RESUMEN
The synthesis mechanisms and function evaluation of selenium(Se)-enriched microorganism remain relatively unexplored. This study unveils that total Se content within A. oryzae A02 mycelium soared to an impressive 8462 mg/kg DCW, surpassing Se-enriched yeast by 2-3 times. Selenium exists in two predominant forms within A. oryzae A02: selenoproteins (SeMet 32.1 %, SeCys 14.4 %) and selenium nanoparticles (SeNPs; 53.5 %). The extensive quantitative characterization of the elemental composition, surface morphology, and size of SeNPs on A. oryzae A02 mycelium significantly differs from those reported for other microorganisms. Comparative RNA-Seq analysis revealed the upregulation of functional genes implicated in selenium transformation, activating multiple potential pathways for selenium reduction. The assimilatory and dissimilatory reductions of Se oxyanions engaged numerous parallel and interconnected pathways, manifesting a harmonious equilibrium in overall Se biotransformation in A. oryzae A02. Furthermore, selenium-enriched A. oryzae A02 was observed to primarily upregulate peroxisome activity while downregulating estrogen 2-hydroxylase activity in mice hepatocytes, suggesting its potential in fortifying antioxidant physiological functions and upholding metabolic balance.
Asunto(s)
Aspergillus oryzae , Selenio , Aspergillus oryzae/metabolismo , Aspergillus oryzae/genética , Selenio/química , Selenio/metabolismo , Selenio/farmacología , Animales , Ratones , Selenoproteínas/metabolismo , Selenoproteínas/biosíntesis , Micelio/metabolismo , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Nanopartículas/químicaRESUMEN
Thyroid hormone (TH) controls the timely differentiation of oligodendrocytes (OLs), and its deficiency can delay myelin development and cause mental retardation. Previous studies showed that the active TH T3 is converted from its prohormone T4 by the selenoprotein DIO2, whose mRNA is primarily expressed in astrocytes in the CNS. In the present study, we discovered that SECISBP2L is highly expressed in differentiating OLs and is required for DIO2 translation. Conditional knock-out (CKO) of Secisbp2l in OL lineage resulted in a decreased level of DIO2 and T3, accompanied by impaired OL differentiation, hypomyelination and motor deficits in both sexes of mice. Moreover, the defective differentiation of OLs in Secisbp2l mutants can be alleviated by T3 or its analog, but not the prohormone T4. The present study has provided strong evidence for the autonomous regulation of OL differentiation by its intrinsic T3 production mediated by the novel SECISBP2L-DIO2-T3 pathway during myelin development.SIGNIFICANCE STATEMENT Secisbp2l is specifically expressed in differentiating oligodendrocytes (OLs) and is essential for selenoprotein translation in OLs. Secisbp2l regulates Dio2 translation for active thyroid hormone (TH) T3 production in the CNS. Autonomous regulation of OLs differentiation via SECISBP2L-DIO2-T3 pathway.
Asunto(s)
Neurogénesis , Oligodendroglía , Selenoproteínas , Animales , Diferenciación Celular , Femenino , Yoduro Peroxidasa , Masculino , Ratones , Vaina de Mielina/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Selenoproteínas/biosíntesis , Selenoproteínas/genética , Hormonas Tiroideas , Yodotironina Deyodinasa Tipo IIRESUMEN
The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNASec) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l-serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.
Asunto(s)
Codón de Terminación , Extensión de la Cadena Peptídica de Translación , Proteínas de Unión al ARN , Ribosomas , Selenocisteína , Selenoproteínas , Codón de Terminación/genética , Microscopía por Crioelectrón , Humanos , Extensión de la Cadena Peptídica de Translación/genética , Conformación Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribosomas/química , Selenocisteína/química , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/biosíntesis , Selenoproteínas/genéticaRESUMEN
Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure-activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.
Asunto(s)
Conformación de Ácido Nucleico , Selenio/química , Selenocisteína/genética , Selenoproteínas/genética , ARN Mensajero/química , ARN Mensajero/genética , Selenocisteína/biosíntesis , Selenocisteína/química , Selenoproteínas/biosíntesis , Selenoproteínas/química , Selenoproteínas/ultraestructura , Relación Estructura-ActividadRESUMEN
Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pseudomonas stutzeri/metabolismo , Ácido Selenioso/metabolismo , Selenoproteínas/biosíntesis , Tiorredoxinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Formiato Deshidrogenasas/metabolismo , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Ácido Selenioso/química , Selenoproteínas/química , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
Selenium (Se) is a micronutrient essential for life. Dietary intake of Se within the physiological range is critical for human health and reproductive functions. Selenium levels outside the recommended range have been implicated in infertility and variety of other human diseases. However, presently it is not clear how different dietary Se sources are processed in our bodies, and in which form or how much dietary Se is optimum to maintain metabolic homeostasis and boost reproductive health. This uncertainty leads to imprecision in published dietary guidelines and advice for human daily intake of Se and in some cases generating controversies and even adverse outcomes including mortality. The chief aim for this review is to describe the sources of organic and inorganic Se, the metabolic pathways of selenoproteins synthesis, and the critical role of selenprotenis in the thyroid gland homeostasis and reproductive/fertility functions. Controversies on the use of Se in clinical practice and future directions to address these challenges are also described and discussed herein.
Asunto(s)
Homeostasis/fisiología , Reproducción/fisiología , Selenio/fisiología , Dieta , Femenino , Fertilidad/fisiología , Humanos , Masculino , Selenio/administración & dosificación , Selenio/deficiencia , Selenoproteínas/biosíntesis , Selenoproteínas/fisiología , Glándula Tiroides/fisiologíaRESUMEN
Obesity is among the most alarming health concerns, impacting public health and causing a socioeconomic challenge, especially in developing countries like Brazil, where approximately one quart of the population presents obesity. As an established risk factor for numerous comorbidities with a multifactorial etiology, obesity is a consequence of energy-dense overfeeding, however with significant undernourishment, leading to excessive adipose tissue accumulation and dysfunction, dyslipidemia, and micronutrient deficiencies. About 60% of patients with obesity take statins, a cholesterol-lowering medication, to curb dyslipidemia, with ~10% of these patients presenting various myopathies as side effects. Statins act upon the rate-limiting enzyme of cholesterol biosynthesis in the liver, which is a pathway providing intermediates to the synthesis of selenoproteins, i.e., enzymes containing the micronutrient selenium. Statins have been postulated to negatively impact selenoprotein synthesis, particularly in conditions of selenium deficiency, and potentially implicated in the myopathies occurring as side effects of statins. The Brazilian population is prone to selenium deficiency, hence could be considered more susceptible to statin side effects. This review examines the specific consequences to the Brazilian population of the harmful intersection between obesity development and concomitant micronutrient deficiencies, particularly selenium, combined with statin treatment in the context of nutrition in Brazil.
Asunto(s)
Dislipidemias/epidemiología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Obesidad/epidemiología , Selenio/deficiencia , Brasil/epidemiología , Dislipidemias/tratamiento farmacológico , Dislipidemias/etiología , Humanos , Hígado/metabolismo , Estado Nutricional , Obesidad/complicaciones , Obesidad/metabolismo , Selenoproteínas/biosíntesisRESUMEN
To investigate if differences in imprinting at tropho-microRNA (miRNA) genomic clusters can distinguish between pre-gestational trophoblastic neoplasia cases (pre-GTN) and benign complete hydatidiform mole (CHM) cases at the time of initial uterine evacuation. miRNA sequencing was performed on frozen tissue from 39 CHM cases including 9 GTN cases. DIO3, DLK1, RTL1, and MEG 3 mRNA levels were assessed by qRT-PCR. Protein abundance was assessed by Western blot for DIO3, DLK1, and RTL1. qRT-PCR and Western blot were performed for selenoproteins and markers of oxidative stress. Immunohistochemistry (IHC) was performed for DIO3 on an independent validation set of clinical samples (n = 42) and compared to normal placenta controls across gestational ages. Relative expression of the 14q32 miRNA cluster was lower in pre-GTN cases. There were no differences in protein abundance of DLK1 or RTL1. Notably, there was lower protein expression of DIO3 in pre-GTN cases (5-fold, p < 0.03). There were no differences in mRNA levels of DIO3, DLK1, RTL1 or MEG 3. mRNA levels were higher in all CHM cases compared to normal placenta. IHC showed syncytiotrophoblast-specific DIO3 immunostaining in benign CHM cases and normal placenta, while pre-GTN cases of CHM lacked DIO3 expression. We describe two new biomarkers of pre-GTN CHM cases: decreased 14q32 miRNA expression and loss of DIO3 expression by IHC. Differences in imprinting between benign CHM and pre-GTN cases may provide insight into the fundamental development of CHM.
Asunto(s)
Progresión de la Enfermedad , Regulación Enzimológica de la Expresión Génica/fisiología , Enfermedad Trofoblástica Gestacional/enzimología , Mola Hidatiforme/enzimología , Yoduro Peroxidasa/biosíntesis , Adolescente , Adulto , Estudios de Cohortes , Femenino , Enfermedad Trofoblástica Gestacional/genética , Enfermedad Trofoblástica Gestacional/patología , Humanos , Mola Hidatiforme/genética , Mola Hidatiforme/patología , Yoduro Peroxidasa/deficiencia , Yoduro Peroxidasa/genética , Embarazo , Selenoproteínas/biosíntesis , Selenoproteínas/deficiencia , Selenoproteínas/genética , Adulto JovenRESUMEN
Selenocysteine (Sec), a rare naturally proteinogenic amino acid, is the major form of essential trace element selenium in living organisms. Selenoproteins, with one or several Sec residues, are found in all three domains of life. Many selenoproteins play a role in critical cellular functions such as maintaining cell redox homeostasis. Sec is usually encoded by an in-frame stop codon UGA in the selenoprotein mRNA, and its incorporation in vivo is highly species-dependent and requires the reprogramming of translation. This mechanistic complexity of selenoprotein synthesis poses a big challenge to produce synthetic selenoproteins. To understand the functions of natural as well as engineered selenoproteins, many strategies have recently been developed to overcome the inherent barrier for recombinant selenoprotein production. In this review, we will describe the progress in selenoprotein production methodology.
Asunto(s)
Ingeniería Genética , Selenocisteína/genética , Selenoproteínas/genética , Homeostasis , Humanos , Oxidación-Reducción , Selenocisteína/metabolismo , Selenoproteínas/biosíntesis , Selenoproteínas/metabolismoRESUMEN
The trace element selenium (Se) is a crucial element for many living organisms, including soil microorganisms, plants and animals, including humans. Generally, in Nature Se is taken up in the living cells of microorganisms, plants, animals and humans in several inorganic forms such as selenate, selenite, elemental Se and selenide. These forms are converted to organic forms by biological process, mostly as the two selenoamino acids selenocysteine (SeCys) and selenomethionine (SeMet). The biological systems of plants, animals and humans can fix these amino acids into Se-containing proteins by a modest replacement of methionine with SeMet. While the form SeCys is usually present in the active site of enzymes, which is essential for catalytic activity. Within human cells, organic forms of Se are significant for the accurate functioning of the immune and reproductive systems, the thyroid and the brain, and to enzyme activity within cells. Humans ingest Se through plant and animal foods rich in the element. The concentration of Se in foodstuffs depends on the presence of available forms of Se in soils and its uptake and accumulation by plants and herbivorous animals. Therefore, improving the availability of Se to plants is, therefore, a potential pathway to overcoming human Se deficiencies. Among these prospective pathways, the Se-biofortification of plants has already been established as a pioneering approach for producing Se-enriched agricultural products. To achieve this desirable aim of Se-biofortification, molecular breeding and genetic engineering in combination with novel agronomic and edaphic management approaches should be combined. This current review summarizes the roles, responses, prospects and mechanisms of Se in human nutrition. It also elaborates how biofortification is a plausible approach to resolving Se-deficiency in humans and other animals.
Asunto(s)
Biofortificación , Ácido Selénico/metabolismo , Selenio/metabolismo , Selenoproteínas/metabolismo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Humanos , Plantas/metabolismo , Ácido Selénico/química , Selenio/química , Selenocisteína/química , Selenocisteína/metabolismo , Selenometionina/química , Selenometionina/metabolismo , Selenoproteínas/biosíntesis , Suelo/químicaRESUMEN
The study investigated biosynthesis of selenoproteins by Saccharomyces. cerevisiae using inorganic selenium. Selenium supplement via two stages was carried out during fermentation and the physicochemical characteristics of selenoproteins and its antioxidant activities were examined through in vitro assessment procedures. After fermentation, dry cells weight (7.47 g/L) and selenium content (3079.60 µg/kg) in the yeast were achieved when fermentation time points at the 6th hour and the 9th hour were chosen to supplement 30% and 70% of 30 µg/mL Na2SeO3 respectively. A maximal yield of selenium content in selenoproteins reached 1013.07 µg/g under optimized culture conditions and was 133-fold higher than the control. One new band with molecular weight of 26.76 KDa appeared in conjugated selenoproteins of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Surface structure of selenoproteins and the control was different by Scanning electron microscopy images. Infrared spectrometry analysis demonstrated that groups of HSe, SeO and C-Se-O involved in selenoproteins were important pieces of evidence showing presence of Se embedded in the protein molecule. Selenoproteins showed strong antioxidant activities on DPPH·, OH and ·O2-, which was much higher than the control proteins. Therefore, the study provided an efficient selenium-enriched culture method of inorganic selenite to organic selenium and basis for selenoproteins applications.
Asunto(s)
Antioxidantes/metabolismo , Antioxidantes/farmacología , Biosíntesis de Proteínas , Saccharomyces cerevisiae/metabolismo , Selenoproteínas/biosíntesis , Selenoproteínas/farmacología , Fenómenos Químicos , Depuradores de Radicales Libres/metabolismo , Depuradores de Radicales Libres/farmacología , Peso Molecular , Selenio/metabolismo , Análisis EspectralRESUMEN
Previously, selenoprotein T (SelT) expression was shown to be induced in nervous, endocrine, and metabolic tissues during ontogenetic and regenerative processes. However, whether SelT plays a critical role in renal diseases remains unclear. Here, we explored the role of SelT in cisplatin-induced acute kidney injury (AKI). Results revealed that SelT was highly expressed in renal tubules, but its expression was significantly reduced in cisplatin-induced AKI. Importantly, knocking down of SelT expression in kidney cells in vitro resulted in cisplatin-induced cell apoptosis, as indicated by the elevation of cleaved-PARP and Bax expression, Caspase-3 activity, and number of TUNEL-positive cells. Moreover, SelT silencing-induced reactive oxygen species (ROS) production, accompanied by a decrease in intracellular superoxide dismutase (SOD) and catalase (CAT) activity and increase in malondialdehyde (MDA) content. Notably, the protein and mRNA levels of Nox4 were increased in response to SelT downregulation. Furthermore, suppression of Nox4 expression by GKT137831 partially alleviated SelT knockdown-induced ROS generation and cell apoptosis in cisplatin-treated kidney cells. Taken together, our findings provide the first evidence that SelT protects against cisplatin-induced AKI by suppression of oxidative stress and apoptosis.
Asunto(s)
Lesión Renal Aguda , Apoptosis/efectos de los fármacos , Cisplatino/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Selenoproteínas/biosíntesis , Reductasa de Tiorredoxina-Disulfuro/biosíntesis , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Animales , Cisplatino/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Ratas , Ratas Endogámicas WKY , Ratas Sprague-DawleyRESUMEN
Cadmium (Cd), a heavy metal contaminant, exists in humans and animals throughout life and closely associate with severe hepatotoxicity. Selenium (Se) has been recognized as an effective chemo-protectant of Cd, but the underlying mechanisms remain unclear. The objective of the present study is to illustrate the antagonistic effect of Se against Cd-induced hepatotoxicity. Primary hepatocytes were cultured in the presence of 5 µM Cd, 1 µM Se and the mixture of 1 µM Se and 5 µM Cd for 24 h. Cell viability and morphology, antioxidant status, endoplasmic reticulum (ER) stress response and selenotranscriptome were assessed. It was observed that Se treatment dramatically alleviated Cd-induced hepatocytes death and morphological change. Simultaneously, Se mitigated Cd-induced oxidative stress by reducing ROS production, increasing reduced glutathione (GSH) level and increasing selenoenzyme (glutathione peroxidase, GPX) activity. Cd induced hepatotoxicity via disordering ER-resident selenoproteins transcription and triggering ER stress and unfolded protein response. Supplementary Se evidently relieved hepatocytes injury via modulating ER-resident selenoproteins transcription to inhibit ER stress. Collectively, our findings showed a potential protection of Se against Cd-induced hepatotoxicity via suppressing ER stress response.
Asunto(s)
Cadmio/toxicidad , Retículo Endoplásmico/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Selenio/farmacología , Selenoproteínas/biosíntesis , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Retículo Endoplásmico/metabolismo , Estrés del Retículo EndoplásmicoRESUMEN
The trace elements iron and selenium play decisive roles in a distinct form of necrotic cell death, known as ferroptosis. While iron promotes ferroptosis by contributing to Fenton-type reactions and uncontrolled lipid autoxidation, the hallmark of ferroptosis, selenium in the form of glutathione peroxidase 4 (GPX4), subdues phospholipid peroxidation and associated cell death. Beyond the canonical cystine/glutamate antiporter system xc-/glutathione/GPX4 nexus, recent studies unveiled the second mainstay in ferroptosis entailing extra-mitochondrial ubiquinone, ferroptosis suppressor protein 1, and NAD(P)H as electron donor. Unlike GPX4, this selenium- and thiol-independent system acts on the level of peroxyl radicals in membranes, thereby restraining lipid peroxidation. Therefore, ferroptosis is a multifaceted cell-death paradigm characterized by several metabolic networks, whereby metabolic dyshomeostasis may cause ferroptotic cell death and organ failure. Here, we discuss the basic features of ferroptosis with a focus on selenium, offering exciting opportunities to control diseases linked to ferroptosis, including transient ischemia/reperfusion and neurodegeneration.
Asunto(s)
Ferroptosis , Selenio/metabolismo , Humanos , Hierro/metabolismo , Peroxidación de Lípido , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Proteína de Unión al Calcio S100A4/metabolismo , Selenio/química , Selenoproteínas/biosíntesis , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The search for potential partners of human SELM in lysates of two cancer cell lines, HT-1080 (fibrosarcoma) and MCF-7 (breast adenocarcinoma), was carried out. Two cytoplasmic actin isoforms-cytoplasmic actin 1 (cytoskeleton ß-actin) and cytoplasmic actin 2 (cytoskeletal γ-actin)-were identified as partners. In addition, the influence of two widely used antitumor selenium compounds (sodium selenite and methylseleninic acid) on the expression of SELM in cancer cells was studied. On the basis of the results obtained by real-time PCR and Western blotting, we concluded that 1 µM and 10 µM sodium selenite did not affect the expression of SELM in fibrosarcoma cells, whereas in breast adenocarcinoma cells 1 µM sodium selenite slightly increased expression and 10 µM sodium selenite significantly (approximately 2 times) decreased it. Methylseleninic acid in both cancer cell lines increased the SELM gene expression; the most pronounced effect was observed when fibrosarcoma cells were treated with 10 µM MSA (the expression of the hSelm gene increased almost 4 times).
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Fibrosarcoma/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Compuestos de Organoselenio/farmacología , Selenoproteínas/biosíntesis , Adenocarcinoma/patología , Neoplasias de la Mama/patología , Fibrosarcoma/patología , Humanos , Células MCF-7RESUMEN
Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3' UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S (SelS). Here we report that the first 91 nucleotides of the SelS 3' UTR contain a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR element greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or an unstructured sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3' UTR reveals a more complex regulation of Sec incorporation than previously realized.
Asunto(s)
Bioensayo , Codón de Terminación/metabolismo , Secuencias Invertidas Repetidas , Terminación de la Cadena Péptídica Traduccional , Selenoproteínas/biosíntesis , Regiones no Traducidas 3' , Animales , Línea Celular Tumoral , Codón de Terminación/química , Secuencia Conservada , Células HEK293 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Espectrometría de Masas , Conformación de Ácido Nucleico , Mutación Puntual , Ratas , Selenocisteína/química , Selenocisteína/metabolismo , Selenoproteínas/genéticaRESUMEN
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.
Asunto(s)
Codón de Terminación , Extensión de la Cadena Peptídica de Translación/genética , Selenocisteína , Selenoproteínas , Animales , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/biosíntesis , Selenoproteínas/genéticaRESUMEN
Recoding 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 RNA-binding 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 recoding 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.
Asunto(s)
Errores Innatos del Metabolismo/genética , Mutación Missense , Proteínas de Unión al ARN/metabolismo , Selenoproteínas/biosíntesis , Animales , Sitios de Unión , Encéfalo/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenotipo , Unión Proteica , Estabilidad Proteica , Proteolisis , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribosomas/metabolismo , Selenocisteína/metabolismoRESUMEN
Selenium is an essential trace element which is incorporated in the form of a rare amino acid, the selenocysteine, into an important group of proteins, the selenoproteins. Among the twenty-five selenoprotein genes identified to date, several have important cellular functions in antioxidant defense, cell signaling and redox homeostasis. Many selenoproteins are regulated by the availability of selenium which mostly occurs in the form of water-soluble molecules, either organic (selenomethionine, selenocysteine, and selenoproteins) or inorganic (selenate or selenite). Recently, a mixture of selenitriglycerides, obtained by the reaction of selenite with sunflower oil at high temperature, referred to as Selol, was proposed as a novel non-toxic, highly bioavailable and active antioxidant and antineoplastic agent. Free selenite is not present in the final product since the two phases (water soluble and oil) are separated and the residual water-soluble selenite discarded. Here we compare the assimilation of selenium as Selol, selenite and selenate by various cancerous (LNCaP) or immortalized (HEK293 and PNT1A) cell lines. An approach combining analytical chemistry, molecular biology and biochemistry demonstrated that selenium from Selol was efficiently incorporated in selenoproteins in human cell lines, and thus produced the first ever evidence of the bioavailability of selenium from selenized lipids.
Asunto(s)
Aceites de Plantas/metabolismo , Ácido Selénico/metabolismo , Ácido Selenioso/metabolismo , Compuestos de Selenio/metabolismo , Selenoproteínas/biosíntesis , Triglicéridos/metabolismo , Línea Celular Tumoral , Células HEK293 , HumanosRESUMEN
Selenium (Se), an essential trace mineral, confers its physiological functions mainly through selenoproteins, most of which are oxidoreductases. Results from animal, epidemiological, and human genetic studies link Parkinson's disease to Se and certain selenoproteins. Parkinson's disease is characterized by multiple motor and non-motor symptoms that are difficult to diagnose at early stages of the pathogenesis. While irreversible, degenerative and age-related, the onset of Parkinson's disease may be delayed through proper dietary and environmental controls. One particular attribute of Se biology is that brain has the highest priority to receive and retain this nutrient even in Se deficiency. Thus, brain Se deficiency is rare; however, a strong body of recent evidence implicates selenoprotein dysfunction in Parkinson's disease. Direct and indirect evidence from mouse models implicate selenoprotein T, glutathione peroxidase 1, selenoprotein P and glutathione peroxidase 4 in counteracting Parkinson's disease through Se transportation to the brain and reduced oxidative stress. It is of future interest to further characterize the full selenoproteomes in various types of brain cells and elucidate the mechanism of their actions in Parkinson's disease.