An introduction to

THE IMPORTANCE OF SELENOPROTEINS

A brief description of selenoproteins

Selenium is one of the nine essential trace elements and it has vital functions regarding to the maintenance of homeostasis. Selenium deficiency may cause physiopathological status while an excess could cause toxicity. Selenoproteins incorporate at least one selenium-containing amino acid (Sec or U), this amino acid is identical to a cysteine but enclose a selenol group instead of a thiol group and it is considered the 21st “naturally occurring” amino acid in the genetic code (Fig1). Selenoproteins are mostly redox enzymes with antioxidant protection capacity, are found in all majors domains of life, even in viruses, suggesting they have an important role and they have been conserved. However, this kind of proteins are completely absent in some phylogenetic groups indicating that selenoproteins are not essential in all living beings. The size of the selenoproteome varies significantly among eukaryotes and the highest amount of selenoproteins is observed in aquatic organisms (both animals and plants).

Fig1. The codon for Sec is UGA which usually performs as a stop codon. Then, in order to transcribe this codon as an amino acid, and prevent translation termination, an special translational machinery is needed. Labunskyy VM, Hatfield DL, Gladyshev VN. Selenoproteins: molecular pathways and physiological roles. Physiol Rev. 2014;94(3):739–777.

Biosynthesis of Selenoproteins

In eukaryotes, the process of synthesising selenoproteins has a high grade of translational control. The translation of the UGA codon is drawn out bt cis-acting factors (SECIS elements and in-frame UGA codon) and trans-acting factors (SPS1, SPS2, SecS, PTSK, eEFsec, SBP2 and the Sec-tRNA[Ser]Sec).

Sec biosynthesis occurs on its own tRNA, which has to suffer biochemical modifications and incorporate selenium in a monoselenophosphate form, this form is obtained from the dietary selenium which is modified by SPS enzymes. The tRNA[Ser]Sec is described as the key molecule and the central component of selenoprotein biosynthesis and it has unique features that makes it different from other tRNAs. It is the longest tRNA sequenced and its length vary from 90 to 93 nucleotides in eukaryotes to well over 100 in bacteria. Because of the unusual transcription of this tRNA, a triphosphate moiety is attached to its 5’-terminal guanosine nucleotide to prevent the processing of the 5’ leader sequence and enable transcription to start at the first nucleotide of the coding sequence.

The gene for tRNA[Ser]Sec is designated as Trsp and its expression is regulated by a 3’ downstream Pol III termination signal and 5’ flanking sequences that include three upstream regulatory elements; a TATA box motif (-30 nt), a proximal sequence element (-70 nt) and a distal sequence element (-200 nt).

In order to incorporate the Sec-tRNA into the peptide under translation, SECIS binding protein 2 (SPB2) has to bind to the SECIS element and interact with the Sec-specific elongation factor (eEFSec) which facilitate the incorporation of Sec into the growing polypeptide. SECIS elements are located on 3’-UTR in eukarya and archaea domains while in bacteria SECIS elements are usually localized immediately downstream near the UGA codon. This sequence contains approximately 60 nucleotides that adopt a stem-loop secondary structure which acts as a cis-element that recognizes trans-acting factors and directs them to the ribosomes. The trans-acting factors are SPB2 which associates with ribosomes and SECIS elements with a high affinity and specificity. Moreover, SPB2 also interacts with the second trans-acting factor, eEFSec, which is implied in the recruitment Sec-tRNA[Ser]Sec and facilitates the incorporation of Selenocysteine in the translated peptide (Fig 2). Usually, none of these proteins which are involved in selenoprotein machinery contain Sec in their amino acid sequence but all of them are essential for the physiological function of the cells. Mutations on the genes that codify for these proteins lead to pathological circumstances. D.rerio contains in its genome 2 homologs for SBP2 and 2 homologs for SECp43.

Fig2. Mechanism of Sec insertion in eukaryotes. Labunskyy VM, Hatfield DL, Gladyshev VN. Selenoproteins: molecular pathways and physiological roles. Physiol Rev. 2014;94(3):739–777.

Background: Selenoproteins in Zebrafish (D.rerio)

We have compared all the 47 proteins of Zebrafish found in SelenoDB, 13 of them were Cys-homologues while the rest contained a selenocysteine and were annotated as selenoproteins. Aquatic organisms have larger selenoproteome than terrestrial living beings. Human selenoproteome is encoded in 25 selenoprotein genes but zebrafish carries 38 known selenoproteins and is the animal model genome we’ve chosen to search for the selenoproteins of Callopanchax toddi because of they phylogenetic closeness.

Teleosts are an infraclass of a Actinopterygii and both D.rerio and C.toddi belong to this group. There is solid evidence that a whole-genome duplication took place in the common ancestor of all teleosts. This event could have lead to the evolution of one of the two newborn paralogues ending with the acquisition of a new selenoprotein function or a new cys-homologue. In fact, D. rerio is considered to have multiplicity of selenoprotein genes that appeared from the whole-genome duplication, followed by the retention of new selenoprotein genes. We have compared all the 47 proteins of Zebrafish found in SelenoDB, 13 of them were Cys-homologues while the rest contained a selenocysteine and were annotated as selenoproteins.

Selenoprotein families

Sec is the main element in charge of the physiological functions of selenoproteins, it is found on the active site of the enzyme and perform redox reactions. Current documented selenoproteins are classified on their function:

Iodothyronine deiodinases (DIO)

The iodothyronine deiodinase family of selenoproteins consist of three paralogous proteins in mammals (DI1, DI2 and DI3) which are involved in reductive deamination, they are essential for the regulation of thyroid hormone activity. These proteins are widely extended, not only along eukaryotes, but also in some other kingdoms as bacteria. D.rerio has four DIO proteins annotated: DIO1, DIO2, DIO3a and DIO3b. DIO1 is present in the cell membrane and is in charge of the metabolization of T4 into T3 hormone in peripheral tissues, this process can be also performed by DIO2 (localized in the endoplasmic reticulum). DIO 3 can only metabolize T4 into reverse triiodothyronine (rT3), which is biologically inactive.

Glutathione peroxidases (GPx)

Glutathione peroxidase family is widespread in all three domains of life. In mammals, there are eight GPx paralogs, from which five (GPx1, GPx2, GPx3, GPx4, and GPx6) contain a Sec residue in their active site while in the other three GPx the active-site Sec is replaced by Cys. D.rerio has 9 GPx selenoproteins: GPx1a, GPx1b, GPx2, GPx3a, GPx3b, GPx4a, GPx4b, GPx7 and GPx8. In this organism, only GPx7 and GPx8 have lost the Sec amino acid and contain Cys.

GPx selenoproteins are responsible of part of hydrogen peroxide signaling, detoxification of hydroperoxides and the maintenance of cellular redox homeostasis. GPx1 in considered an important antioxidant molecule and belongs to a group of stress-related selenoproteins. It is regulated by selenium availability, in fact, during selenium deficiency, the level of GPx1 is reduced. However, GPx4 is less affected by this metal availability maybe because it is a housekeeping selenoprotein. Specifically, GPx1 it is in charge of the catalysis of glutathione-dependent reduction of hydrogen peroxide to water. As hydrogen peroxide is known to bé toxic to the cells, GPx1 is considered an important antioxidant molecule. GPx2 has been shown to play a role in the development of cancer.

Thioredoxin reductases (TXNRDs)

Thioredoxin reductases (TXNRDs) are oxidoreductases that combined with thioredoxin (Trx), comprise the major disulfide reduction system of the cell. All TR protein isoforms contain Sec in mammals and also in D.rerio. TRs are flavoenzymes enrolled in reducing thioredoxins and have an important role in redox homoeostasis. There are three TRs which are present in all vertebrates despite of Danio rerio and other fish species, in which TXNRD1 is not found.

Methionine sulfoxide reductases A (MsrA)

Msr A is highly conserved. Its main function is to catalyze the enzymatic reduction of the amino acid methionine-S-sulfoxide by using thioredoxin. Despite MsrA and MSRB families are structurally different and do not present similarities in their sequences, they have complementary functions and share part of the name.

Selenoprotein R (MSRB family)

Methionine-R-sulfoxide reductase 1 (MSRB1) is a zinc-containing selenoprotein that was previously identified as Selenoprotein R (SelR) and Selenoprotein X (SelX). MSRB selenoproteins function is the stereospecific reduction of methionine-R-sulfoxide. MSRB1 is the main MSRB protein present in mammals and is localized in the cytosol and in the nucleus. Nevertheless, two additional homologs (MSRB2 and MSRB3) show lower levels of expression but their catalytic efficiency is similar to MSRB1. MSRB2 is located in mitochondrias, whereas MSRB3 is targeted to the ER. MSRB1a, MSRB1b, MSRB2 and MSRB3 have been annotated in D. rerio.

15-kDa selenoprotein (Sel15)

Sel15 is known in mammals as Sep15, which is together with Selenoprotein M (SelM) thioredoxin-like fold ER-resident protein. This Sep15 is known to form a complex with an ER-resident chaperone (UGGT) which is involved in calnexin regulation cycle, which is essential for the folding process of some glycoproteins in the ER, and its expression is induced by misfolded proteins in ER.

Selenoprotein E (SELENOE)

SELENOE coding gene is part of SEP15 family. This selenoprotein has been described only in fish. Probably, this gene arose from a gene duplication but its function remains unknown.

Selenoprotein H (SELENOH)

SELENOH is a stress-related selenoprotein. This protein is widespread over different domains and encodes for a nucleolar protein from the SelWTH family. In mammals, it has been shown to protect neurons against UVB damage, promote mitochondrial biogenesis and suppress cellular senescence.

Selenoprotein I (SELENOI)

SELENOI is one of the most recent evolved selenoproteins and it can only be found in vertebrates. It is a multi-pass transmembrane protein encoded by a CDP-alcohol phosphatidyltransferase class-I family gene. Its main function is to produce phosphatidylethanolamine which is necessary for the formation and maintenance of vesicular membranes. It is also involved in lipid metabolism and in protein folding.

Selenoprotein J (SELENOJ)

SELENOJ is one of the vertebrate-specific selenoproteins and is highly restricted in phylogeny, being found only in actinopterygian fishes and sea urchin. It has a potential role during embryogenesis, preferentially expressed in the eye lens. In Danio rerio two homologs have been described SELENOJ1 and SELENOJ1.2.

Selenoprotein K (SELENOK)

SELENOK is known to be related with SelS, based on their topology. It is a transmembrane protein located in the endoplasmic reticulum. It is involved in mechanisms in charge of endoplasmic reticulum associated degradation of proteins that are glycosylated and misfolded. It also protects cells against endoplasmic reticulum stress-induced apoptosis.

Selenoprotein L (SELENOL)

SELENOL is encoded by a gene that belongs to a subfamily of selenoprotein L, within a superfamily of thioredoxin (Txr). It is restricted in aquatic organisms and, among vertebrates it is only found in fishes. In SELENOL, the UxxU motif replaces the catalytic CxxC motif in thioredoxins (it possesses a thioredoxin-like fold), and that suggests a redox function for this protein family. Then it is important to notice that typically Selenoprotein L contains two Sec residues.

Selenoprotein M (SELENOM)

SELENOM is, together with Sel15, a thioredoxin-like folds ER-resident proteins. In fact, SELENOM is a distant homolog of Sel15 which is encoded by genes that belong to selenoprotein M/SEP15 family. It is present in vertebrates but is specially highly expressed in mammals. Its function remains unknown but it may be involved in maintaining redox homeostasis.

Selenoprotein N (SELENON)

SELENON is a glycoprotein localized in the endoplasmic reticulum. It is implied in redox regulation of calcium homeostasis and in cell protection against oxidative stress. It has an important role in muscle organization in early development in zebrafish.

Selenoproteins O (SELENOO)

This protein is localized in the mitochondria, and it is the largest selenoprotein present in mammals. In Danio rerio two homologs have been described SELENOO1 and SELENOO2.

Selenoproteins P (SELENOP)

SELENOP is a protein that is abundantly expressed and secreted to the plasma (accounts for approximately 50% of the total Se of the plasma). This protein has recently evolved and SelP homologous are found predominantly in vertebrates. Danio rerio shows two different homologs: SELENOP1 and SELENOP2. This selenoprotein is unique because it contains 17 Sec residues per polypeptide in zebrafish. It is implicated as an extracellular antioxidant.

Selenoprotein S (SELENOS)

SELENOS could be assigned to a single SelK/SelS family (related by topology). This protein encodes for a transmembrane protein that is localized in the endoplasmic reticulum. It is involved in the degradation process of misfolded proteins and it can also have a role in inflammation control in higher eukaryotes.

Selenoproteins T (SELENOT)

SELENOT proteins belong to the Rdx family together with SelW, SelH and selV. This proteins are found in the endoplasmic reticulum. As the name of the family already indicates, it possesses a thioredoxin-like fold and a conserved CxxU motif. In Danio rerio three homologs have been described: SELENOT1, SELENOT1b,SELENOT2.

Selenoproteins U (SELENOU)

SELENOU proteins are a family within a peroxiredoxin-like FAM213 superfamily. SELENOU is phylogenetically restricted in vertebrates such as chicken and fish, while other vertebrate members contain cysteine-containing homologs. SELENOU contains UxxC motif where the cysteine-containing homologs have CxxC (catalytic site). In Danio rerio three homologs have been described: SELENOU1a, SELENOU2 and SELENOU3.

Selenoproteins W (SELENOW)

SELENOW1 belongs to SelWTH family, which possesses a thioredoxin-like fold and a conserved CxxU motif, that suggests a redox function of this protein. In Zebrafish, this protein is expressed in sensory organs.

Selenophosphate synthetases (SEPHS)

SEPHS is responsible of the synthesis of selenophosphate (the active selenium donor). Two SEPHS paralogues (SEPHS1 and SEPHS2) are common in some eukaryotes but, only SEPHS2 shows catalytic activity during selenophosphate synthesis. However, SEPHS1 sems to play an essential role in regulating cellular physiology. In Danio rerio two homologs have been annotated: SEPHS and SEPHS2.