Redox Active Motifs in Selenoproteins

Selenoproteins use the rare amino acid selenocysteine (Sec) to act as the first line of defense against oxidants, which are linked to aging, cancer, and neurodegenerative diseases. Many selenoproteins are oxidoreductases in which the reactive Sec is connected to a neighboring Cys and able to form a ring. These Sec-containing redox motifs govern much of the reactivity of selenoproteins. To study their fundamental properties, we have used Se-77 NMR spectroscopy in concert with theoretical calculations to determine the conformational preferences and mobility of representative motifs. This use of Se-77 as a probe enables the direct recording of the properties of Sec as its environment is systematically changed. We find that all motifs have several ring conformations in their oxidized state. These ring structures are most likely stabilized by weak, nonbonding interactions between the selenium and the amide carbon. To examine how the presence of selenium and ring geometric strain governs the motifs\u27 reactivity, we measured the redox potentials of Sec-containing motifs and their corresponding Cys-only variants. The comparisons reveal that for C-terminal motifs the redox potentials increased between 20-25 mV when the selenenylsulfide bond was changed to a disulfide bond. Changes of similar magnitude arose when we varied ring size or the motifs\u27 flanking residues. This suggests that the presence of Sec is not tied to unusually low redox potentials. The unique roles of selenoproteins in human health and their chemical reactivities may therefore not necessarily be explained by lower redox potentials, as has often been claimed

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-R-sulfoxide reductase 1) 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

Formation and Spatio-Temporal Evolution of Periodic Structures in Lipid

bution functions of the domain lattices. Such analysis is employed to estimate domain interaction forces. The morphology and dynamics of superstructures in lipid bilayers were monitored in giant unilamellar vesicles (GUVs), composed of an equimolar ternary mixture of sphingomyelin, cholesterol, and dioleoylphosphatidylcholine (DOPC). This composition is widely used as a model raft system based on the constituents of the cell membrane detergent insoluble fraction. Cholesterol and sphingolipids interact favorably, creating a liquid-ordered phase in which mobility is reduced relative to the DOPC-enriched phase. Strong adhesion of closed-form GUVs to a silica substrate, coated with a conventional supported lipid bilayer, leads to asymmetric shapes with relatively high surface area-to-volume ratios, similar to shapes observed previously under thermal expansion and osmotic deflation. 10,11 After adhesion, the bound shape gradually transforms to a lowest-energy shape under the adhesio

Contribution of Selenocysteine to the Peroxidase Activity of Selenoprotein S

Selenoprotein S (SelS, VIMP) is an intrinsically disordered enzyme that utilizes selenocysteine to catalyze the reduction of disulfide bonds and peroxides. Here it is demonstrated that selenocysteine is the residue oxidized by the peroxide substrate. It is possible to trap the reaction intermediate selenenic acid when the resolving cysteine is mutated. The selenocysteine allows SelS to rapidly re-form its selenenylsulfide bond following its reduction, and to resist inactivation by H₂O₂. We propose that SelS’s peroxidase mechanism is similar to that of atypical 2-Cys peroxiredoxin and that selenocysteine allows SelS to sustain activity under oxidative stress

Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations

Employing selenocysteine-containing protein fragments to form the amide bond between respective protein fragments significantly extends the current capabilities of the widely used protein engineering method, expressed protein ligation. Selenocysteine-mediated ligation is noteworthy for its high yield and efficiency. However, it has so far been restricted to solid-phase synthesized seleno-peptides and thus constrained by where the selenocysteine can be positioned. Here we employ heterologously expressed seleno-fragments to overcome the placement and size restrictions in selenocysteine-mediated chemical ligation. Following ligation, the selenocysteine can be deselenized into an alanine or serine, resulting in nonselenoproteins. This greatly extends the flexibility in selecting the conjugation site in expressed protein ligations with no influence on native cysteines. Furthermore, the selenocysteine can be used to selectively introduce site-specific protein modifications. Therefore, selenocysteine-mediated expressed protein ligation simplifies incorporation of post-translational modifications into the protein scaffold

Genetic Incorporation of ϵ‐N‐Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl‐tRNA Synthetase

Post-translational modifications regulate protein structure and function. Lysine benzoylation is a newly discovered histone modification with unique physiological relevance. To construct proteins with this modification site-specifically, we generated orthogonal tRNAPyl -MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl-tRNA synthetase, allowing the genetic incorporation of ϵ-N-benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation