6

Selenoprotein T

Yannick Tanguy, Sébastien Arthaud, Anthony Falluel-Morel, Destiny-Love

Manecka, Abdeslam Chagraoui, Isabelle Lihrmann, Youssef Anouar

INSERM U982, Neuronal and Neuroendocrine Differentiation and Communication

Laboratory, IFRMP23, University of Rouen, 76821 Mont-Saint-Aignan, France

E-mail: youssef.anouar@univ-rouen.fr

Selenoprotein T (SelT) has been recently identified as a member of the redoxin

protein family, based on the occurrence in its primary structure of a “thioredoxin-

like fold” containing a selenocystein. Few studies have been reported on the

distribution of SelT, showing its low expression in adult tissues and its abundance

during embryogenesis. A pangenomic microarray analysis allowed us to identify

SelT as a gene stimulated by a trophic neuropeptide, the pituitary adenylate

cyclase-activating polypeptide, during neuronal differentiation. It was shown that

SelT is mainly localized in the endoplasmic reticulum and participates actively to

intracellular Ca2+

homeostasis. Other genomic studies revealed that SelT gene

expression is stimulated upon tissue injury, suggesting that the selenoprotein could

also play an important role in protection against oxidative stress.

6.1� Introduction

In 1999, Kryukov and collaborators reported the identification of two new

selenoproteins, selenoprotein R and selenoprotein T (SelT). This discovery was

made possible by the development of a specific computer program, named

SECISearch, which recognizes selenoprotein genes by identifying selenocystein

insertion sequences (SECIS). This bioinformatic tool identifies the quartet consensus

motif of the SECIS element in all expressed sequence tag (EST) libraries, and

evaluates the potential of correct folding as well as the energetic stability of this

J. Liu et al. , Selenoproteins and Mimics© Zhejiang University Press, Hangzhou and Springer-Verlag Berlin Heidelberg 2011

(eds.)

6� Selenoprotein T 90

particular sequence. Added to the presence of a UGA codon in the open-reading

frame of the putative ESTs, these criteria allowed the identification of SelT [1]

.

6.2� Sequence Analysis of SelT

Molecular cloning allowed us to characterize SelT cDNA sequence which

encompasses 970 nucleotides encoding a protein of 195 amino acid residues with

a calculated mass of 22.3 kDa [1, 2]

. The selenocystein (U) residue of SelT is

present in its N-terminal part, and is separated from a cystein (C) residue by two

amino acids, thus forming a powerful redox center (CVSU) which is found in

other selenoproteins [1]

. Moreover, this molecular signature is comprised between

predicted β-strand and α-helix [3]

secondary structures (Fig. 6.1), a necessary

conformation found in other redox proteins with a traditional thioredoxin fold

(CXXC), such as thioredoxins, glutaredoxins and disulfide isomerases [4]

. Owing

to these structural properties, SelT, along with five other selenoproteins, SelM,

Sel15, SelV, SelH and SelW, belong to a new redoxin protein family named the

the thioredoxin-like family, whose members exhibit these domains [3, 5]

.

Fig. 6.1.� Structure of SelT. Bioinformatic analyzes permitted us to identify α-helix and β-

strand secondary structures

and other specific domains such as a signal peptide a “thioredoxin

like domain” and a transmembrane domain (TMD)

The CXXC or CXXU sequences are key motifs for various functions of

selenoproteins [6]

. Among these, some “thioredoxin-like” selenoproteins possess a

glutathione peroxidase activity, as was demonstrated for SelH [7]

, or a protein

folding activity which was shown for SelW or Sel15 [8]

. The protein SelT also

exhibits a hydrophobic amino acid stretch which may represent a transmembrane

domain (TMD) [2]

. Computer-based, sequence comparative analyses revealed the

presence of SelT homologous sequences in plants, protozoans, zebrafish and other

mammals [1]

, the SelT protein sequence being extremely well conserved during

evolution (Fig. 6.2).

6.3� Tissue-distribution and Regulation 91

Fig. 6.2.� Comparison of SelT sequences. Comparison of SelT protein sequences showing a

high conservation during evolution, since 65% of SelT amino acids are conserved at 90% or

more. The putative redox center is boxed, and the amino acids are represented according to their

nature (hydrophobic, in red; hydrophilic, in green; acid, in blue; and basic, in pink)

In protozoa, zebrafish and plants, SelT homologs, contain a C instead of a U

residue in the thioredoxin motif. In zebrafish, three SelT orthologs were identified [9]

.

Pairwise sequence alignment analyses of mammalian SelW, SelT, SelH and SelV

selenoenzymes indicated no significant similarities between these proteins which

seem to be distant homologs [3]

. Nonetheless, these proteins are structurally related

since they exhibit a similar pattern of predicted secondary structures, with an

additional central α-helix domain in SelT [3]

.

6.3� Tissue-distribution and Regulation

As demonstrated for different selenoproteins, selenium is a key factor for their

biosynthesis, and its supplementation in a culture medium increases SelT levels in

mammalian cells [1, 10]

. However, SelT expression is probably very limited in adult

human tissues since its incidence in EST clones is particularly weak [1]

. Indeed, in a

dbEST library, only the infant brain, melanocytes and placenta displayed SelT

clones with an incidence of 1 per 10,000 ESTs. However, studies by RT-PCR [2]

or

Northern blot [3]

, of a broad range of adult rat tissues revealed SelT mRNA

expression in all analyzed samples. Other results obtained by Western blot analyses

showed that SelT is only detected in the brain, kidney, liver and testis of Sec-tRNA

overexpressing transgenic mice [3]

. By contrast, SelT is strongly and ubiquitously

expressed in proliferating and differentiating cells. Indeed, in situ hybridization

experiments showed that SelT mRNAs are abundantly expressed in all embryonic

tissues, from the earlier to the later stages [2]

. In zebrafish, the three SelT orthologs,

6� Selenoprotein T 92

SePT1a, SePT1b and SePT2, were all detected in embryos. The SePT1b and SePT2

forms exhibited a large tissue-distribution, whereas SePT1a was restricted to

certain neurectoderma tissues, such as olfactory vesicles, photoreceptor cell layer,

retina and epiphysis [11]

.

The expression of SelT was strongly induced during PC12 neuronal

differentiation [12, 13]

. Thus, under the effect of the trophic factor Pituitary Adelynate

Cyclase-Activated Polypeptide (PACAP), SelT mRNA and protein levels were

significantly stimulated during differentiation of PC12 pheochromocytoma cells

toward a neuronal phenotype [2, 14]

. This observation suggested that SelT, among

other PACAP-responsive genes, could play a role in this important cellular

process [14]

.

Although SelT levels are low in adult tissues, its expression could be induced

in pathophysiological conditions. Thus, a gene expression profiling experiment

revealed that SelT mRNA could be induced in hypoxic lungs [15]

. Another study

showed that SelT gene expression is stimulated following a prolonged cerebral

hypoxia [16]

. It is known that the neurodegeneration associated with cerebral

ischemia evokes the release of toxic molecules, like reactive oxygen species (ROS)

or glutamate, which amplifies the neuronal death. Under such conditions, it was

previously shown that the expression of another selenoprotein, GPx4, is stimulated

in reactive astrocytes [17]

. Owing to the potential redox activity of SelT and its

stimulation in stress conditions, it is possible that this selenoprotein may participate

to the defense response during tissue injury.

6.4� Function

Our previous studies suggested a role of SelT in PC12 neuronal differentiation.

Immunocytochemistry experiments revealed that SelT is mainly localized in the

endoplasmic reticulum [2, 4]

, in line with the work of others showing that the

protein is located not only in this compartment but also in the Golgi apparatus and

the mitochondria [3]

. These observations are supported by the presence of a signal

peptide and a transmembrane domain which may allow the integration of SelT in

the membrane of these organelles [2]

.

In the PC12 cell model, it was also demonstrated that PACAP is the only

peptide factor tested that was able to stimulate SelT gene expression [2]

. The use of

specific chemical blockers showed that mobilization of intracellular Ca2+

pools

and the activation of the protein kinase A pathway are key transduction

mechanisms implicated in SelT gene stimulation in response to PACAP [2]

. The

importance of Ca2+

in this action and the subcellular localization of SelT were

motivating arguments to investigate the implication of SelT in Ca2+

mobilization

from intracellular pools. When PC12 cells were transfected with a SelT-

expressing plasmid and analyzed by microfluorimetry, they exhibited a higher

cytosolic Ca2+

level compared to control cells [2]

. This effect was dependent on the

6.4� Function 93

selenium-containing center of SelT since the replacement of the Sec residue to an

Ala residue abolished the activity [2]

. Moreover, cell treatment with thapsigargin,

an inhibitor of intracellular Ca2+

reuptake in the ER, confirmed the action of SelT

in this compartment [2]

. The use of SelT-specific silencing RNA further established

this activity of SelT, which exclusively occurred in PACAP-differentiated cells.

Indeed, contrary to control cells, the PACAP-differentiated, SelT-deficient cells

were unable to sustain SelT action on intracellular Ca2+

concentration following

PACAP stimulation. Probably as a consequence, it was also shown that SelT

deficiency affects the regulated secretory activity of PACAP-treated PC12 cells

(Fig. 6.3) [2]

.

Fig. 6.3.� Intracellular function of SelT in PACAP-differentiated PC12 cells. During PC12 cell

differentiation, PACAP increases cytosolic Ca2+

concentration and stimulates the release of

catecholamines (green arrows). This effect could be reinforced by the stimulation of SelT gene

expression, since its presence in the endoplasmic reticulum permitted amplification of Ca2+

mobilization from the intracellular pools, probably through an interaction with Ca2+

channel

receptors (purple arrows)

6� Selenoprotein T 94

6.5� Conclusion

Although the investigations on the physiological role of SelT are still in their

infancy, the data obtained so far underline the importance of this selenoprotein for

the establishment of a differentiated neuronal phenotype. The first results

indicated that, like other selenoproteins, the redox selenium-containing center of

SelT is responsible for the effects of the protein. It has recently been shown that

selenoprotein N (SelN), which is mutated in certain dystrophies, is able to

modulate the activity of Ca2+

release channels through a redox mechanism [18]

.

Similarly, SelT could modulate, via the Sec active center, Ca2+

release channel

activity [2]

.

Interestingly, SelT displays many similarities with another selenoprotein,

selenoprotein W (SelW), which is also linked to muscle disease. The two

selenoproteins exhibit a similar expression pattern during embryogenesis and

brain development. SelW expression is low in white muscle of animals with

calcified skeletal and cardiac muscles due to a default in Ca2+

sequestration in the

sarcoplasmic reticulum [18, 19]

. In addition, SelW is complexed with glutathione in

the cytosol and is strongly expressed in proliferating myoblasts, in which the

selenoprotein participates in the degradation of ROS [20]

. Furthermore, it was

recently shown that the genetic invalidation of SelT in the fibroblastic NIH3T3

cell line leads to the up-regulation of many factors involved in redox regulation,

including SelW[21]

. The commonalities between SelT, SelN and SelW reinforce

the idea that SelT could play an important role in the control of Ca2+

homeostasis

and oxidative stress. Induction of SelT during tissue injury and the regulation of

its expression by the trophic factor PACAP, which exerts protective effects [21]

, are

strong arguments for such a role. These possibilities open new perspectives for the

characterization of the physiological and pathophysiological functions of SelT.

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