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Biological Trace Element Research Nov 2019The fact that selenocysteine (Sec) is delivered to the elongating ribosome by a tRNA that recognizes a UGA stop codon makes it unique and a thorn in the side of what was... (Review)
Review
The fact that selenocysteine (Sec) is delivered to the elongating ribosome by a tRNA that recognizes a UGA stop codon makes it unique and a thorn in the side of what was originally thought to be a universal genetic code. The mechanism by which this redefinition occurs has been slowly coming to light over the past 30 years, but key questions remain. This review seeks to highlight the prominent mechanistic questions that will guide the direction of work in the near future. These questions arise from two major aspects of Sec incorporation: (1) novel functions for the Sec insertion sequence (SECIS) that resides in all selenoprotein mRNAs and (2) the myriad of RNA-binding proteins, both known and yet to be discovered, that act in concert to modify the translation elongation process to allow Sec incorporation.
Topics: Animals; Codon, Terminator; Humans; Peptide Chain Elongation, Translational; RNA-Binding Proteins; Selenocysteine; Selenoproteins
PubMed: 31342342
DOI: 10.1007/s12011-019-01827-y -
Biochimie Jul 2015The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNA(Sec) recognizes a UGA codon. This... (Review)
Review
The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNA(Sec) recognizes a UGA codon. This sets up an intriguing interplay between translation elongation, translation termination and the complex machinery that marks mRNAs that contain premature termination codons for degradation, leading to nonsense mediated mRNA decay (NMD). In this review we discuss the intricate and complex relationship between this key quality control mechanism and the process of Sec incorporation in mammals.
Topics: Animals; Codon; Humans; Nonsense Mediated mRNA Decay; Peptide Elongation Factors; Protein Biosynthesis; RNA-Binding Proteins; Selenocysteine
PubMed: 25622574
DOI: 10.1016/j.biochi.2015.01.007 -
Methods in Enzymology 2022Selenoproteins, which contain the 21st amino acid selenocysteine, play roles in maintaining cellular redox homeostasis. Many open questions remain in the field of...
Selenoproteins, which contain the 21st amino acid selenocysteine, play roles in maintaining cellular redox homeostasis. Many open questions remain in the field of selenoprotein biology, including the functions of a number of uncharacterized human selenoproteins, and the properties of selenocysteine compared to its analogous amino acid cysteine. The mechanism of selenocysteine incorporation involves an intricate machinery that deviates from the mechanism of incorporation for the canonical 20 amino acids. As a result, recombinant expression of selenoproteins has been historically challenging, and has hindered a deeper evaluation of selenoprotein biology. Genetic code expansion methods, which incorporate protected analogs of selenocysteine, allow the endogenous selenocysteine incorporation mechanism to be bypassed entirely to facilitate selenoprotein expression. Here we present a method for incorporating a photocaged selenocysteine amino acid (DMNB-Sec) into human selenoproteins directly in mammalian cells. This approach offers the opportunity to study human selenoproteins in their native cellular environment and should advance our understanding of selenoprotein biology.
Topics: Animals; Cysteine; Genetic Code; Humans; Mammals; Protein Biosynthesis; Selenocysteine; Selenoproteins
PubMed: 35101208
DOI: 10.1016/bs.mie.2021.10.015 -
Advances in Protein Chemistry and... 2012Like all rules, even the genetic code has exceptions: these are generically classified as "translational recoding." Almost every conceivable mode of recoding has been... (Review)
Review
Like all rules, even the genetic code has exceptions: these are generically classified as "translational recoding." Almost every conceivable mode of recoding has been documented, including signals that redefine translational reading frame and codon assignation. While first described in viruses, it is becoming clear that sequences that program elongating ribosomes to shift translational reading frame are widely used by organisms in all domains of life, thus expanding both the coding capacity of genomes and the modes through which gene expression can be regulated at the posttranscriptional level. Instances of programmed ribosomal frameshifting and stop codon reassignment are opening up new avenues for treatment of numerous inborn errors of metabolism. The implications of these findings on human health are only beginning to emerge.
Topics: Animals; Gene Expression Regulation; Humans; Lysine; Protein Biosynthesis; Ribosomes; Selenocysteine
PubMed: 22243583
DOI: 10.1016/B978-0-12-386497-0.00004-9 -
Microbial Cell Factories Oct 2023Seleno-methylselenocysteine (SeMCys) is an effective component of selenium supplementation with anti-carcinogenic potential that can ameliorate neuropathology and...
BACKGROUND
Seleno-methylselenocysteine (SeMCys) is an effective component of selenium supplementation with anti-carcinogenic potential that can ameliorate neuropathology and cognitive deficits. In a previous study, a SeMCys producing strain of Bacillus subtilis GBACB was generated by releasing feedback inhibition by overexpression of cysteine-insensitive serine O-acetyltransferase, enhancing the synthesis of S-adenosylmethionine as methyl donor by overexpression of S-adenosylmethionine synthetase, and expressing heterologous selenocysteine methyltransferase. In this study, we aimed to improve GBACB SeMCys production by synthesizing methylmethionine as a donor to methylate selenocysteine and by inhibiting the precursor degradation pathway.
RESULTS
First, the performance of three methionine S-methyltransferases that provide methylmethionine as a methyl donor for SeMCys production was determined. Integration of the NmMmt gene into GBACB improved SeMCys production from 20.7 to 687.4 μg/L. Next, the major routes for the degradation of selenocysteine, which is the precursor of SeMCys, were revealed by comparing selenocysteine hyper-accumulating and non-producing strains at the transcriptional level. The iscSB knockout strain doubled SeMCys production. Moreover, deleting sdaA, which is responsible for the degradation of serine as a precursor of selenocysteine, enhanced SeMCys production to 4120.3 μg/L. Finally, the culture conditions in the flasks were optimized. The strain was tolerant to higher selenite content in the liquid medium and the titer of SeMCys reached 7.5 mg/L.
CONCLUSIONS
The significance of methylmethionine as a methyl donor for SeMCys production in B. subtilis is reported, and enhanced precursor supply facilitates SeMCys synthesis. The results represent the highest SeMCys production to date and provide insight into Se metabolism.
Topics: Selenocysteine; Bacillus subtilis; Vitamin U; Cysteine; Selenium
PubMed: 37853389
DOI: 10.1186/s12934-023-02203-1 -
International Journal of Molecular... Dec 2021Selenium is a fascinating element that has a long history, most of which documents it as a deleterious element to health. In more recent years, selenium has been found... (Review)
Review
Selenium is a fascinating element that has a long history, most of which documents it as a deleterious element to health. In more recent years, selenium has been found to be an essential element in the diet of humans, all other mammals, and many other life forms. It has many health benefits that include, for example, roles in preventing heart disease and certain forms of cancer, slowing AIDS progression in HIV patients, supporting male reproduction, inhibiting viral expression, and boosting the immune system, and it also plays essential roles in mammalian development. Elucidating the molecular biology of selenium over the past 40 years generated an entirely new field of science which encompassed the many novel features of selenium. These features were (1) how this element makes its way into protein as the 21st amino acid in the genetic code, selenocysteine (Sec); (2) the vast amount of machinery dedicated to synthesizing Sec uniquely on its tRNA; (3) the incorporation of Sec into protein; and (4) the roles of the resulting Sec-containing proteins (selenoproteins) in health and development. One of the research areas receiving the most attention regarding selenium in health has been its role in cancer prevention, but further research has also exposed the role of this element as a facilitator of various maladies, including cancer.
Topics: Animals; Diet; Genetic Code; Health; Humans; RNA, Transfer, Amino Acid-Specific; Selenium; Selenocysteine; Selenoproteins
PubMed: 35008430
DOI: 10.3390/ijms23010005 -
Advances in Nutrition (Bethesda, Md.) Mar 2011The biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec... (Review)
Review
The biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA([Ser]Sec), is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase to form O-phosphoseryl-tRNA([Ser]Sec). Sec synthase (SecS) then uses O-phosphoseryl-tRNA([Ser]Sec) and the active donor of selenium, selenophosphate, to form Sec-tRNA([Ser]Sec). Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with O-phosphoseryl-tRNA([Ser]Sec) in the presence of SecS to form cysteine-(Cys-)tRNA([Ser]Sec). This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium.
Topics: Animals; Archaea; Cysteine; Eukaryota; Genetic Code; Genomics; Mice; Selenocysteine; Selenoproteins
PubMed: 22332041
DOI: 10.3945/an.110.000265 -
Angewandte Chemie (International Ed. in... Apr 2016Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code...
Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.
Topics: Bacteria; Base Sequence; Codon, Terminator; Evolution, Molecular; Genetic Code; Genome, Bacterial; Metagenome; Selenocysteine
PubMed: 26991476
DOI: 10.1002/anie.201511657 -
Journal of the American Chemical Society Jul 2018Selenoproteins containing the 21 amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The...
Selenoproteins containing the 21 amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low p K, high reactivity, and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remains unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNA-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNA-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.
Topics: Codon, Terminator; Escherichia coli; Escherichia coli Proteins; Genetic Code; Glutathione Peroxidase; HeLa Cells; Humans; Models, Molecular; Palladium; Protein Biosynthesis; Protein Engineering; Selenocysteine; Selenoproteins; Thioredoxins; Glutathione Peroxidase GPX1
PubMed: 29984990
DOI: 10.1021/jacs.8b04603 -
The Journal of Biological Chemistry Aug 2023Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing...
Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.
Topics: Humans; Lyases; Selenium; Selenocysteine; Selenoprotein P; Selenoproteins; Jurkat Cells
PubMed: 37406814
DOI: 10.1016/j.jbc.2023.105009