemic phenotype in KO mice. Furthermore, the bioinformatics analysis showed that Selenot-KO-induced DEPs were mainly related to lipid metabolism, cancer, peroxisome proliferator-activated receptor (PPAR) signaling pathway, complement and coagulation cascades, and protein digestion and absorption. Overall, these findings provide a holistic perspective into SELENOT function and novel insights into the role of SELENOT in glucose and lipid metabolism, and thus, enhance our understanding of SELENOT function.

hsa-miR-33-5p as a Therapeutic Target Promotes Apoptosis of Breast Cancer Cells via Selenoprotein T

Objective: Increasing evidence suggests that microRNA (miRNA) participates in regulating tumor cell apoptosis. We aimed to observe the effect of hsa-miR-33-5p on the apoptosis of breast cancer cells and to explore its regulatory relationship with selenoprotein T (SelT).Methods: RT-qPCR was used to examine the expression of hsa-miR-33-5p and SelT both in breast cancer tissues and cells. MCF-7 and MDA-MB-231 cells were transfected with hsa-miR-33-5p mimics or si-SelT. Then, a flow cytometry assay was carried out to examine the apoptosis of cells. Furthermore, SelT and apoptosis-related proteins including caspase-3, caspase-8, caspase-9, Bax, and Bcl-2 were detected via RT-qPCR and western blot. A luciferase reporter assay was utilized for assessing whether SelT was targeted by hsa-miR-33-5p.Results: Downregulated hsa-miR-33-5p was found both in breast cancer tissues and cells. After its overexpression, MCF-7 cell apoptosis was significantly promoted. Furthermore, our data showed that miR-33-5p elevated apoptosis-related protein expression in MCF-7 cells. Contrary to hsa-miR-33-5p, SelT was upregulated both in breast cancer tissues and cells. SelT expression was significantly inhibited by hsa-miR-33-5p overexpression. The luciferase reporter assay confirmed that SelT was a direct target of hsa-miR-33-5p. SelT overexpression could ameliorate the increase in apoptosis induced by hsa-miR-33-5p mimics.Conclusion: Our findings revealed that hsa-miR-33-5p, as a potential therapeutic target, could accelerate breast cancer cell apoptosis.

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 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 cell against the toxic effects of seleium compounds during selenocysteine synthesis.

Selenoprotein T Promotes Proliferation and G1-to-S Transition in SK-N-SH Cells: Implications in Parkinson's Disease

ABSTRACT Background Selenium is prioritized to the brain mainly for selenoprotein expression. Selenoprotein T (SELENOT) protects dopaminergic, postmitotic neurons in a mouse model of Parkinson's disease (PD). Objective We hypothesized a proliferative role of SELENOT in neural cells. Methods To assess SELENOT status in PD, sedated male C57BL/6 mice at 10–12 wk of age were injected with 6-hydroxydopamine in neurons, and human peripheral blood mononuclear cells were isolated from 9 healthy subjects (56% men, 68-y-old) and 11 subjects with PD (64% men, 63-y-old). Dopaminergic neural progenitor–like SK-N-SH cells with transient SELENOT overexpression or knockdown were maintained in the presence or absence of the antioxidant N-acetyl-l-cysteine and the calcium channel blocker nimodipine. Cell cycle, proliferation, and signaling parameters were determined by immunoblotting, qPCR, and flow cytometry. Results SELENOT mRNA abundance was increased (P < 0.05) in SK-N-SH cells treated with 1-methyl-4-phenylpyridinium iodide (3.5-fold) and peripheral blood mononuclear cells from PD patients (1.6-fold). Likewise, SELENOT was expressed in tyrosine hydroxylase–positive dopaminergic neurons of 6-hydroxydopamine–injected mice. Knockdown of SELENOT in SK-N-SH cells suppressed (54%; P < 0.05) 5-ethynyl-2′-deoxyuridine incorporation but induced (17–47%; P < 0.05) annexin V–positive cells, CASPASE-3 cleavage, and G1/S cell cycle arrest. SELENOT knockdown and overexpression increased (88–120%; P < 0.05) and reduced (37–42%; P < 0.05) both forkhead box O3 and p27, but reduced (51%; P < 0.05) and increased (1.2-fold; P < 0.05) cyclin-dependent kinase 4 protein abundance, respectively. These protein changes were diminished by nimodipine or N-acetyl-l-cysteine treatment (24 h) at steady-state levels. While the N-acetyl-l-cysteine treatment did not influence the reduction in the amount of calcium (13%; P < 0.05) by SELENOT knockdown, the nimodipine treatment reversed the decreased amount of reactive oxygen species (33%; P < 0.05) by SELENOT overexpression. Conclusions These cellular and mouse data link SELENOT to neural proliferation, expanding our understanding of selenium protection in PD.