Endoplasmic reticulum-resident selenoproteins as regulators of calcium signaling and homeostasis

Abstract

The human selenoprotein family contains 25 members that share the common feature of containing the amino acid, selenocysteine (Sec). Seven selenoproteins are localized to the endoplasmic reticulum (ER) and exhibit different structural features contributing to a range of cellular functions. Some of these functions are either directly or indirectly related to calcium (Ca²⁺) flux or homeostasis. The presence of the unique Sec residue within these proteins allows some to exert oxidoreductase activity, while the function of the Sec in other ER selenoproteins remains unclear. Some functional insight has been achieved by identifying domains within the ER selenoproteins or through the identification of binding partners. For example, selenoproteins K and N (SELENOK AND SELENON) have been characterized through interactions detected with the inositol 1,4,5-triphosphate receptors (IP3Rs) and the SERCA2b pump, respectively. Others have been linked to chaperone functions related to ER stress or Ca²⁺ homeostasis. This review summarizes the details gathered to date regarding the ER-resident selenoproteins and their effect on Ca²⁺ regulated pathways and outcomes in cells.

Graphical abstract

Introduction

Selenium is an essential micronutrient that plays an important role in several aspects of human health. Deficient intake of selenium can lead to a wide variety of health disorders ranging from impaired gestational development, poor immunity, cognitive and neurodegenerative problems, and endocrine imbalances [1]. The biological effects of selenium are exerted mainly through the actions of selenoproteins, which contain this element in the form of the 21^st amino acid selenocysteine (Sec) [2]. There are 25 selenoproteins in humans [3] that exhibit a wide range of biological functions with nearly half of the members still uncharacterized in terms of their function. Animal models have demonstrated the essential role of selenoproteins, as mice deficient in tRNA^Sec, the selenocysteine-specific tRNA required for selenoprotein synthesis, do not survive early embryogenesis [4]. The most studied selenoproteins include those that function as enzymes. The significance of these enzymes and other members of the selenoprotein family to human health have been demonstrated in patients with errors in the synthesis process or in the selenoproteins themselves [5]. A number of studies in humans have found that defects in expression or function of selenoproteins can impinge upon a variety of physiological processes involving the muscular and skeletal systems, respiratory system, neurological system, and endocrine system. Perhaps the best example of how selenoproteins affect multiple systems is the syndrome in which there is defective production or function of one protein involved in selenoprotein synthesis, the SECIS binding protein 2 (SECISBP2). Mutations in SECISBP2 give rise to a spectrum of health problems depending on the patient and/or type of mutation present [6].

There are 25 genes encoding selenoproteins in humans that were revealed in a 2003 hallmark study by the Gladyshev laboratory following the completion of the human genome project [3]. While several new members of the family were identified in that study, some selenoproteins had already been characterized as redox regulating enzymes containing the Sec residue located within the catalytic site. The defining characteristic of selenoproteins is the presence of the 21^st amino acid Sec, which itself has special properties. The Sec residue is isosteric to cysteine (Cys) with selenium in place of sulfur. This results in a highly reactive site in the enzyme that is better at nucleophilic exchange reactions compared to Cys [7]. The lower pKa of the selenol side chain of Sec compared to thiol of Cys means that Sec is mostly deprotonated at physiological pH [8], and this further enhances its oxidoreductase activity.

Subsequent to the complete identification of the selenoprotein family members in 2003, the nomenclature was recently revised [9]. Selenoprotein enzymes with known functions are designated according to these functions. These include the glutathione peroxidases (GPXs), the thioredoxin reductases (TXNRDs), iodothyronine deiodinases (DIOs), methionine-R-sulfoxide reductase 1 (MSRB2) and selenophosphate synthetase 2 (SEPHS2). Those selenoproteins without a clearly demonstrated enzymatic function are named with the root symbol SELENO followed by a letter. A list of selenoproteins with the characteristics of each is shown in Table 1. As indicated in this table, several selenoproteins have been associated with a cellular function involving Ca²⁺, either directly or indirectly, and the remainder of this review will elaborate on ER-resident selenproteins and what is known regarding their Ca²⁺ relevant functions.

Table 1.

Summary of Selenoprotein Functions

Selenoprotein	Abbreviation(s)	Function
Cytosolic glutathione peroxidase	GPX1	Reduces cellular H2O2.
Gastrointestinal glutathione peroxidase	GPX2	Reduces peroxide in gut.
Plasma glutathione peroxidase	GPX3	Reduces peroxide in blood.
Phosholipid hydroperoxide glutathione peroxidase	GPX4	Reduces phospholipid peroxide.
Olfactory glutathione peroxidase	GPX6	Importance unknown.
Thioredoxin reductase Type I	TRXRD1, TR1	Localized to cytoplasm and nucleus; regenerates reduced thioredoxin.
Thioredoxin reductase Type II	TRXRD2, TR3	Localized to mitochondria; regenerates reduced thioredoxin.
Thioredoxin reductase Type III	TRXRD3, TR2, TGR	Testes-specific expression; regenerates reduced thioredoxin.
Iodothyronine Deiodinase Type I	DIO1	Important for systemic active thyroid hormone levels.
Iodothyronine Deiodinase Type II	DIO2	ER enzyme important for local active thyroid hormone levels.
Iodothyronine Deiodinase Type III	DIO3	Inactivates thyroid hormone.
Methionine sulfoxide reductase B1	MSRB1, SelR	Reduces sulfoxidated methionines on proteins.
Selenoprotein F	SELENOF, Sep15	ER thiol-disulfide oxidoreductase possibly involved in protein folding.
Selenoprotein H	SELENOH, SelH	Nuclear localization; involved in redox sensing and transcription.
Selenoprotein I	SELENOI, SelI, EPT1	Possibly involved in phospholipid biosynthesis.
Selenoprotein K	SELENOK, SelK	Transmembrane protein localized to ER and involved in calcium flux in immune cells and ER associated degradation in cell lines.
Selenoprotein M	SELENOM, SelM	Thioredoxin-like ER-resident protein that may be involved in regulation of body weight and energy metabolism.
Selenoprotein N	SELENON, SelN, SEPN1, SepN	Transmembrane protein localized to ER; mutations lead to multiminicore disease and other myopathies.
Selenoprotein O	SELENOO, SelO	Mitochondrial protein that contains a C-X-X-U motif suggestive of redox function.
Selenoprotein P	SELENOP, SelP, Sepp1	Secreted into plasma for selenium transport to tissues.
Selenoprotein S	SELENOS, SelS, SEPS1, SELENOS, VIMP	Transmembrane protein found in ER involved in ER associated degradation.
Selenoprotein T	SELENOT, SelT	ER protein involved in calcium mobilization, unclear exactly how.
Selenoprotein V	SELENOV, SelV	Testes-specific expression.
Selenoprotein W	SELENOW, SelW, SEPW1	Putative antioxidant role, perhaps important in muscle growth.
Selenophosphate synthetase	SPS2	Involved in synthesis of all selenoproteins, including itself.

Calcium Homeostasis and ER Stress

Within the cell, cytosolic levels of free Ca²⁺ are kept low under baseline conditions by the synchronized action of a variety of channels, pumps, exchangers, and Ca²⁺-binding proteins. The endoplasmic reticulum (ER) represents the primary storage site for intracellular Ca²⁺, with concentrations reaching high micromolar levels in this organelle, whereas resting cytosolic concentrations typically range from 50 to 200 nM [10]. Seven of the twenty-five members of the selenoprotein family are localized to the ER, including SELENOF, SELENOK, SELENOM, SELENON, SELENOS, SELENOT, and the type-2 iodothyronine deiodinase [11]. Structural features of these ER selenoproteins are shown in Figure 1. Cytosolic Ca²⁺ is maintained at nanomolar levels by plasma membrane Ca²⁺ ATPases and SR/ER Ca²⁺ ATPase b (SERCAb) that pump Ca²⁺ into the extracellular space and the ER lumen, respectively. In response to various stimuli, Ca²⁺ is released from the ER by means of two classes of channels, the inositol 1,4,5-trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs). For example, immune cells stimulated through cell surface receptors generate inositol 1,4,5-trisphosphate (IP3) that binds to the IP3Rs in the ER membrane to release Ca²⁺ stored in the lumen of this organelle. Loss of Ca²⁺ from ER stores is sensed by stromal interaction molecule 1 (STIM1). This alters the structure of STIM1 and causes its oligomerization and it then engages the Ca²⁺ channel protein Orai1 or transient receptor potential canonical 1 (TRPC1) in the plasma membrane to open and allow influx of millimolar levels of Ca²⁺ [12]. This process is referred to as store operated Ca²⁺ entry (SOCE) and is crucial for cell proliferation, migration and other functions. It is important to note that SOCE is emerging as a potential target for cancer therapeutics [13]. When different cell types transform and progress to metastatic tumor cells, Ca²⁺ homeostasis and signaling is altered in a process referred to as Ca²⁺ remodeling [14]. Certain selenoproteins contribute to SOCE and some may also be involved in Ca²⁺ remodeling in cancer, and these issues that are discussed in greater detail below.

Figure 1.

Domain organization of ER selenoproteins. This diagram illustrates the relative position of Sec residues (red), signal peptides (green), transmembrane domains (yellow), thioredoxin-like motifs (pink), EF hand motifs (orange), and ER retention signals (white triangles). Established binding/interaction sites are denoted by three-quarter circle symbols.

In addition to its integral role in Ca²⁺ signaling, the ER is also critically important for the folding, assembly, and post-translational modification of nascent proteins destined for secretion or residence in the cell membrane. Newly synthesized proteins destined for modification in the ER are marked by an N-terminal signal peptide that tags them for insertion into the Sec61 translocon spanning the ER membrane. Within the ER, a host of chaperones and enzymes act to ensure that proteins mature and reach their optimal conformation. These include glucose-regulated protein (GRP) chaperones, glucosidases, transferases, protein disulfide isomerases (PDIs), and the Ca²⁺-regulated chaperones, calnexin and calreticulin. ER homeostasis can be disrupted by a variety of insults, such as the accumulation of misfolded proteins [15], elevated levels of reactive oxygen species (ROS) [16, 17], and abnormalities in Ca²⁺ signaling [18, 19], which are all generically referred to as ER stress. These disturbances trigger the unfolded-protein response (UPR), a protective counter-measure that acts to reestablish balance and promote survival by increasing the production of chaperones involved in protein folding, inhibiting global translation, and eliminating chronically misfolded proteins. The UPR is conveyed by three ER stress sensors: PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase 1-alpha (IRE1α), and activation of transcription factor 6 (ATF6) that are negatively regulated by the chaperone GRP78 under baseline conditions [15, 20]. Increasing levels of misfolded proteins causes GRP78 to dissociate from these sensors, resulting in activation of the UPR. Proteins that fail to properly fold are eliminated by means of ER-associated degradation (ERAD), a multistep process in which misfolded proteins are selected, retrotranslocated across the ER membrane, ubiquitinated, and finally degraded by cytosolic proteasomes. In cases of severe and/or sustained ER stress where the UPR fails to restore homeostasis, apoptotic cell signaling pathways are activated [21]. In particular, sustained UPR activation of Jun amino-terminal kinase (JNK), a downstream mediator of the UPR, has been demonstrated to be pro-apoptotic in a wide variety of cell types and contexts [22–24]. Furthermore, severe ER stress promotes apoptosis by increasing ER-to-mitochondria Ca²⁺ transfer at mitochondria-associated membranes, leading to disturbances in the mitochondrial membrane potential and release of cytochrome c [25]. Of clinical importance, chronic ER stress is implicated in the pathogenesis of many chronic diseases, including type 2 diabetes [26, 27], cardiovascular disease [28] and multiple neurodegenerative disorders [29].

There is substantial evidence indicating that Ca²⁺ homeostasis, protein folding, and oxidative stress are extensively intertwined. Redox imbalance and ER stress typically increase cytosolic Ca²⁺ levels and diminish ER Ca²⁺ stores by a variety of mechanisms. For example, in addition to its interactions with the aforementioned ER stress sensors, GRP78 also binds to the Sec61 translocon and prevents Ca²⁺ leak from the ER to cytosol under unstressed condtions [30, 31]. Under conditions of ER stress, GRP78 dissociates from Sec61 and increased ER Ca²⁺ leak ensues. Similarly, oxidation of cysteine residues on RyRs promotes ER Ca²⁺ leakage [32, 33], whereas ROS-dependent thiol modification of IP3Rs increases ligand affinity and thereby promotes Ca²⁺ efflux [34, 35]. In particular, studies in cell culture have reported that selenium deprivation augments IP3 triggered ER Ca²⁺ release [36]. Furthermore, the redox environment of the ER is more oxidizing than the surrounding cytosol [37, 38], and this appears to be influenced, at least in part, by ER Ca²⁺ levels [39]. When ER Ca²⁺ stores become depleted, the redox state in this organelle becomes more reducing and modulates redox-sensitive proteins via disruption of intramolecular disulfide bonds. In particular, the SERCA2b pump contains a pair of cysteine residues in the fourth lumen loop that forms a disulfide bond when ER Ca²⁺ levels are high, which serves to inactivate the pump [40]. When ER Ca²⁺ levels decline, this disulfide bond is reduced and the pump becomes fully active.

Regulation of Ca²⁺ signaling by ER-resident selenoproteins

Various ER-resident selenoproteins have been shown to modulate Ca²⁺ flux into and out of the ER lumen, promote protein folding, and respond to ER luminal redox status. The following section will review what is currently known regarding these individual selenoproteins and how they impact Ca²⁺-related processes such as proliferation, survival, and apoptosis. The structural and topological features of the seven ER-resident selenoproteins suggest diversity even within this subgroup (Figure 1). How the domains and topologies may be related to potential functions is discussed in detail below.

Selenoprotein F (SELENOF, previously Sep15)

SELENOF, formerly known as SEP15, is a 15 kDa selenoprotein that was first identified in 1998 by Gladyshev and colleagues in a human T-cell line [41]. It shares a common evolutionary origin with SELENOM, and together they comprise a distinct clade within the thioredoxin superfamily. The primary sequence of SELENOF contains a cleavable N-terminal signal peptide, a thioredoxin-like fold, and a C-x-U redox motif. It also possesses a distinct cysteine-rich region in its N-terminus that is required for interaction with its binding partner, UDP-glucose:glycoprotein glucosyltransferase (UGGT) [42, 43]. Due to the fact that its sequence does not contain an ER retention signal, the localization of SELENOF to the ER is thought to be dependent upon UGGT binding. Within the ER lumen, SELENOF is believed to mediate disulfide bond formation on a distinct set of glycoproteins that serve as UGGT substrates. Studies performed on Drosophila SELENOF determined that the equilibrium redox potential was −225 mV, a value that falls between the established redox potentials of protein disulfide isomerase (−175 mV) and cytosolic thioredoxin (−270 mV) [44].

Evidence to date has largely implicated SELENOF in protein folding due to its documented interaction with UGGT. UGGT is an essential component of the calnexin/calreticulin cycle, a quality control pathway that promotes folding of N-linked glycoproteins within the ER. SELENOF associates tightly with UGGT in a 1:1 complex that glucosylates misfolded protein domains, tagging them for another round of protein folding. Calnexin itself is a Ca²⁺-binding ER-resident chaperone that can function in protein quality control or Ca²⁺ signaling, depending upon its palmitoylation status [45]. When palmitoylated, calnexin is preferentially distributed to mitochondria-associated membranes (MAMs) and regulates ER Ca²⁺ levels via interactions with the SERCA2b pump. Moreover, ER stress reduces calnexin palmitolyation, allowing it to migrate away from MAMs into the ER lumen, where it acts as a chaperone.

Whereas SELENOF has been linked to protein folding, it has yet to be directly associated with Ca²⁺ homeostasis. Interestingly, induction of ER stress with the SERCA pump inhibitor, thapsigargin, was observed to trigger rapid degradation of SELENOF, whereas administration of the ER stress agents, tunicamycin or brefeldin A, upregulated SELENOF expression [46]. This indicates that SELENOF may be preferentially targeted to disturbances related to protein folding rather than Ca²⁺ dysregulation.

Expression of SELENOF is highly sensitive to levels of dietary Se, and is most prevalent in tissues with secretory functions, such as liver, kidney, thyroid, testes, and prostate [44]. This protein has been of significant interest to cancer researchers, as altered levels have been reported in several cancers and the gene is located on chromosome 1p31, a region often deleted or mutated in cancer. One of the initial reports on SELENOF found that expression levels were reduced in lung cancer, hepatocarcinoma, and a prostate cancer cell line [47]. Conversely, subsequent studies have reported SELENOF upregulation in human liver cancer cell lines, HepG2 and Huh7 [48], and shown that SELENOF can promote colon cancer [49]. In particular, knockdown of SELENOF inhibited growth of cultured colon cancer cells [50] and SELENOF KO mice were less susceptible to chemically-induced colon cancer than control mice [51].

Constitutive KO mice for SELENOF have been developed and initially characterized. They were observed to be viable and fertile with normal brain morphology [52]. Early development of cataracts was the primary defect found to result from SELENOF deletion. The authors of this paper observed enriched SELENOF expression during lens development and suggested that formation of cataracts were a consequence of impaired folding of lens proteins due to SELENOF deficiency. SELENOF KO mice also displayed elevated levels of oxidative stress in liver, yet when ER stress pathways were assessed in this tissue, no significant differences were observed. Earlier studies conducted in NIH3T3 cells, also found that SELENOF knockdown did not increase baseline ER stress, suggesting that SELENOF is either non-essential or its loss can be compensated for by similar proteins [46].

Selenoprotein K (SELENOK)

This small (~10 kDa) selenoprotein is a single spanning transmembrane protein localized to ER membrane [53]. It contains a highly disordered cytosolic region that contains a Sec residue located near the C-terminus and it is expressed in most tissues with highest levels detected in immune cells [53–55]. Two cellular functions have emerged for SELENOK including participation in the ER associated protein degradation (ERAD) pathway and regulation of Ca²⁺ flux from the ER. The role in ERAD was initially pursued based on evidence that SELENOK was transcriptionally upregulated during ER stress [56], and this led to the identification of an ER stress response element (ERSE) in the promoter of human SELENOK [57]. Subsequent studies showed that SELENOK is bound within complexes in the ER membrane along with SELENOS, valosin-containing protein (VCP; p97), and the Derlins [57, 58]. One may expect that a deletion in SELENOK may lead to increased ER stress and enhanced apoptosis, but perhaps due to redundancy or compensation this has not turned out to be the case. In fact, SELENOK knockout mice appear healthy and fertile, and cells from these mice show no signs of ERAD defects or ER stress [53].

Closer examination of the immune cells from SELENOK knockout mice found that receptor mediated Ca²⁺ flux from ER stores, or SOCE as described above, was impaired in SELENOK deficient immune cells. The reason for this impairment was eventually found to be due to the role SELENOK plays as a cofactor along with the enzyme, DHHC6, in palmitoylating the IP3Rs on particular cysteine residues [59]. This was shown to stabilize the IP3Rs and promote efficient SOCE. Thus, lower SELENOK levels caused by either lower dietary selenium intake or genetic factors leads to less IP3Rs in the ER membrane and lower capacity for SOCE in immune cells. While this does not appear to render cells more sensitive to stress or apoptosis, several immune cell functions that rely on efficient SOCE are compromised in SELENOK deficient immune cells. These include proliferation, migration, cytokine secretion, and protection against pathogens [53].

Exactly how the Sec residue within SELENOK is involved in Ca²⁺ flux or ERAD functions is not well understood. Unlike the selenoezymes that are well characterized, SELENOK does not have a thioredoxin-fold motif or oxidoreductase catalytic domain itself. For catalyzing palmitoylation of the IP3Rs, it is conceivable that the Sec residue in the SELENOK cofactor is utilized by the catalytic domain of the DHHC6 enzyme to more efficiently transfer the palmitoyl group to the cysteine residues of the IP3Rs. In fact, the redox chemistry of the Sec residue is consistent with promoting acyl transferase reactions [60]. Interestingly, the Sec residue in this selenoprotein exhibits some peroxidase activity in vitro [61], but whether this contributes to in vivo function has not yet been determined. Overall, SELENOK plays at least two important roles in the ER membrane related to cellular Ca²⁺. Reducing its expression or interfering with its function does not result in altered ER Ca²⁺ stores, but does hinder Ca²⁺ flux in a manner that does not induce increased cell stress or death. SELENOK deficiency leads to impaired functions of immune cells and perhaps other cell-types yet to be fully scrutinized (e.g. neurons, myocytes, etc.). It also has been reported that knockdown of SELENOK in a mouse T cell line affects calcium homeostasis in a manner that may involve calcium homeostasis endoplasmic reticulum protein (CHERP) [62]. Although the mechanism linking SELENOK and CHERP was not determined, this study underscores the notion that the IP3Rs are likely not the only calcium related protein related to SELENOK protein levels. Finally, it is worth noting that SELENOK may be important in cancer progression due to its role in promoting SOCE. While there is limited data regarding levels of SELENOK in tumors compared to healthy tissues, our laboratory has found in unpublished studies that SELENOK contributes to growth and migration of melanoma cells consistent with its crucial role in SOCE. Work is on-going to determine the precise role SELENOK plays in primary and secondary tumor formation and whether it may serve as an effective therapeutic target.

Selenoprotein M (SELENOM)

Selenoprotein M (SELENOM) is a thiol-disulfide oxidoreductase with enriched expression in the brain that is ancestrally related to SELENOF [63]. Major structural features include an active site consisting of a Sec-containing thioredoxin-like motif and an ER retention tetrapeptide in the C-terminal domain. The C-terminal region of SELENOM does not assume a defined secondary structure and is speculated to participate in substrate interactions [43]. Immunohistochemical analysis of SELENOM expression in the mouse brain showed high levels in several discrete regions, including the arcuate and paraventricular nuclei of the hypothalamus and the CA2/CA3 region of the hippocampus [64]. Furthermore, SELENOM levels in brain are highly responsive to Se supplementation [65]. Functionally, SELENOM has been documented to be neuroprotective and promote Ca²⁺ homeostasis. Overexpression of SELENOM in neuronal cell culture was found to decrease superoxide production, reduce cytosolic Ca²⁺ flux, and prevent apoptosis induced by oxidative challenge with hydrogen peroxide [66]. In contrast, SELENOM knockdown resulted in decreased cell viability in conjunction with increased ROS production and elevated baseline levels of cytosolic Ca²⁺.

The initial characterization of the phenotypic effects of SELENOM deletion in mice led to the unexpected finding that SELENOM exerts a significant influence on energy homeostasis [64]. Indeed, SELENOM KO mice displayed increased body weight, elevated white adipose tissue levels, and reduced hypothalamic leptin sensitivity relative to wild-type counterparts. Yet, SELENOM KO mice showed no apparent deficits in motor coordination, anxiety-like behavior, or cognition. Nonetheless, it should be noted that these behavioral tests were all conducted on young adult mice fed a Se-adequate diet. Thus, it is unclear whether further perturbations may arise when mice age or are challenged with Se-deficiency.

While the current literature on SELENOM is limited, several studies suggest that it may be protective against Alzheimer’s disease. For example, SELENOM expression levels were found significantly reduced in transgenic mice overexpressing a human mutant presenilin 2 (PS2) gene that causes early onset familial Alzheimer’s disease (FAD) [67]. Importantly, FAD-linked presenilin-2 mutants are known to disrupt Ca²⁺ homeostasis, as they have been documented to inhibit SERCA2b activity, increase ER Ca²⁺ leakage, and promote Ca²⁺ shuttling from the ER to mitochondria [68, 69]. Furthermore, with respect to Alzheimer’s disease, elevated levels of amyloid beta (Aβ) are known to promote ER Ca²⁺ release and apoptosis [70, 71], and in vitro studies have found that SELENOM protects against Aβ-induced toxicity [72, 73]. One of these studies reported that SELENOM attenuates Aβ-induced toxicity by binding the transition metals Zn²⁺ and Cd²⁺ in its thioredoxin-like active site [73]. Yet, this study was performed using a Sec-to-Cys SELENOM mutant and zinc-binding has yet to be verified with endogenous Sec-containing SELENOM. In addition, rats overexpressing SELENOM display increased activation of the ERK pathway and elevated glutathione peroxidase and superoxide dismutase activity in brain [74]. When combined with Se supplementation, SELENOM overexpression decreased γ-secretase activity, which drives production of Aβ42, the most pathogenic form of the Aβ peptide implicated in the development of Alzheimer’s. More recent studies on cortical tissue utilized two-dimensional electrophoresis to identify which proteins were most affected by SELENOM overexpression [75]. These analyses determined that five proteins were significantly elevated: brain-type creatine kinase (CKB), E3 ubiquitin ligase RING1 (RING1), lactate dehydrogenase B (LDHB), synaptotagmin-15 (SYT15), and eukaryotic initiation factor 4H (EIF4H). Both creatine kinase and lactate dehydrogenase are established enzymes in energy metabolism, providing additional corroborating evidence for the influence of SELENOM on this process.

The mechanistic basis of how SELENOM regulates Ca²⁺ homeostasis has yet to be elucidated. It presumably acts by reducing disulfide bonds on proteins involved in Ca²⁺ shuttling within the ER lumen, with prospective partners including the IP3Rs, RyRs, SERCA2b, and PS2. Further studies are warranted to: 1) unveil the precise molecular nature of how SELENOM influences Ca²⁺ homeostasis and 2) determine the signaling pathways affected by SELENOM that contribute to obesity.

Selenoprotein N (SELENON)

This 70 kDa transmembrane protein is localized to the ER and SELENON mRNA is expressed highest during development with lower, ubiquitous expression detected in adult tissues [76, 77]. There are two isoforms of the SELENON gene product, one corresponding to the full-length transcript and the second excluding exon 3 via splicing. Both transcripts are detected in skeletal muscle, brain, lung and placenta, with isoform 2 being the predominant transcript [78]. The full-length transcript has two potential in-frame Sec incorporation sites, whereas isoform 2 codes for only one Sec residue. Experimental evidence indicates that only the single Sec-containing isoform 2 is successfully translated [85]. The SELENON amino acid sequence contains a redox motif S-C-U-G that is similar to the G-C-U-G motif in TXNRDs, which suggests it can exert oxidoreductase activity. However, no enzymatic activity or specific biological function has yet been identified for SELENON. This is quite surprising given the abundance of evidence in humans and rodent models implicating defective SELENON expression in congenital myopathy characterized by muscle weakness, spinal rigidity, and respiratory insufficiency due to impaired diaphragm function [79, 80]. The diseases attributed to mutations in the SELENON gene include rigid spine muscular dystrophy (RSMD1), multi-minicore disease, rare cases of congenital fiber type disproportion, and desmin-related myopathy with Mallory body-like inclusions, all of which are collectively referred to as SEPN1-relative myopathies [81]. These muscle-related disorders that may involve respiratory insufficiency, but SEPN1-relative myopathies exhibit varied outcomes with a wide spectrum of severity and the onset can range from infancy to adulthood.

The topology of SELENON features its Sec located within the ER lumen [82]. Thus, the potential oxidoreductase activity of SELENON may be directed at the contents of the ER and may involve redox sensing, protein-folding, chaperoning, or some combination of these functions. One potential role for SELENON has been uncovered with evidence that it can reverse H₂O₂ induced inhibition of SERCA2b. In particular, SELENON may reduce disulfide bonds between a L4 cysteine pair, allowing SERCA2b driven Ca²⁺ pumping activity to proceed [83]. Consistent with this notion, SELENON-deficient myotubes showed higher basal Ca²⁺ levels in the cytosol and lower Ca²⁺ levels in the ER [84]. Furthermore, the absence of SELENON was associated with abnormal susceptibility to H₂O₂ induced oxidative stress, demonstrated by increased cell death. A healthy cell phenotype was restored by pretreatment with the antioxidant N-acetylcysteine. There likely are other proteins that are targeted by SELENON for reduction of disulfide bonds, perhaps Ca²⁺ handling proteins or proteins in the process of conformational folding. Also, SELENON is not the only redox regulator of L4 cysteine pair in SERCA2b [85], so other activities by this putative selenoenzyme are likely to be important for preventing SEPN1-relative myopathies. These muscle-related disorders that may involve respiratory insufficiency, likely due to defects in diaphragm function, and muscle dysfunctions are the common feature for the wide variety of disorders associated with mutations in the SELENON gene [86]. The molecular mechanisms by which the different mutations affect SELENON expression or function remains to be determined, but it may be related to SERCA2b function as described in more detail below.

An interesting feature of SELENON is the presence of at least one EF hand motif located in a position predicted to be within the ER lumen [87]. EF hand motifs bind Ca²⁺, and proteins containing these motifs exhibit structural changes when transitioning between the bound or unbound state [88]. In this manner, SELENON may sense Ca²⁺ within the ER lumen and switch between Ca²⁺ bound and unbound states. The presence or absence of Ca²⁺ bound to SELENON’s EF hand motif are likely to induce conformational changes in the protein, although experimental evidence for this is lacking. This may be related to the finding in zebrafish that SELENON interacts with the ryanodine receptor 1 (RyR1), which is the major component of the RyR intracellular Ca²⁺ release channel [89]. This is important given that mutations affecting SELENON and RyR1 result in an overlapping spectrum of congenital myopathies. The relevance of this model is evident in the fact that selenoprotein N function causes disruption of muscle architecture in the zebrafish embryo [90]. Interestingly, loss of function of either SELENON or RyR1 in developing zebrafish led to defects in Ca²⁺ mobilization in the embryo as measured by free Ca²⁺ levels around the Kupffer's Vesicle, a site used for Ca²⁺ flux measurement in the zebrafish embryo. Thus, the evidence from this embryonic model strongly supports the notion that SELENON along with RyR1 regulate Ca²⁺ flux to support muscle tissue development. It is frustrating that a specific molecular or enzymatic activity has not yet been elucidated to explain this effect, but the S-C-U-G and/or EF hand motifs are likely to be involved and further investigation may reveal how sometime in the future.

Selenoprotein S (SELENOS)

SELENOS is another of the many selenoproteins discovered in silico by the Gladyshev laboratory [3]. Prior to its identification as a selenoprotein, it was known in the literature as TANIS, a novel gene found upregulated in response to fasting in the obese/diabetic animal model Pssamomys obesus [91]. In humans, the SELENOS gene encodes two alternative transcripts that differ in their 3’ UTR sequences [92]. Experiments conducted in vitro revealed that read-through of the UGA/Sec codon occurred in one variant, whereas the other transcript encoded a truncated protein that lacked Sec. Full-length SELENOS is predominantly localized to the ER membrane and consists of 189 amino acids. Although not ancestrally related to SELENOK, SELENOS shares several important topological features worth noting. These include a single-pass ER transmembrane domain and proline residues located in a glycine-rich C-terminal region that mediates binding with VCP [93]. Due to its interaction with VCP, SELENOS has also been referred to in the literature as VIMP, an acronym for VCP-interacting membrane protein [94]. VCP is a particularly important ATPase at the ER membrane that uses the energy of ATP hydrolysis to move or segregate proteins within complexes [95]. The VCP chaperone binds to both SELENOS and SELENOK, but how the interactions occur and whether the cytosolic-facing Sec residues located near the C-terminus are involved is presently unclear. For SELENOS, the Sec residue (position 188) is known to bond with an adjacent Cys residue (position 174), to yield a Cys174-Sec188 selenosulfide bond [96, 97]. Furthermore, studies using a recombinant SELENOS where the Sec residue was replaced by Cys, observed a reduction potential of −200 mV and determined it to be a substrate of thioredoxin [98].

SELENOS is widely expressed, with highest levels reported in pancreas, adipose tissue, testis, and stomach. Its promoter contains an ER stress response element and two binding sites for nuclear factor kappa B [99]. Likewise, various studies have observed SELENOS upregulation in response to inflammatory cytokines [100], ER stress, and glucose deprivation [101]. Among humans, genetic variation of SELENOS has been linked to inflammation, as a single nucleotide polymorphism in the promoter region (−105G»A) was identified that significantly associated with inflammatory cytokine levels and exhibited diminished responsiveness to challenge with ER stress [100]. Additional experiments in macrophages demonstrated that SELENOS knockdown increased release of IL-6 and TNF-α, corroborating the role of SELENOS in inflammation. The link to inflammation is further supported by prior studies that identified the acute-phase inflammatory protein, serum amyloid A, as an interaction partner of SELENOS by yeast two-hybrid screening [91].

In terms of function, several studies have established SELENOS as an essential player in ERAD. It is an important component of a multiprotein complex that translocates misfolded proteins across the ER membrane for cytosolic degradation by the ubiquitin-proteasome system. In addition to SELENOS, this complex includes VCP, the Derlin chaperone proteins, SELENOK, and E3 ubiquitin ligases [58, 94]. Recent studies demonstrated that formation of this retrotranslocation complex is contingent upon SELENOS-mediated recruitment of VCP to the ER membrane [58]. Moreover, experiments in cell culture by another group found that SELENOS knockdown impairs cell viability and increases tau phosphorylation in response to agents that cause ER stress [102]. These findings, along with the observation of pronounced SELENOS expression in neurofibrillary tangles in postmortem human brain, suggest that SELENOS may be protective against neurodegeneration. To date, there is no evidence directly implicating SELENOS in Ca²⁺ regulation. Nonetheless, it is highly likely that SELENOS exerts an indirect influence on Ca²⁺ signaling by maintaining ER homeostasis through its documented role in ERAD.

Selenoprotein T (SELENOT)

SELENOT is a thioredoxin-like enzyme localized to the ER membrane that was initially identified using an algorithm that searched for mRNAs containing a selenocysteine insertion element (SECIS) in the 3’ untranslated region (UTR) [103]. Structurally, SELENOT contains a signal peptide, a thioredoxin-like fold with a Sec residue in the active site, and two hydrophobic stretches (residues 87–102 and 125–143) indicative of transmembrane domains [104]. It is presumed to modulate ER thiol redox balance and recent in vitro studies have shown that it exhibits thioredoxin reductase-like activity [105].

SELENOT is widely expressed at high levels during embryogenesis and early development, but expression levels are very low postnatally, with the exception of endocrine tissues such as the pancreas, pituitary, testis, and thyroid [106]. In adult tissues, SELENOT expression is most pronounced in secretory cells, such as pancreatic islets and testicular Leydig cells, which produce insulin and testosterone, respectively [106, 107]. Although expression levels are typically low in non-endocrine tissues, exposure to noxious stimuli leads to significant upregulation. In the mouse brain, SELENOT is barely detectable under baseline conditions but is significantly elevated in the nigrostriatal pathway in response to treatment with the neurotoxin MPTP [105]. Moreover, increased SELENOT expression was also found in the striatum of postmortem samples from individuals with Parkinson’s disease. In addition, while SELENOT levels are normally undetectable in liver, expression was reported to be strongly induced during the acute phase of liver regeneration [106]. One of the first papers describing SELENOT identified it as a target gene of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) during differentiation of PC12 cells [108]. This paper determined that SELENOT upregulation was dependent upon increased levels of cAMP and intracellular Ca²⁺ flux in response to PACAP. More recent studies investigating the nature of the PACAP-induced signal activating SELENOT gene transcription unveiled a pathway involving protein kinase A, AMP kinase, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) that is coupled to mitochondrial biogenesis [104].

Along with having its expression regulated by Ca²⁺, SELENOT itself contributes to Ca²⁺ homeostasis. Silencing of SELENOT was observed to impair Ca²⁺ mobilization induced by PACAP, whereas SELENOT overexpression elevated baseline cytosolic Ca²⁺ levels [108]. Later studies performed on gastric smooth muscle by a different group, reported that siRNA-mediated SelT knockdown diminished baseline cytosolic Ca²⁺ content and significantly reduced the enzymatic activity of myosin light chain kinase, a key regulator of muscle contraction [109]. It is currently speculated that SELENOT modulates Ca²⁺ signaling by means of a redox mechanism involving thiol groups on Ca²⁺ channels and pumps, but the precise nature of how this occurs has yet to be elucidated.

With respect to function, SELENOT is one of only four selenoproteins known to cause embryonic lethality in mice (GPX4, TXNRD1, and TXNRD2 being the others) and among this group, it is the only one present in the ER [105, 110]. Conditional knockout mice have been generated that ablate SELENOT expression in pancreatic β-cells [107] and nervous tissue [105, 110]. In mice where SELENOT knockout was restricted to pancreatic β-cells, researchers observed alterations in the size and number of pancreatic islets, along with impairments in glucose-stimulated insulin secretion and glucose tolerance. Two recent papers have bred floxed SELENOT mice with another strain expressing Cre under the control of the Nestin promoter (Nestin-Cre) in order to selectively silence SELENOT in neural cells. These mice were reported to exhibit reduced brain volume during the first postnatal week that was associated with increased apoptotic death of immature neurons [110]. Brain size normalized as the mice matured and the predominant behavioral phenotype in adulthood was hyperactivity in the open field apparatus. Subsequent studies showed that these mice were more vulnerable to MPTP-induced neurotoxicity, as they exhibited increased levels of oxidative stress, extensive neurodegeneration in dopaminergic neurons, and severe motor deficits in comparison to controls [105]. This demonstrates that although SELENOT is minimally expressed in the adult brain, it does protect dopaminergic neurons against toxic insults.

Type 2 Iodothyronine Deiodinase (DIO2)

Thyroid hormone (TH) is regulated by the actions of three selenoproteins: iodothyronine deiodinase 1 and 2 (DIO1 and 2) that catalyze the conversion of the pro-hormone, T4, to the active hormone T3, and iodothyronine deiodinase 3 (DIO3) that catalyzes the deactivation of the hormone [111]. DIO2 is the predominant form in adult tissues that generates active T3 in local tissues [112], and consists of a single transmembrane protein with the Sec near the C-terminus located in the cytosol similar to DIO1 [113]. The activity of DIO2 is regulated at many levels, but degradation through ERAD is particularly interesting. In mammals, DIO2 is ubiquitinated by the E3 ubiquitin ligases, WSB-1 [114] and TEB4 [115]). Ubiquitinated DIO2 interacts with VCP and can be retrotranslocated to the cytosol for proteasomal degradation [116]. However, the degradation of DIO2 is prevented by two ubiquitin-specific peptidases, USP20 and USP33, that promote deubiquitination and counteract the catabolic effects of WSB-1 and TEB4 [117]. In addition, ER stress has been shown to rapidly reduce DIO2 activity [118]. Surprisingly, this observed reduction in activity was not associated with decreased mRNA levels or increased proteasomal degradation, but rather resulted from diminished de novo DIO2 synthesis due to p-eIF2α-mediated inhibition of translation. So, both the synthesis and degradation of DIO2 appears to be dynamically regulated by environmental conditions to control local levels of T3. Also influenced by DIO2 is SERCA2b, which has been found in both slow twitch skeletal muscles and human myocardium. Levels of SERCA2b mRNA are regulated by TH through TH response elements in the promoter of the SERCA2b gene [119]. This has implications for cardiomyocytes, in that active T3 produced by the activity of the DIO2 (and possibly DIO1) can accelerate sarcoplasmic reticulum Ca²⁺ uptake, thereby improving cardiomyocyte relaxation and overall cardiac diastolic function [120]. A transgenic mouse model was developed in which human DIO2 was highly expressed in the heart and myocardial thyrotoxicity was detected in isolated hearts [121]. Thus, there appears to be an upper limit to the beneficial effects of more active TH available to the cardiomyocytes. Hence, DIO2 may control Ca²⁺ related functions in cells in a manner that is controlled by ERAD directed turnover of DIO2 levels.

Conclusions

Several selenoproteins are localized to the ER but exhibit diverse structural features and different topologies, with some found in the membrane and others in the lumen. This is consistent with the notion that they play different roles, which has been borne out by reports showing some to be involved in sensing and regulating redox tone, and others regulating protein folding and the chaperoning of misfolded proteins during UPR (Figure 2). In some cases, roles have been demonstrated for ER selenoproteins in regulating Ca²⁺ flux or homeostasis. Exactly how selenoproteins are involved in these processes is only beginning to be understood. Given that none of these ER selenoproteins are considered ion channels and only one (SELENON) has a Ca²⁺ binding EF hand motif, it is likely that most play indirect roles in regulating Ca²⁺ homeostasis and signaling. Examples include SELENON and SELENOK, which indirectly regulate ER Ca²⁺ levels by modifying the SERCA2b pump and IP3Rs, respectively. In addition to the ER resident selenoproteins discussed above, other well characterized selenoproteins that function as cytosolic or mitochondrial enzymes in redox based reactions are likely to influence Ca²⁺ homeostasis and Ca²⁺-dependent signaling. Overall, it remains to be determined exactly how the ER selenoproteins impinge upon Ca²⁺ homeostasis and Ca²⁺-dependent signaling, and there is a need for further dissection of molecular mechanisms by which they operate.

Figure 2.

Major topological features of ER selenoproteins. This illustration details the topology of Sec residues and transmembrane domains in ER selenoproteins. Also shown are key proteins involved in Ca²⁺ homeostasis, protein folding, and ERAD known to interact with individual ER selenoproteins. U represents Sec residues.

Highlights.

• Seven of twenty-five selenoproteins are localized to the ER but exhibit diverse structural features and different topologies, with some found in the membrane and others in the lumen.

• Some ER selenoproteins have been shown to be involved in sensing and regulating redox tone, and others regulating protein folding and the chaperoning of misfolded proteins during unfolded protein response, and others in regulating Ca²⁺ flux and homeostasis.

• Examples of molecular mechanisms involving these ER selenoproteins include SELENON and SELENOK, which indirectly regulate ER Ca²⁺ levels by modifying the SERCA2 pump and IP3R, respectively.

• The role of the selenocysteine (Sec) residue within the seven ER selenoproteins in some cases involves its oxidoreductase potential, while in others remains unclear.