Chemical chaperones in metabolic fitness beyond protein folding.

Chemical chaperones are small molecules that improve protein folding, alleviating aberrant pathological phenotypes due to protein misfolding. Recent reports suggest that, in parallel with their role in relieving endoplasmic reticulum (ER) stress, chemical chaperones rescue mitochondrial function and insulin signaling. These effects may underlie their pharmacological action on metabolically demanding tissues.

Association of COVID-19 mortality with serum selenium, zinc and copper: Six observational studies across Europe.

Introduction: Certain trace elements are essential for life and affect immune system function, and their intake varies by region and population. Alterations in serum Se, Zn and Cu have been associated with COVID-19 mortality risk. We tested the hypothesis that a disease-specific decline occurs and correlates with mortality risk in different countries in Europe. Methods: Serum samples from 551 COVID-19 patients (including 87 non-survivors) who had participated in observational studies in Europe (Belgium, France, Germany, Ireland, Italy, and Poland) were analyzed for trace elements by total reflection X-ray fluorescence. A subset (n=2069) of the European EPIC study served as reference. Analyses were performed blinded to clinical data in one analytical laboratory. Results: Median levels of Se and Zn were lower than in EPIC, except for Zn in Italy. Non-survivors consistently had lower Se and Zn concentrations than survivors and displayed an elevated Cu/Zn ratio. Restricted cubic spline regression models revealed an inverse nonlinear association between Se or Zn and death, and a positive association between Cu/Zn ratio and death. With respect to patient age and sex, Se showed the highest predictive value for death (AUC=0.816), compared with Zn (0.782) or Cu (0.769). Discussion: The data support the potential relevance of a decrease in serum Se and Zn for survival in COVID-19 across Europe. The observational study design cannot account for residual confounding and reverse causation, but supports the need for intervention trials in COVID-19 patients with severe Se and Zn deficiency to test the potential benefit of correcting their deficits for survival and convalescence.

Mosaic Evolution of the Phosphopantothenate Biosynthesis Pathway in Bacteria and Archaea.

Phosphopantothenate is a precursor to synthesis of coenzyme A, a molecule essential to many metabolic pathways. Organisms of the archaeal phyla were shown to utilize a different phosphopantothenate biosynthetic pathway from the eukaryotic and bacterial one. In this study, we report that symbiotic bacteria from the group Candidatus poribacteria present enzymes of the archaeal pathway, namely pantoate kinase and phosphopantothenate synthetase, mirroring what was demonstrated for Picrophilus torridus, an archaea partially utilizing the bacterial pathway. Our results not only support the ancient origin of the coenzyme A pathway in the three domains of life but also highlight its complex and dynamic evolution. Importantly, this study helps to improve protein annotation for this pathway in the C. poribacteria group and other related organisms.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Selenoprotein Gene Nomenclature.

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Selenoprotein N in skeletal muscle: from diseases to function.

Selenoprotein N (SelN) deficiency causes several inherited neuromuscular disorders collectively termed SEPN1-related myopathies, characterized by early onset, generalized muscle atrophy, and muscle weakness affecting especially axial muscles and leading to spine rigidity, severe scoliosis, and respiratory insufficiency. SelN is ubiquitously expressed and is located in the membrane of the endoplasmic reticulum; however, its function remains elusive. The predominant expression of SelN in human fetal tissues and the embryonic muscle phenotype reported in mutant zebrafish suggest that it is involved in myogenesis. In mice, SelN is also mostly expressed during embryogenesis and especially in the myotome, but no defect was detected in muscle development and growth in the Sepn1 knock-out mouse model. By contrast, we recently demonstrated that SelN is essential for muscle regeneration and satellite cell maintenance in mice and humans, hence opening new avenues regarding the pathomechanism(s) leading to SEPN1-related myopathies. At the cellular level, recent data suggested that SelN participates in oxidative and calcium homeostasis, with a potential role in the regulation of the ryanodine receptor activity. Despite the recent and exciting progress regarding the physiological function(s) of SelN in muscle tissue, the pathogenesis leading to SEPN1-related myopathies remains largely unknown, with several unsolved questions, and no treatment available. In this review, we introduce SelN, its properties and expression pattern in zebrafish, mice, and humans, and we discuss its potential roles in muscle tissue and the ensuing clues for the development of therapeutic options.

Misfolded human tRNA isodecoder binds and neutralizes a 3' UTR-embedded Alu element.

Several classes of small noncoding RNAs are key players in cellular metabolism including mRNA decoding, RNA processing, and mRNA stability. Here we show that a tRNA(Asp) isodecoder, corresponding to a human tRNA-derived sequence, binds to an embedded Alu RNA element contained in the 3' UTR of the human aspartyl-tRNA synthetase mRNA. This interaction between two well-known classes of RNA molecules, tRNA and Alu RNA, is driven by an unexpected structural motif and induces a global rearrangement of the 3' UTR. Besides, this 3' UTR contains two functional polyadenylation signals. We propose a model where the tRNA/Alu interaction would modulate the accessibility of the two alternative polyadenylation sites and regulate the stability of the mRNA. This unique regulation mechanism would link gene expression to RNA polymerase III transcription and may have implications in a primate-specific signal pathway.

Increased muscle stress-sensitivity induced by selenoprotein N inactivation in mouse: a mammalian model for SEPN1-related myopathy.

Selenium is an essential trace element and selenoprotein N (SelN) was the first selenium-containing protein shown to be directly involved in human inherited diseases. Mutations in the SEPN1 gene, encoding SelN, cause a group of muscular disorders characterized by predominant affection of axial muscles. SelN has been shown to participate in calcium and redox homeostasis, but its pathophysiological role in skeletal muscle remains largely unknown. To address SelN function in vivo, we generated a Sepn1-null mouse model by gene targeting. The Sepn1(-/-) mice had normal growth and lifespan, and were macroscopically indistinguishable from wild-type littermates. Only minor defects were observed in muscle morphology and contractile properties in SelN-deficient mice in basal conditions. However, when subjected to challenging physical exercise and stress conditions (forced swimming test), Sepn1(-/-) mice developed an obvious phenotype, characterized by limited motility and body rigidity during the swimming session, as well as a progressive curvature of the spine and predominant alteration of paravertebral muscles. This induced phenotype recapitulates the distribution of muscle involvement in patients with SEPN1-Related Myopathy, hence positioning this new animal model as a valuable tool to dissect the role of SelN in muscle function and to characterize the pathophysiological process.

Satellite cell loss and impaired muscle regeneration in selenoprotein N deficiency.

Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.

Compartmentalization and regulation of mitochondrial function by methionine sulfoxide reductases in yeast.

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Selenoprotein N is dynamically expressed during mouse development and detected early in muscle precursors.

BACKGROUND: In humans, mutations in the SEPN1 gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as SEPN1-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount in situ hybridization, we characterized in detail the spatio-temporal expression pattern of the murine Sepn1 gene during development, focusing particularly on skeletal muscles. RESULTS: In whole embryos, Sepn1 transcripts were detected as early as E5.5, with expression levels peaking at E12.5, and then strongly decreasing until birth. In isolated tissues, only mild transcriptional variations were observed during development, whereas a striking reduction of the protein expression was detected during the perinatal period. Furthermore, we demonstrated that Sepn1 is expressed early in somites and restricted to the myotome, the sub-ectodermal mesenchyme and the dorsal root ganglia at mid-gestation stages. Interestingly, Sepn1 deficiency did not alter somitogenesis in embryos, suggesting that SelN is dispensable for these processes in mouse. CONCLUSION: We characterized for the first time the expression pattern of Sepn1 during mammalian embryogenesis and we demonstrated that its differential expression is most likely dependent on major post-transcriptional regulations. Overall, our data strongly suggest a potential role for selenoprotein N from mid-gestation stages to the perinatal period. Interestingly, its specific expression pattern could be related to the current hypothesis that selenoprotein N may regulate the activity of the ryanodine receptors.

Selenoprotein function and muscle disease.

The crucial role of the trace element selenium in livestock and human health, in particular in striated muscle function, has been well established but the underlying molecular mechanisms remain poorly understood. Over the last decade, identification of the full repertoire of selenium-containing proteins has opened the way towards a better characterization of these processes. Two selenoproteins have mainly been investigated in muscle, namely SelW and SelN. Here we address their involvement in muscle development and maintenance, through the characterization of various cellular or animal models. In particular, mutations in the SEPN1 gene encoding selenoprotein N (SelN) cause a group of neuromuscular disorders now referred to as SEPN1-related myopathy. Recent findings on the functional consequences of these mutations suggest an important contribution of SelN to the regulation of oxidative stress and calcium homeostasis. Importantly, the conclusions of these experiments have opened new avenues of investigations that provide grounds for the development of therapeutic approaches.

Loss of selenoprotein N function causes disruption of muscle architecture in the zebrafish embryo.

Mutations in the gene coding for selenoprotein N (SelN), a selenium containing protein of unknown function, cause different forms of congenital muscular dystrophy in humans. These muscular diseases are characterized by early onset of hypotonia which predominantly affect in axial muscles. We used zebrafish as a model system to understand the function of SelN in muscle formation during embryogenesis. Zebrafish SelN is highly homologous to its human counterpart and amino acids corresponding to the mutated positions in human muscle diseases are conserved in the zebrafish protein. The sepn1 gene is highly expressed in the somites and notochord during early development. Inhibition of the sepn1 gene by injection of antisense morpholinos does not alter the fate of the muscular tissue, but causes muscle architecture disorganization and greatly reduced motility. Ultrastructural analysis of the myotomes reveals defects in muscle sarcomeric organization and in myofibers attachment, as well as altered myoseptum integrity. These studies demonstrate the important role of SelN for muscle organization during early development. Moreover, alteration of myofibrils architecture and tendon-like structure in embryo deficient for SelN function provide new insights into the pathological mechanism of SelN-related myopathy.

A single homozygous point mutation in a 3'untranslated region motif of selenoprotein N mRNA causes SEPN1-related myopathy.

Mutations in the SEPN1 gene encoding the selenoprotein N (SelN) have been described in different congenital myopathies. Here, we report the first mutation in the selenocysteine insertion sequence (SECIS) of SelN messenger RNA, a hairpin structure located in the 3' untranslated region, in a patient presenting a classical although mild form of rigid spine muscular dystrophy. We detected a significant reduction in both mRNA and protein levels in the patient's skin fibroblasts. The SECIS element is crucial for the insertion of selenocysteine at the reprogrammed UGA codon by recruiting the SECIS-binding protein 2 (SBP2), and we demonstrated that this mutation abolishes SBP2 binding to SECIS in vitro, thereby preventing co-translational incorporation of selenocysteine and SelN synthesis. The identification of this mutation affecting a conserved base in the SECIS functional motif thereby reveals the structural basis for a novel pathological mechanism leading to SEPN1-related myopathy.

Diversity and functional plasticity of eukaryotic selenoproteins: identification and characterization of the SelJ family.

Selenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. Consistent with its potential role as a structural crystallin, SelJ has preferential and homogeneous expression in the eye lens in early stages of zebrafish development. A structural role for SelJ would be in contrast to the majority of known selenoenzymes. The unusually highly restricted phylogenetic distribution of SelJ, its specialization, and the comparative analysis of eukaryotic selenoproteomes reveal the diversity and functional plasticity of selenoproteins and point to a mosaic evolution of the use of Sec in proteins.

Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution.

While the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome-wide prediction of genes with in-frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteine-containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. In contrast, mammals, worms and land plants contained cysteine homologues. We demonstrated selenium incorporation into chicken SelU and characterized the SelU expression pattern in zebrafish embryos. Our data indicate a scattered evolutionary distribution of selenoproteins in eukaryotes, and suggest that, contrary to the picture emerging from data available so far, other taxa-specific selenoproteins probably exist.

Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish.

Selenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function.

Selenoprotein N: an endoplasmic reticulum glycoprotein with an early developmental expression pattern.

Rigid spine muscular dystrophy and the classical form of multiminicore disease are caused by mutations in SEPN1 gene, leading to a new clinical entity referred to as SEPN1-related myopathy. SEPN1 codes for selenoprotein N, a new member of the selenoprotein family, the function of which is still unknown. In a previous study, two isoforms were deduced from SEPN1 transcript analyses. Using polyclonal antibodies directed against SEPN1 and cDNA constructs encoding for the two isoforms, we show that the main SEPN1 gene product corresponds to a 70 kDa protein, containing a single selenocysteine residue. Subcellular fractionation experiments and endoglycosidase H sensitivity indicate that SEPN1 is a glycoprotein-localized within the endoplasmic reticulum. Immunofluorescence analyses confirm this subcellular localization and green fluorescent protein fusion experiments demonstrate the presence of an endoplasmic reticulum-addressing and -retention signal within the N-terminus. SEPN1 is present at a high level in several human fetal tissues and at a lower level in adult ones, including skeletal muscle. Its high expression in cultured myoblasts is also down-regulated in differentiating myotubes, suggesting a role for SEPN1 in early development and in cell proliferation or regeneration.