Structural and biophysical characterization of the selenoprotein SelW1 from Chlamydomonas reinhardtii.

Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas. The U14C mutant of SelW1 was isolated and biophysically characterized. It contains an intramolecular disulfide bond and is thermally stable up to 70 °C. NMR resonance assignment of reduced and oxidized SelW1 showed that SelW1 adopts a thioredoxin fold. Interestingly, both forms show two additional sets of resonance for amino acid residues near the termini and have basically identical dynamic behavior. Since SelW1 from Chlamydomonas resembles the ancestor of mammalian selenoproteins in certain aspects, this study lays the basis for future characterization of SelW1 function and possible interaction partners.

Selenoprotein W redox-regulated Ca2+ channels correlate with selenium deficiency-induced muscles Ca2+ leak.

Selenium (Se) deficiency induces Ca2+ leak and calcification in mammal skeletal muscles; however, the exact mechanism is still unclear. In the present study, both Se-deficient chicken muscle models and selenoprotein W (SelW) gene knockdown myoblast and embryo models were used to study the mechanism. The results showed that Se deficiency-induced typical muscular injuries accompanied with Ca2+ leak and oxidative stress (P < 0.05) injured the ultrastructure of the sarcoplasmic reticulum (SR) and mitochondria; decreased the levels of the Ca2+ channels, SERCA, SLC8A, CACNA1S, ORAI1, STIM1, TRPC1, and TRPC3 (P < 0.05); and increased the levels of Ca2+ channel PMCA (P < 0.05). Similarly, SelW knockdown also induced Ca2+ leak from the SR and cytoplasm; increased mitochondrial Ca2+ levels and oxidative stress; injured SR and mitochondrial ultrastructure; decreased levels of SLC8A, CACNA1S, ORA1, TRPC1, and TRPC3; and caused abnormal activities of Ca2+ channels in response to inhibitors in myoblasts and chicken embryos. Thus, both Se deficiency and SelW knockdown induced Ca2+ leak, oxidative stress, and Ca2+ channel reduction. In addition, Ca2+ levels and the expression of the Ca2+ channels, RyR1, SERCA, CACNA1S, TRPC1, and TRPC3 were recovered to normal levels by N-acetyl-L-cysteine (NAC) treatment compared with SelW knockdown cells. Thus, with regard to the decreased Ca2+ channels, SelW knockdown closely correlated Se deficiency with Ca2+ leak in muscles. The redox regulation role of SelW is crucial in Se deficiency-induced Ca2+ leak in muscles.

Cloning and expression of selenoprotein W from pearl mussels Cristaria plicata.

Selenoprotein W (SelW) is a selenocysteine containing protein with redox activity involved in the antioxidant response. In this study, a selenoprotein W was cloned from pearl mussel Cristaria plicata (designated as CpSelW), and the expression patterns were characterized in tissues after Aeromonas hydrophila challenged. The full-length cDNA of cpSelW was of 858bp, containing a 5' untranslated region (UTR) of 145bp, a 3' UTR of 455bp with a poly (A) tail, and an open reading frame (ORF) of 258bp encoding a polypeptide of 85 amino acids with the predicted molecular mass of 9.277kDa, which shared 61% identity with SelW from Gallus gallus. A tertiary structure model generated for the CpSelW displayed a ß-α-ß-ß-ß-α secondary structure pattern, which was similar to mouse SelW protein 3D structure. The mRNA of CpSelW was constitutively expressed in tested tissues of healthy mussel, including mantle, gill, hemocytes, muscle, and hepatopancreas, and it was highly expressed in hepatopancreas. After mussels were stimulated by A. hydrophila, the mRNA expression of CpSelW in hemocytes at 6, 12 and 24h, in gill at 12h and in hepatopancreas at 24h was significantly down-regulated.

Molecular cloning, characterization and mRNA expression analysis of a novel selenoprotein: avian selenoprotein W from chicken.

In this study, a novel avian selenoprotein W (SelW) gene was cloned from chicken cerebral tissue. The complete nucleotide sequence of the gene contained a 258 bp open reading frame encoding 85 amino acids. Bioinformatics approaches identified the chicken SelW protein is characterized by a ß-α-ß-ß-ß-α secondary structure pattern, wherein ß1 and ß2 are parallel strands forming a classical ß1-α1-ß2 motif, which is also observed in thioredoxin-like fold proteins. The protein has a candidate CXXU redox motif which is located in the loop (residues 10-13) between ß1 and α1. The 3D structural similarity between mouse and chicken SelW protein suggests that the proteins may exhibit similar functions. Additionally, a selenocysteine insertion sequence (SECIS) element was found in the 3'-untranslated region of the SelW mRNA. The SECIS element was classified as form II. Moreover, we analyzed the mRNA expression of SelW genes in 36 different tissues of 60-day-old chickens; the expression of SelW was detected in all tissues, indicating that SelW is expressed widely in chicken tissue. Hence, we suggest that SelW might play an important role in the biochemical functions of Se in birds.

Interaction of selenoprotein W with 14-3-3 proteins: a computational approach.

SelW, a protein containing a selenocysteine (Sec) in a conserved Cys-X-X-Sec motif, has been suggested to have an antioxidant role in cell metabolism. SelW is known to specifically interact with different isoforms of 14-3-3 proteins. The latter are involved in several cellular processes such as regulation of the cell cycle, metabolism control, apoptosis, protein trafficking, and gene transcription. 14-3-3 proteins feature a conserved solvent-exposed cysteine residue, in a surface environment prone to induce chemical modifications of the thiol functionality following oxidative stress. The structures of 12 homologous complexes between SelW and 14-3-3 were calculated using sequential alignments, molecular modeling, and docking algorithms guided by known experimental NMR data. These structures reveal the viability of a protein complex in which the conserved Sec residue on SelW approaches the conserved exposed Cys on 14-3-3, making a plausible Sec-Se-S-Cys bond. On the basis of the structural information derived from these calculations, we propose a working hypothesis that entails a role for SelW as a physiological partner of 14-3-3 proteins, able to facilitate a redox-based regulation mechanism.

Solution structure of selenoprotein W and NMR analysis of its interaction with 14-3-3 proteins.

Selenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13-->Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins.

SelT, SelW, SelH, and Rdx12: genomics and molecular insights into the functions of selenoproteins of a novel thioredoxin-like family.

Selenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins.

Chemical forms of selenium for cancer prevention.

Cancer is becoming an increasingly significant disease worldwide. Currently, more than 7 million people die each year from cancer. With the existing knowledge, at least one-third of worldwide cancer cases could be prevented. Searching for naturally occurring agents in routinely consumed foods that may inhibit cancer development, although challenging, constitutes a valuable and plausible approach to the control and prevention of cancer. To date, the use of the micronutrient selenium (Se) in human clinical trials is limited, but the outcome indicates that Se is among the most promising agents. Although it is convenient to describe the effects of Se in terms of the element, it must always be kept in mind that the chemical form of Se and the dose are determinants of its biological activities. Hyphenated techniques based on coupling chromatographic separation with inductively coupled plasma mass spectrometric (ICP-MS) detection are now established as the most realistic and potent analytical tools available for real-life speciation analysis. These speciation investigations provide evidence that the Se compounds, which can generate monomethylated Se (e.g., Se-methylselenocysteine and methylseleninic acid), are more efficacious than other Se compounds because of their chemoprevention activity.