Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode.
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ABSTRACT:
SUBMITTER: Liao P
PROVIDER: S-EPMC8360269 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl2, thus facilitating the synthesis of multi-milligrams of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.
| S-EPMC9159075 | biostudies-literature
Project description:Selenium is biologically active through the functions of selenoproteins that contain the amino acid selenocysteine. This amino acid is translated in response to in-frame UGA codons in mRNAs that include a SECIS element in its 3' untranslated region, and this process requires a unique tRNA, referred to as tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as a termination signal, is a candidate restriction point for the regulation of selenoprotein synthesis by selenium. A specialized reporter construct was used that permits the evaluation of SECIS-directed UGA translation to examine mechanisms of the regulation of selenoprotein translation. Using SECIS elements from five different selenoprotein mRNAs, UGA translation was quantified in response to selenium supplementation and alterations in tRNA([Ser]Sec) levels and isoform distributions. Although each of the evaluated SECIS elements exhibited differences in their baseline activities, each was stimulated to a similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using a dominant-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be limiting for UGA translation under conditions of high selenoprotein mRNA in both a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during selenoprotein translation indicate that it is this isoform that is translationally active and that selenium-induced tRNA methylation is a mechanism of regulation of the synthesis of selenoproteins.
| S-EPMC1370604 | biostudies-literature
Project description:We have re-examined the utility of native chemical ligation at -Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites without side reactions. After optimization, ligation at a -Glu-Cys- site could also be used as a ligation site, with minimal levels of byproduct formation. However, -Asp-Cys- is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of β-linked byproduct. The feasibility of native chemical ligation at -Gln-Cys- enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high-resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.
| S-EPMC3838204 | biostudies-literature
Project description:Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.
| S-EPMC134693 | biostudies-literature
Project description:Quantum key distribution - the establishment of information-theoretically secure keys based on quantum physics - is mainly limited by its practical performance, which is characterised by the dependence of the key rate on the channel transmittance R(η). Recently, schemes based on single-photon interference have been proposed to improve the key rate to [Formula: see text] by overcoming the point-to-point secret key capacity bound with interferometers. Unfortunately, all of these schemes require challenging global phase locking to realise a stable long-arm single-photon interferometer with a precision of approximately 100 nm over fibres that are hundreds of kilometres long. Aiming to address this problem, we propose a mode-pairing measurement-device-independent quantum key distribution scheme in which the encoded key bits and bases are determined during data post-processing. Using conventional second-order interference, this scheme can achieve a key rate of [Formula: see text] without global phase locking when the local phase fluctuation is mild. We expect this high-performance scheme to be ready-to-implement with off-the-shelf optical devices.
| S-EPMC9262923 | biostudies-literature
Project description:Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.
| S-EPMC8583801 | biostudies-literature
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.
2021-11-17 | GSE183923 | GEO
Project description:Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol-disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one-pot chemical synthesis of the thioredoxin-like domain of SELENOF, highlighted by the use of Fmoc-protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)-like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non-native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER.
| S-EPMC9304195 | biostudies-literature
Project description:Although targeted delivery mediated by ligand modified or tumor microenvironment sensitive nanocarriers has been extensively pursued for cancer chemotherapy, the efficiency is still limited by premature drug release after systemic administration. Herein we report a highly blood-stable, tumor-adaptable drug carrier made of disulfide (DS) bonded mPEG-(Cys)(4)-PDLLA micelles. Intravenously injected disulfide bonded micelles stably retained doxorubicin in the bloodstream and efficiently delivered the drug to a tumor, with a 7-fold increase of the drug in the tumor and 1.9-fold decrease in the heart, as compared with self-assembled (SA), non-crosslinked mPEG-PDLLA micelles. In vivo administration of disulfide bonded micelles led to doxorubicin accumulation in cancer cell nuclei, which was not observed after administration of self-assembled micelles. With a doxorubicin dose as low as 2 mg/kg, disulfide bonded micelles almost completely suppressed tumor growth in mice.
| S-EPMC3489991 | biostudies-literature
Project description:Selenium and copper are essential trace elements for humans, needed for the biosynthesis of enzymes contributing to redox homeostasis and redox-dependent signaling pathways. Selenium is incorporated as selenocysteine into the active site of redox-relevant selenoproteins including glutathione peroxidases (GPX) and thioredoxin reductases (TXNRD). Copper-dependent enzymes mediate electron transfer and other redox reactions. As selenoprotein expression can be modulated e.g. by H2O2, we tested the hypothesis that copper status affects selenoprotein expression. To this end, hepatocarcinoma HepG2 cells and mice were exposed to a variable copper and selenium supply in a physiologically relevant concentration range, and transcript and protein expression as well as GPX and TXNRD activities were compared. Copper suppressed selenoprotein mRNA levels of GPX1 and SELENOW, downregulated GPX and TXNRD activities and decreased UGA recoding efficiency in reporter cells. The interfering effects were successfully suppressed by applying the copper chelators bathocuproinedisulfonic acid or tetrathiomolybdate. In mice, a decreased copper supply moderately decreased the copper status and negatively affected hepatic TXNRD activity. We conclude that there is a hitherto unknown interrelationship between copper and selenium status, and that copper negatively affects selenoprotein expression and activity most probably via limiting UGA recoding. This interference may be of physiological relevance during aging, where a particular shift in the selenium to copper ratio has been reported. An increased concentration of copper in face of a downregulated selenoprotein expression may synergize and negatively affect the cellular redox homeostasis contributing to disease processes.
| S-EPMC7567034 | biostudies-literature