A human Y334C mutation in selenocysteine synthase causes cardio-respiratory failure and perinatal death in mice which can be rescued by selenium-independent GPX4
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ABSTRACT: Selenoproteins are a small family of proteins containing the trace element selenium in form of the rare amino acid selenocysteine (Sec), which is decoded by the UGA codon. In humans, a number of pathogenic mutations in genes encoding distinct selenoproteins or selenoprotein biosynthesis factors have been identified. Pathogenic mutations in selenocysteine synthase (SEPSECS), which catalyzes the last step in Sec-tRNA[Ser]Sec biosynthesis, were reported in children suffering from progressive cerebello-cerebral atrophy. To understand the underlying mechanisms of SEPSECS mutations, we generated a novel mouse model recapitulating the respective human pathogenic Y334C mutation in the murine Sepsecs gene (SepsecsY334C). Unlike in patients, homozygous mutant pups died perinatally with signs of cardio-respiratory failure. Perinatal death is reminiscent of the Sedaghatian spondylometaphyseal dysplasia disorder in humans, which is caused by inactivating mutations of the gene encoding the selenoprotein and key ferroptosis regulator glutathione peroxidase 4 (GPX4). Protein expression levels of distinct selenoproteins in SepsecsY334C/Y334C mice were found to be generally reduced in brain and isolated cortical neurons, while transcriptomics analysis uncovered an upregulation of NRF2-regulated genes. Crossbreeding of SepsecsY334C/Y334C mice with mice harboring a targeted mutation of the catalytically active site Sec to Cys in GPX4 surprisingly rescued perinatal death of SepsecsY334C/Y334C mice, showing that the cardio-respiratory defects of Sepsecs-mutant mice were caused by the lack of GPX4. Like in SepsecsY334C/Y334C mice, selenoprotein expression levels remained low and NRF2-regulated genes remained highly expressed in these compound mutant mice, indicating that selenium-independent GPX4, along with a sustained antioxidant response are sufficient to compensate for dysfunctional Sec-tRNA[Ser]Sec biosynthesis. Our findings imply that children with mutations in SEPSECS or GPX4 may benefit from treatments partially compensating for impaired GPX4 activity.
Project description:Comparative analysis of gene expression in bone marrow-derived macrophages (BMDM) from trsp knockout mice (Trspfl/fl-LysM-Cre+/-) and Control (Trspfl/fl-LysM-Cre-/-) mice. Selenium, a micronutrient whose deficiency in the diet causes immune dysfunction and inflammatory disorders, exerts its physiological effects partly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins, is mediated by Sec tRNA[Ser]Sec. To identify macrophage-specific selenoprotein function, we generated mice with the Sec tRNA[Ser]Sec gene specifically deleted in myeloid cells. These mutant mice were devoid of the selenoproteome in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. Therefore, selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages.
Project description:Comparative analysis of gene expression in bone marrow-derived macrophages (BMDM) from trsp knockout mice (Trspfl/fl-LysM-Cre+/-) and Control (Trspfl/fl-LysM-Cre-/-) mice. Selenium, a micronutrient whose deficiency in the diet causes immune dysfunction and inflammatory disorders, exerts its physiological effects partly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins, is mediated by Sec tRNA[Ser]Sec. To identify macrophage-specific selenoprotein function, we generated mice with the Sec tRNA[Ser]Sec gene specifically deleted in myeloid cells. These mutant mice were devoid of the selenoproteome in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. Therefore, selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages. We have generated mice in which we have selectively removed the selenocysteine tRNA gene (trsp) in macrophages under the control of LysM-Cre promoter. Microarray analysis was performed on RNA samples taken from bone marrow-derived macrophages in knockout and control mice. 1. Control unstimulated 2. Knockout unstimulated 3. Control lipopolysaccharide (LPS) stimulated (4h) 4. Knockout LPS stimulated (4h). Three replicates for each condition. Thus, a total of 12 samples.
Project description:Expression of selenoproteins requires the co-translational incorporation of selenocysteine (Sec) in response to an in-frame UGA codon. The machinery of UGA/Sec re-coding is complex and many factors affect the hierarchy of expression among selenoproteins, including modification of tRNA[Ser]Sec. Its hyper-modification in the anticodon stem loop is influenced by selenium bioavailability, and a mutation in adenosine 37 (A37) that abrogates isopentenylation, has a profound effect on selenoprotein expression in mice. Patients with mutations in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder and hence we wondered whether mutations in TRIT1 negatively affected the expression of selenoproteins. Fibroblasts from a patient carrying a pathogenic R323Q mutation in TRIT1 in homozygosity did not show decreased selenoprotein expression, although recombinant TRIT1R323Q had significantly reduced activity in vitro towards anticodon stem-loop substrates. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Selenoprotein expression as assessed by western blotting, 75Se metabolic labeling, and ribosomal profiling was not decreased despite the general reduction of N6-isopentenyl-adenosine in tRNAs. We show that 5-methylcarboxymethylation and 2’O-methylation of U34 occur independently of isopentenylation of A37 in tRNA[Ser]Sec. Reanalyzing previously published ribosomal profiling datasets, we demonstrate that (i) failure of 5-carboxymethylation at U34 is associated with reduced expression of GPX1, but not GPX4, and that (ii) FTSJ1 is not the elusive U34-2’O-methyltransferase involved in the methylation of tRNA[Ser]Sec.
Project description:Comparative analysis of gene expression in the liver of the Trsp-knockout mice (Trspfl/fl-AlbCre+/+) and A34 (Trspfl/fl-AlbCre+/+-A34t/t), G37L (Trspfl/fl-AlbCre+/+-G37t/t; 2 copies), G37H (Trspfl/fl-AlbCre+/+-G37t/t; 16 copies) transgenic mice with gene expression of wild type mice (Trsp+/+-AlbCre+/+). Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes. Trsp vs DTrsp; Trsp vs A34; Trsp vs G37L; Trsp vs G37H. Biological replicates from littermates. One replicate per array.
Project description:Comparative analysis of gene expression in the liver of the Trsp-knockout mice (Trspfl/fl-AlbCre+/+) and A34 (Trspfl/fl-AlbCre+/+-A34t/t), G37L (Trspfl/fl-AlbCre+/+-G37t/t; 2 copies), G37H (Trspfl/fl-AlbCre+/+-G37t/t; 16 copies) transgenic mice with gene expression of wild type mice (Trsp+/+-AlbCre+/+). Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.
Project description:Re-coding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3’ UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNAbinding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA re-coding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2 .
Project description:Selenium (Se) is an important trace element for many organisms and is incorporated into selenoproteins as selenocysteine (Sec). In eukaryotes, selenophosphate synthetase SPS2 is essential for Sec biosynthesis. In recent years, genetic disruptions of both Sec biosynthesis genes and selenoprotein genes have been investigated in different animal models, which provide important clues for understanding the Se metabolism and function in these organisms. However, a systematic study on the knockdown of SPS2 has not been performed in vivo. Herein, we conducted microarray experiments to study the transcriptome of fruit flies with knockdown of SPS2 in larval and adult stages. Several hundred differentially expressed genes were identified in each stage.
Project description:Selenium (Se) is an important trace element for many organisms and is incorporated into selenoproteins as selenocysteine (Sec). In eukaryotes, selenophosphate synthetase SPS2 is essential for Sec biosynthesis. In recent years, genetic disruptions of both Sec biosynthesis genes and selenoprotein genes have been investigated in different animal models, which provide important clues for understanding the Se metabolism and function in these organisms. However, a systematic study on the knockdown of SPS2 has not been performed in vivo. Herein, we conducted microarray experiments to study the transcriptome of fruit flies with knockdown of SPS2 in larval and adult stages. Several hundred differentially expressed genes were identified in each stage. Genes expression profiles of 12 fruit fly samples corresponding to larval and adult stages of SPS2 knockdown and control groups (3 replicates for each group) were analyzed using an Affymetrix Drosophila genome microarray.
Project description:Comparative analysis of gene expression in cultured primary keratinocytes isolated from newborn control (K14-cre; GPx4fl/+) and knockout (K14-cre; GPx4fl/fl) mice. Selenoproteins are essential for skin function, as targeted abolition of selenoproteins in epidermal tissue results in newborn mice manifesting gross abnormalities of skin and hair, accompanied by retarded growth and premature death. To investigate whether lack of a single selenoprotein could induce similar phenotypic effect in mice, we generated keratinocyte-specific knockout mice lacking glutathione peroxidase 4 (GPx4), an essential selenoprotein in skin, to examine phenotypic changes resulting from the lack of GPx4 in skin. Ablation of GPx4 results in focal alopecia and disturbed hair follicle morphogenesis, with GPx4 being essential during early stages of hair follicle morphogenesis as well as for keratinocyte adhesion and proliferation in culture. We have generated mice with selective removal of the GPx4 gene in keratinocytes under the control of Keratin-14-cre (K14-cre) promoter. Comparative microarray analysis was performed on RNA samples taken from pooled primary keratinocytes from knockout and control mice from the same litter. Array replicates were performed using RNA samples from three different litters.
Project description:Trace element selenium (Se) is incorporated as the 21st amino acid, selenocysteine (Sec), into selenoproteins through tRNA[Ser]Sec. Selenoproteins act as gatekeepers of redox homeostasis and modulate immune function to effect anti-inflammation and resolution. However, mechanistic underpinnings involving metabolic reprogramming during inflammation and resolution remain poorly understood. Endotoxin lipopolysaccharide (LPS) activation of murine bone marrow-derived macrophages (BMDMs) cultured in the presence or absence of Se (as selenite) was used to examine temporal changes in the proteome and metabolome by multiplexed tandem mass tag-quantitative proteomics, metabolomics, and machine-learning approaches. Se-dependent modulation of glycolytic, TCA and PPP pathways predisposed BMDMs to preferentially increase OXPHOS to efficiently regulate inflammation and its timely resolution. Use of macrophages lacking selenoproteins, indicated that all three metabolic nodes were sensitive to selenoproteome expression. Furthermore, inhibition of SDH with dimethylmalonate affected the pro-resolving effects of Se by increasing the resolution interval in a murine peritonitis model.