Selenoprotein T is a key player in ER proteostasis, endocrine homeostasis and neuroprotection

Author’s Accepted Manuscript Selenoprotein T is a key player in ER proteostasis, endocrine homoeostasis and neuroprotection Anouar Youssef, Isabelle Lihrmann, Anthony Falluel-Morel, Loubna Boukhzar www.elsevier.com

PII: DOI: Reference:

S0891-5849(18)30909-2 https://doi.org/10.1016/j.freeradbiomed.2018.05.076 FRB13778

To appear in: Free Radical Biology and Medicine Received date: 5 February 2018 Revised date: 18 May 2018 Accepted date: 20 May 2018 Cite this article as: Anouar Youssef, Isabelle Lihrmann, Anthony Falluel-Morel and Loubna Boukhzar, Selenoprotein T is a key player in ER proteostasis, endocrine homoeostasis and neuroprotection, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.05.076 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Selenoprotein T is a key player in ER proteostasis, endocrine homoeostasis and neuroprotection Anouar Youssefa,b,*, Isabelle Lihrmanna,b, Anthony Falluel-Morela,b, Loubna Boukhzara,b a

Rouen-Normandie University, UNIROUEN, INSERM, U1239, Neuronal and

Neuroendocrine Differentiation and Communication Laboratory, 76821 Mont-SaintAignan, France b

Institute for Research and Innovation in Biomedicine of Normandy, 76000 Rouen,

France

Keyword: Selenoprotein, Endoplasmic reticulum stress, Hormone regulation, Brain development, Protein processing

*Corresponding author at: Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, INSERM U1239, Institute for Research and Innovation in Biomedicine, Rouen-Normandie University, Mont-Saint-Aignan, France Email address: [email protected]

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Highlights: Selenoprotein T is a key regulator of ER homeostasis Selenoprotein T is involved in tissue development and homeostasis Selenoprotein T plays specific functions in endocrine cells Selenoprotein T exerts neuroprotective and regenerative actions Selenoprotein T could play a role in OST and GPI protein tagging

Abbreviations: ACTH, adrenocorticotropin hormone; Ca2+, calcium; cAMP, cyclic AMP; CRF, corticotropin-releasing factor; DAT, dopamine transporter; ER, endoplasmic reticulum; OST, Oligosaccharyl transferase; PACAP, pituitary adenylate cyclase-activating polypeptide; PD, Parkinson disease; PK, protein kinase; POMC, proopiomelanocortin; NRF, nuclear respiratory factor; Sec, selenocysteine, SECIS, selenocysteine insertion sequence; UPR, unfolded protein response.

Acknowledgments: The authors were supported by INSERM, Rouen-Normandie University, Conseil Régional de Normandie, the FEDER program of the European Union.

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ABSTRACT Selenoprotein T (SELENOT, SELT) is a thioredoxin-like enzyme anchored at the endoplasmic reticulum (ER) membrane, whose primary structure is highly conserved during evolution. SELENOT is abundant in embryonic tissues and its activity is essential during development since its gene knockout in mice is lethal early during embryogenesis. Although its expression is repressed in most adult tissues, SELENOT remains particularly abundant in endocrine organs such as the pituitary, pancreas, thyroid and testis, suggesting an important role of this selenoprotein in hormone production. Our recent studies showed indeed that SELENOT plays a key function in insulin and corticotropin biosynthesis and release by regulating ER proteostasis. Although SELENOT expression is low or undetectable in most cerebral structures, its gene conditional knockout in brain provokes anatomical alterations that impact mice behavior. This suggests that SELENOT also plays an important role in brain development and function. In addition, SELENOT is induced after injury in brain or liver and exerts a cytoprotective effect. Thus, the data gathered during the last ten years of intense investigation of this newly discovered thioredoxin-like enzyme point to an essential function during development and in adult endocrine organs or lesioned brain, most likely by regulating ER redox circuits that control homeostasis and survival of cells with intense metabolic activity.

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1. Introduction Given the essential role of selenoproteins such as the glutathione peroxidases, iodothyronine deiodinases or thioredoxin reductases in cell homeostasis, the emphasis was placed recently on several novel selenoproteins with no known function so far, in order to establish their physiological and pathophysiological relevance. In pioneering studies, Gladyshev and co-workers [1,2] identified new selenoproteins using an algorithm that searches for primary and secondary structures as well as free energy requirements to form a stem-loop structure only present in the 3’-untranslated sequence of the mRNAs encoding selonoproteins. This structure named the selenocysteine (Sec) insertion sequence (SECIS) allows the decoding of an in-frame UGA stop codon as a Sec in selenoproteins. Using this bioinformatics approach, two new members of the selenium-containing protein family, with completely unknown function have been uncovered in databases of human expressed sequence tags from various tissues [1]. The first one is selenoprotein R (SELENOR) which is currently known as methionine sulforeductase B [3] and which has been linked to methionine redox control in different proteins [4], including actin for the regulation of its assembly/disassembly [5]. Selenoprotein T (SELENOT) was the second newly identified selonoprotein using the bioinformatics; however, this selenoprotein has not received as much attention as SELENOR until we identified its transcript in a microarray study as an upregulated gene during neuroendocrine cell differentiation in response to the neurotrophic factor pituitary adenylate cyclase-activating polypeptide (PACAP) [6]. Since this transcriptomic analysis was performed on a mouse embryonic-placental cDNA library, it was clear that SELENOT ought to have a function during embryonic development, and to contribute to the effects of trophic factors during ontogenesis, like PACAP which has antioxidant, prosurvival and prodifferentiating actions in various developing tissues [6-8]. The present review is intended to update and extend the knowledge of the findings that have been previously described for SELENOT during the last years [9].

2. Structure and evolution of SELENOT In mammals, the SELENOT gene is present on chromosome 3 in mouse and human, and on chromosome 2 in rat, and includes 6 exons, with exon 2 containing the Sec-encoding TGA triplet, exon 5 containing the TAG stop codon and exons 5-6 containing the 3’untranslated region including the stem-loop SECIS structure (Fig. 1A).

Molecular and

bioinformatics analyses revealed that the SELENOT transcript encompasses 970 nucleotides

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encoding a preprotein of 195 amino acids with a calculated mass of 22.3 kDa [10]. The preprotein contains a 19-amino acid signal peptide and a highly hydrophobic stretch of 16 amino acids at positions 87-102 [10,11] which may represent a transmembrane domain (Fig. 1B, C). The Sec residue is located at position 49 in the N-terminal part of the protein and is separated from an upstream cysteine by two amino acids, to form a CVSU motif which represents a putative redox center as found in several other redox active selonoproteins. The CxxC or CxxU motifs are key for various functions of selenoproteins [12]. In SELENOT, this redox site is comprised between predicted β-strand and α-helix secondary structures (Fig. 1C, D), a topology also found in other redox proteins with a classical thioredoxin fold (CxxC), such as thioredoxins (Fig. 1D), glutaredoxins and disulfide isomerases [13,14]. Based on the similarity of their thioredoxin-like domain, SELENOT and five other selonoproteins, i.e. SELENOM, SELENOF, SELENOV, SELONOH and SELENOW have been grouped in a new subclass named redoxins [13,15]. Sequence alignment analysis of SELENOT with other redoxins such as SELENOW and SELENOH showed no significant amino acid sequence homologies, albeit these proteins exhibit a similar pattern of predicted secondary structures, with an additional central α-helix domain in SELENOT, indicating that redoxins are probably distant homologs [13]. The nucleotide sequence of SELENOT has been strongly conserved during evolution since there is 97-100% homology between human and other mammalian species, 95% with chicken and 89% with frog SELENOT [11,16]. Interestingly, the tetraploid goldfish, Carassius auratus, has three selenoprotein paralogs with a Sec motif in the N-terminal part of the proteins as in mammals [17], while the nematode C. elegans possesses two genes encoding SELENOT protein paralogues, which lack Sec and contain a CXXC redox motif. The appearance of these two SELENOT paralogs is due to two major evolutionary events corresponding to the replacement of the Sec by a Cys and a gene duplication that occurred in the nematode lineage [18]. In fact, SELENOT seems to be one of the most ancient selonoproteins in vertebrates, and the comparison of its sequence in several model organisms reveals the occurrence of homologs with Cys instead of Sec like in protozoans, terrestrial and aquatic plants and insects, suggesting that this selenoprotein may have a more ancient origin in living organisms [11].

3. Subcellular localization and activity of SELENOT In our initial studies, we suggested that the hydrophobic sequence at positions 87-102 could represent a transmembrane domain required for partial integration of SELENOT in the endoplasmic reticulum (ER) membrane since its deletion resulted in a diffuse cytoplasmic labeling of a transfected protein in PC12 cells [10]. Other studies using bioinformatics 5

algorithms also predicted that SELENOT is a membrane protein [19]. Shchedrina et al. suggested the existence of two hydrophobic domains within the sequence 87-102 that may allow SELENOT to cross the ER membrane twice, although the putative helices would be very short compared to the typical length required to span the bilayer [20]. Alternatively, it is also possible that SELENOT is anchored to the membrane without spanning the whole lipid bilayer. Many membrane proteins are permanently anchored to membranes via amphipathic helices, i.e. monotopic proteins. Such membrane proteins are embedded at the lipid head group region. Indeed, modeling studies suggest that the hydrophobic segment of SELENOT contains amphipathic helices that interface with the ER membrane allowing partial binding and insertion of the protein [21]. Along with our confocal and electron microscopy studies (Fig. 2), these observations indicate that SELENOT is localized to the ER membrane where it can interact with and modify other thiol-containing proteins through its Sec active moiety [10,22,23]. Because SELENOT amino acid sequence is extremely well conserved during evolution, this selenoprotein ought to have an important enzymatic activity through its thioredoxin-like domain. Selenoproteins with a thioredoxin-like fold may exert gluthatione perixodase activity as demonstrated for SELENOH [24] or protein folding activity as shown for SELENOW and SELENOF [25]. Using a recombinant protein, we have recently shown that SELENOT exerts a thioredoxin reductase-like activity since it was able to reduce 5,5’-dithio-bis (2dinitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid in the presence of NADPH. This activity was dependent on the redox center of SELENOT and was inhibited by aurothiomalate, a specific inhibitor of thioredoxin reductase activity [26]. In contrast, SELENOT did not display a glutathione peroxidase activity assayed using the Cayman’s assay. This initial study established the oxidoreductase activity of SELENOT in vitro but its substrates and mode of action in vivo remain largely unknown. In particular, the reducing agents and coupling proteins that would allow endogenous SELENOT, through its thioredoxin fold with the Sec residue, to act as a reductase in the ER remain to be identified. 4. Tissue-distribution and regulation of gene expression In the course of its identification by bioinformatics, a screen of expressed sequence tags of adult human tissues showed a weak incidence of SELENOT clones, reflecting a low expression level of this selenoprotein [1]. Only infant brain and placenta databases displayed an incidence of 1 per 10,000 for SELENOT ESTs. This is consistent with the fact that SELENOT was identified in a mouse embryonic cDNA library in our microarray analysis of PACAP-stimulated genes [7]. A histochemical in situ hybridization study carried out on rat 6

embryonic tissues confirmed the high and widespread expression of SELENOT during development and its strong expression as early as E7 [10]. This analysis at the level of the gene expression was confirmed by immunohistochemistry and western blot using specific antibodies, which revealed the abundance of SELENOT during embryonic ontogenesis [27]. In contrast, SELENOT levels profoundly decreased in most tissues after birth, except in endocrine organs such as the pituitary, thyroid, testis and pancreas [22,27] where a strong SELENOT immunoreactive signal was maintained (Fig. 2). In endocrine glands, SELENOT is strongly expressed in secretory cells as revealed by its colocalization with secretory granule markers, i.e. chromogranins or insulin [22,27]. In the pituitary gland, SELENOT was present in all hormone-producing cells [23]. In the testis, SELENOT is found in the testosteroneproducing Leydig cells but also in the proliferating and differentiating spermatogenic cells. In line with these observations in endocrine tissues, high amounts of selenium have been found in secretory cells of the thyroid, and selenium supplementation has been shown to be protective for Leydig cells [28]. In the adult rat brain, the overall level is low but SELENOT is highly expressed in few cells endowed with plasticity such as Bergmann glial cells in the cerebellum or the rostral migratory stream glial cells [27]. The molecular mechanisms underlying the differential expression of SELENOT in various tissues and leading to overall low expression of the gene in adult tissues or on the contrary high expression in embryonic and endocrine tissues remain undetermined. We have previously shown that SELENOT expression could be regulated by cAMP and Ca2+ levels which are stimulated by the trophic peptide PACAP in neuroendocrine cells to trigger their secretory activity [10]. This finding demonstrates that SELENOT gene expression is induced by regulatory cues in particular conditions (Fig. 3). Nonetheless, the molecular mechanisms underlying the transcriptional regulation of the different selenoprotein genes in general remain largely unknown. We recently characterized the signaling pathways converging at the SELENOT gene promoter in order to trigger its transcription in response to PACAP stimulation and cAMP elevation in the neuroendocrine pheochromocytoma PC12 cells derived from the adrenal gland. These studies revealed that cAMP stimulates major regulators of metabolic pathways, which activate the NRF transcription factor to ultimately induce SELENOT gene transcription (Fig. 4). Interestingly, PACAP, acting through its PAC-1 receptor, and cAMP use the same pathway to promote mitochondriogenesis in these cells, thus coupling mitochondrial biogenesis and the antioxidant response involving SELENOT (Abid et al., manuscript in preparation) (Fig. 4). We also observed that SELENOT gene transcription is induced in neurons and astrocytes upon neurotoxin treatment and in oxidative 7

stress condition [26], but the molecular mechanisms involved in these stimulatory effects remain completely unknown. It is interesting to note that the three goldfish paralogs and the two C. elegans orthologs of SELENOT are also regulated by environmental stressors including heavy metals, heat stress or rotenone; the different forms exhibiting differential response patterns [17,18]. Elucidating the mechanisms underlying these effects in the different species could enhance our understanding of the cell response to metabolic activation and oxidative/ER stress.

5. Function of SELENOT Several studies are currently ongoing to address the role of SELENOT in the ER in different tissues. The fact that SELENOT gene knockout leads to early embryonic lethality argues for a pivotal role of this selenoprotein during embryogenesis [26]. It is interesting to note that so far only 4 selenoproteins i.e. two TrxR, one GPx and SELENOT were found essential on the basis of gene knockout studies [29-31]. The thioredoxin-like structure and the potent oxidoreductase activity of a recombinant protein [26] strongly suggest that SELENOT exerts a key redox function that controls protein processing in the ER, allowing cells to cope with oxidative stress and to ensure ER homeostasis. As suggested for other selenoproteins present in the ER, such as SELENOK, SELENOS or SELENOF [32-34], SELENOT may affect through its thioredoxin-like fold, thiol redox circuits including thiol-disulfide oxidoreductases (e.g., ERp57 and protein disulfide isomerase), and folding proteins such as different chaperones (e.g., BiP, calnexin, calreticulin, and glucose-regulated protein GRP94), to alter various cellular processes impacting differentiation, survival and homeostasis. We recently reported using endocrine cells, a cell type where SELENOT expression persists in the adult, that the protein is required for adaptation to the stressful conditions of high hormone level production. Indeed, SELENOT knockdown in corticotrope cells promoted unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulumǦassociated protein degradation (ERAD) and adrenocorticotropin hormone (ACTH) production. Using a screen in yeast for SELENOTǦmembrane protein interactions, we sorted keratinocyteǦassociated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST), which is responsible for protein N-glycosylation. KCP2 or Krtcap2 (GID:142382643) is an ER 14-kDa protein comprising 136-aa and three transmembrane spans that was initially characterized from solubilized actively engaged ribosomes within the OST complex [35]. Noteworthy, it is abundant in active secretory tissues [36]. In fact, we found that SELENOT interacts not only with KCP2 but also with other subunits of the AǦtype OST complex which are depleted after 8

SELENOT knockdown, leading to NǦglycosylation defects of POMC, the precursor of ACTH [23]. This study identified SELENOT as a novel subunit of the AǦtype OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones (Fig. 5). Our results showed that SELENOT could participate in the Nglycosylation of endogenous glycoproteins. The nature of its substrates is still unclear but several observations indicate that SELENOT could specifically participate to the Nglycosylation of proteins harboring disulfide bridges. Indeed, SELENOT expression persists in cells which produce glycoprotein hormones with disulfide bonds, such as adult pancreatic insulin- and somatostatin-producing cells, but not glucagon-producing cells [22], suggesting that it may participate to the N-glycosylation of acceptor sites in cystein-rich protein domains before disulfide bond formation, in the same way as the proteins MagT1/IAP and TUSC33/N33 in the B-type OST complex [37]. Since N-glycosylation is a key process for endocrine activity [38] and since its deficiency is at the origin of a family of diseases referred to as congenital disorders of glycosylation (CDG) associated with hormonal disturbances [39], it is clear that the role of SELENOT in these processes is still underappreciated. Furthermore, it has recently been shown that SELENOT is among seven ER proteins identified through a genetic screen, which are necessary for efficient processing of glycosylphosphatidylinositol (GPI) anchoring of proteins [40]. This important process is involved in the biosynthesis of 150 mammalian proteins anchored to the cell membrane, and requires N-Glycan–dependent protein folding and endoplasmic reticulum retention before targeting of GPI-attached proteins to the membrane. GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertilization, and immune system [41]. Mutations in genes involved in remodeling of the GPI lipid moiety cause human diseases characterized by neurological abnormalities in accordance with the importance of GPI-anchored proteins in neurological development and function [42]. It is tempting to speculate that SELENOT which plays a key function during embryogenesis and brain development [26,43] could exert its effects through regulation of GPI-anchoring protein processing. Additional studies will be required to determine to what extent SELENOT and GPI-anchored proteins essential for embryogenesis and brain ontogenesis and function are associated. In any case, these observations highlight the molecular actions that SELENOT could exert in the ER, probably through its impact on protein maturation and which could influence the biosynthesis of key intracellular, membrane-attached or extracellular actors.

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5.1. Role of SELENOT in Ca2+ regulation and cell adhesion Because SELENOT was identified as a PACAP-stimulated gene during PC12 cell differentiation, we reasoned that this selenoprotein could be involved in the well-known prodifferentiating and prosurvival effects exerted by this neurotrophic factor [7, 10]. Its localization in the ER, a major source of intracellular Ca2+, the involvement of this intracellular messenger in SELENOT gene expression and the fact that Ca2+ is involved in the effects exerted by PACAP during neuronal cell differentiation [8], prompted us to investigate the role of SELENOT in Ca2+ regulation. Using Ca2+ microfluorimetry analysis, we showed that SELENOT overexpression increases intracellular Ca2+ levels and, inversely, that silencing of the SELENOT gene impairs PACAP-induced Ca2+ mobilization from intracellular and extracellular sources [10]. These results suggested that SELENOT may interact directly or indirectly with thiol groups and/or glycosylation moieties of intracellular Ca2+ channels and pumps to regulate their activity through a redox mechanism (Fig. 3). This hypothesis is increasingly supported by studies showing the involvement of different selenoproteins present in the ER such as SELENON, SELENOK or SELENOM in the regulation of Ca2+ homeostasis and Ca2+ channel function [44-48]. In mouse fibroblast cells, SELENOT knockdown altered cell adhesion and decreased the expression of genes involved in cell morphology and oxidative stress, suggesting the involvement of SELENOT in cell adhesion and the control of redox homeostasis [49]. It remains to be established whether ROS including different compounds not known yet and/or Ca2+ regulation are involved in this effect of SELENOT.

5.2. Role of SELENOT in brain The role of SELENOT was intensely investigated in the brain during development and in the adult. SELENOT is particularly abundant in the brain at different embryonic stages but is almost undetectable in the adult. During brain development, SELENOT is present at high levels in both proliferating and differentiating neuronal precursors and its expression is maintained in immature neurogenic and gliogenic cells at different stages [27]. SELENOT is particularly concentrated in the forebrain (neocortex and thalamus), the midbrain, and the hindbrain. An important immunopositive signal is observed in the olfactory bulb, particularly in the mitral cell layer, in the cerebral periventricular cells or in the cerebral neocortex [27]. In the cerebellum, SELENOT is detected at all the postnatal stages examined, including adult. It is expressed in neurons of the external germinative layer, while an intense SELENOT immunoreactive signal is observed in the developing Purkinje cell layer, particularly in 10

Bergmann cells. SELENOT is present in the ER of immature and early differentiating neural cells, but is absent in most adult differentiated nerve cells, such as adult Purkinje cells for instance [27]. The high expression of SELENOT in the developing brain suggests an important role of this protein during brain ontogenesis. Therefore, we used a conditional knockout mouse line in which SELENOT gene was specifically disrupted in nerve cells to decipher its function in the brain. A morphometric analysis revealed that these mice have reduced volumes of different brain structures, a phenomenon that was observed early during the first postnatal week. This phenotype could be ascribed to the loss of immature neurons, but not glial cells, and the elevated levels of intracellular reactive oxygen species that were observed in these immature neurons. However, developmental compensatory mechanisms probably operate later during development to attenuate the brain volume alterations in adult mice. Nevertheless, SELENOT knockout mice exhibit a hyperactive behavior, indicating that SELENOT deficiency during development leads to cerebral malfunction in adulthood [43]. Thus, SELENOT exerts a neuroprotective effect during brain development and is required later for proper behavior in adult, two phenomena that are probably related since alteration of neuronal differentiation may disrupt the circuits that control brain functions. These findings are reminiscent of the phenotype of mice lacking PACAP, the neuropeptide that stimulates SELENOT gene expression [10,27], suggesting a possible role of SELENOT in mediating, at least partially, the neurotrophic activities of the neuropeptide [43]. Because SELENOT is associated with oxidative stress control and neuronal survival, we reasoned that it may be involved in neurodegenerative diseases. To test this hypothesis, we used mice models of Parkinson’s disease (PD) and cell cultures of dopaminergic neurons [26]. In PD, oxidative stress is central to the process of dopaminergic neurondegeneration, and a role of selenoproteins in the protection of dopaminergic neurons has been suggested [50,51]. In the dopaminergic SH-SY5Y cell model, SELENOT promotes cell survival and inhibits oxidative stress provoked by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). In mice, treatment

with

PD-inducing

neurotoxins

such

as

1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP) or rotenone triggers SELENOT expression in dopaminergic neurons and fibers, as well as astrocytes of the nigrostriatal pathway, indicating that SELENOT is probably recruited in both cell types to prevent neurodegeneration. In fact, SELENOT gene disruption in brain of knockout mice provokes rapid and severe parkinsonian-like motor impairment, which is associated with marked oxidative stress and dopaminergic neurodegeneration. Motor deficit of the knockout mice is most likely due to reduced tyrosine hydroxylase (TH) activity and dopamine levels observed in the nigrostriatal 11

system after neurotoxin administration. Altered TH activity is impacted by oxidative stress for instance through tyrosine nitrosylation which is dramatically increased in SELENOTdeficient mice compared to wild-type mice after neurotoxin treatment. These data show that SELENOT protects dopaminergic neurons against oxidative stress and prevents their degeneration and dopamine content decline (Fig. 6). In a pilot study using PD patient brain specimen, we observed a significant increase of SELENOT mRNA and protein levels in the caudate putamen tissue, suggesting that this selenoprotein may also be involved in human PD [26]. This observation needs to be substantiated in large cohorts, which may lead to establish SELENOT as a marker of disease progression and/or a therapeutic target in this and other neurodegenerative diseases.

5.3. Role of SELENOT in pancreas As mentioned above, tissue distribution studies showed the persistence of high expression of SELENOT in adult endocrine organs including pituitary, pancreas, testis and thyroid [26]. In the pancreas, SELENOT is particularly abundant in insulin- and somatostatinproducing cells, but is undetectable in glucagon-producing cells. In order to determine the role of SELENOT in b-pancreatic cells, we generated conditional pancreatic b-cell SELENOT knockout mice [22]. These mice display impaired glucose tolerance, which is likely due to altered insulin synthesis or release as revealed by the higher glucose to insulin ratio in knockout compared to wild-type littermates. In contrast, SELENOT deficiency did no modify insulin sensitivity as indicated by the similar levels of plasma glucose in mutant and wild-type mice following insulin injection. In fact, SELENOT is not only involved in blood glucose homeostasis but also in the structural integrity of the endocrine pancreas since islet morphology is altered and displays a smaller size in knockout compared to wild-type mice [22]. These results revealed for the first time the involvement of this thioredoxin-like protein in the regulation of insulin production and secretion and hence in the control of glucohomoeostasis. Our findings are in line with the data reported by Labunskyy et al. [52], which showed that both overexpression and deficiency of selenoproteins can promote diabetes development. Although several studies pointed to the role of thioredoxins in the control of b -cell function by protecting the pancreatic cells against oxidative injury [53-55], the interplay of different thioredoxin-related proteins including SELENOT in the regulation of glucose homeostasis is not fully understood and deserves further investigation.

5.4. Role of SELENOT in liver 12

Liver displays the particular characteristic of regenerating after partial hepatectomy. Hepatocytes, which make 80% of the liver parenchyma, proliferate after tissue resection to regenerate the missing tissue. SELENOT was not detected in normal liver but was strongly induced during the acute phase of liver regeneration, in line with the high expression of this selenoprotein in proliferating and differentiating cells [27]. This is reminiscent of the induction of the selenoenzyme deiodinase III in regenerating liver, which converts thyroid hormones to inactive entities in order to inhibit their pro-differentiating activities and to facilitate thereby the regenerative process [56]. The role of SELENOT during regeneration remains to be elucidated. The fact that its induction occurs mainly in Kupffer cells in liver suggests that this selenoprotein could contribute actively to the production and secretion of paracrine factors from these cells such as the cytokines TNF-a, IL-6 or TGF-b1 which modulate hepatocyte proliferation during liver regeneration [57].

5.5. Role of SELENOT in smooth muscle The pattern of SELENOT expression has been investigated in smooth muscle from different tissues after feeding rats diets with low or high selenium content [58]. It was found that SELENOT is expressed at low levels in the smooth muscle tissue of blood vessels, esophagus, bronchus, stomach and intestine. The SELENOT gene expression is very sensitive to dietary selenium in these tissues. It was thereafter shown that SELENOT is involved in the stimulation of the myosin light chain kinase (MLCK) in the gastric smooth muscle in the rat [59]. Since MLCK plays a key role in the contraction process of smooth muscle in a Ca2+/calmodulin-dependent manner, these results suggest that SELENOT could be involved in smooth muscle activity through Ca2+ regulation.

5.6. Role of SELENOT in immune organs and in infection The expression and role of SELENOT in the immune system has been investigated only in chicken so far. It appears that SELENOT expression in spleen, thymus and bursa of fabricius is dependent on selenium content [60]. These studies suggest that SELENOT is involved in the regulation of oxidative stress in immune organs and in the response of the immune system [61]. Also, a recent study showed that SELENOT is required for C. elegans in order to avoid the bacterial pathogens S. marcescens and P. aeruginosa, indicating its possible involvement in innate immunity [18].

6. Conclusions and future directions 13

SELENOT seems to be one of the most important selonoproteins as revealed by the high conservation of the protein in various species and the early embryonic lethality caused by its gene knockout in mice. The cause of this lethality is not known yet and requires further investigation. Given the intense SELENOT expression in rat embryos as well as placenta, as early as at E7 [10], it will be important to decipher how SELENOT potentially affects the implantation and the development of embryos. Recent studies indicate that SELENOT is involved in the development of various organs such as the brain and the pancreas, and exerts functions as diverse as Ca2+ regulation, hormone secretion, neuroprotection, cardioprotection (62) and innate immunity in adults. The redox activity of SELENOT in the ER could be central to its various effects (Fig. 7). Further studies are warranted first to establish the impact of SELENOT on cellular processes such as differentiation, migration or survival in order to help organs to develop or to regenerate, and second to determine how SELENOT fulfils such diverse functions in different organs in physiological and pathophysiological conditions (Fig. 7). The molecular mechanisms underlying the effects of SELENOT are beginning to be understood and the data obtained so far point to an important role in ER proteostasis, at least in endocrine cells [23]. This effect may involve an enzymatic activity of its Sec-containing thioredoxin-like domain which could regulate various posttranslational modifications that require disulfide bridge formation in proteins in transit through the ER, such as the Nglycosylation process [22]. Thus, SELENOT may affect thiol redox circuits that could impinge on the synthesis and modification of essential cues, whose impairment in the absence of SELENOT would lead to dysfunctional redox control, free radical accumulation and cell death. High SELENOT expression in proliferating and differentiating cells during development, like in the brain, or in adult cells endowed with some plasticity such as endocrine cells implies that this selenoprotein is associated with intense cell metabolism and confers an advantage to these cells to cope with oxidative stress associated with this state. In addition, the SELENOT gene is stimulated in dopaminegic neurons undergoing degeneration [26], liver cells after hepatectomy [27] and cerebral cells under hypoxia [63], indicating that SELENOT is required to protect different cell types against injury. It appears that SELENOT gene activation recruits major metabolic regulators that also control mitochondrial enzyme gene activation and mitochondriogenesis (our unpublished results). This finding along with the abundance of SELENOT in cells with high metabolic activity indicate that this selenoprotein is integrated in cell metabolic pathways which couple energy supply and the antioxidant response and which are essential for cell survival and homoeostasis.

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Legends to figures:

Fig. 1. Structure of SELENOT. A) Structure of the mouse SELENOT gene which contains 6 exons (Ex). The translation start site (ATG), the Sec (TGA) and the stop (TAG) codons are indicated. Exon 6 contains the 3’non-coding sequence of the mRNA where the SECIS motif is present. B) Amino-acid primary sequence of SELENOT with the signal peptide (green), βsheets (blue), the CVSU (red) and α helices (purple). C) Schematic representation showing the location of the different motifs and secondary structures present in SELENOT. D) Comparison of the secondary structures of SELENOT with those of SELENOW and the thioredoxin showing the similarities in gene organization and secondary structures. The three proteins display the thioredoxin fold β-α-β-β-β-α. SELENOT has 4-5 additional a-helices (adapted from [10]).

Fig. 2. Distribution of SELENOT immunoreactivity in the adult rat pituitary gland. A) SELENOT immunoreactivity is located in anterior (AP) but not posterior pituitary (PP). B) In pituitary cells, SELENOT is located in the endoplasmic reticulum (arrow) as indicated by electron microscopy analysis. Fig. 3. Ca2+ Regulation by SELENOT. PACAP stimulates SELENOT gene expression through cAMP induction and protein kinase A (PKA) activation. SELENOT in turn regulates Ca

2+

stores in the endoplasmic reticulum, in response to PACAP, probably via IP3 receptor

or SERCA pump control (adapted from [9]).

Fig. 4. Signaling pathways involved in PACAP- and cAMP-induced SELENOT gene transcription in PC12 cells. SELENOT gene transcription is activated through major metabolic regulators and is coupled to mitochondriogenesis and oxidative stress to promote PACAP-induced PC12 cell survival and differentiation.

Fig. 5. Role of SELENOT in ER protein processing. SELENOT is a novel subunit of the oligosaccharyl transferase (OST) complex, involved in ER homeostasis by contributing to hormone N-glycosylation, folding and secretion, in endocrine cells. SELENOT interacts with the subunit Kcp2 to promote the stability of the A-type OST complex which is essential for the N-glycosylation of specific proteins.

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Fig. 6. Schematic representation of the effect of SELENOT in dopaminergic neurons following neurotoxin administration in mice. The neurotoxin MPTP is taken from blood to astrocytes where it is converted to MPP+ by monoamine oxydase B (MAOB). MPP+ is transported to neurons via dopamine transporters (DAT) where it blocks mitochondrial complex I, thus leading to oxidative stress. Compounds related to ROS which are inhibited by SELENOT provoke the accumulation of 3-nitrotyrosine (3-NT), leading to tyrosine hydroxylase inactivation and dopamine content decline. SELENOT protects dopaminergic neurons in these conditions.

Fig. 7. Schematic representation of the various effects exerted by SELENOT in the ER potentially through its redox activity. The redoxin activity of SELENOT contributes to the regulation of Ca2+ homeostasis, the cell antioxidant capacity and ER protein folding and maturation, which impact cell survival, differentiation and secretory activity.

Highlights: Selenoprotein T is a key regulator of ER homeostasis Selenoprotein T is involved in tissue development and homeostasis Selenoprotein T plays specific functions in endocrine cells Selenoprotein T exerts neuroprotective and regenerative actions Selenoprotein T could play a role in OST and GPI protein tagging

21

22

Figure 1

A

Promoter

Ex1

Ex2 Ex3

Ex4

Ex5 Ex6

SELENOT ATG

B MRLLLLLLVAASAMVRSEASANLGGVPSKRLKMQYATGPLLKFQICVSU GYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASFLSVFKLVLIGLIIV GKDPFAFFGMQAPSIWQWGQENKVYACMMVFFLSNMIENQCMSTGA FEITLNDVPVWSKLESGHLPSMQQLVQILDNEMKLNVHMDSIPHHRS

C 1

69 72

40 64

NH3+

78

109 117

COO87 102

D

SELENOW

Thioredoxin

β1

α1

125 143

β2

CxxU α1

β1

β2

CxxC

α1

β1 CxxU

β3

β4

α2

β3

β4

α3

α2

α2

SELENOT

167 178 195

145 149 158

Transmembran segment

α3

α4

β2

β3

α5 β4

α6/7

Signal peptide Signal peptide

CXXC/U Motif

β Strand

α Helix

Hydrophobic segment

Figure 1

Figure 2

B

A AP

PP

20 μm

Figure 2

Figure 3

50µm

75µm

cAMP

50µm

SELENOT

Endoplasmic reliculum

Figure 3

Figure 4

PACAP

AC

ATP

Gq

Gs

PLC

Endoplasmic reticulum

cAMP

IP3

SELENOT P

PK SELT P

Ma Matabolic re regulator P

Me Metabolic regulator

Nucleus

NRF

Mitochondria

Figure 4

Figure 5

Figure 5

Figure 6

Mitochondria MPP+ MPP+

&

SELENOT

SELT SELENOT Endoplasmic reticulum

Dopamine

Figure 6

Figure 7

SELENOT

ER

Ca2+ homeostasis

Antioxidant activity ER proteostasis Cell survival and differentiation

Cell survival and Secretory activity Cell survival and Secretory activity

Figure 7