Overcoming Challenges with Biochemical Studies of Selenocysteine and Selenoproteins

Abstract

Selenocysteine (Sec) is an essential amino acid that distinguishes itself from cysteine by a selenium atom in place of a sulfur atom. This single change imparts distinct chemical properties to Sec which are crucial for selenoprotein (Sec-containing protein) function. These properties include a lower pK_a, enhanced nucleophilicity, and reversible oxidation. However, studying Sec incorporation in proteins is a complex process. While we find Sec in all domains of life, each domain has distinct translation mechanisms. These mechanisms are unique to canonical translation and are composed of Sec-specific enzymes and an mRNA hairpin to drive recoding of the UGA stop codon with Sec. In this review, we highlight the obstacles that arise when investigating Sec insertion, and the role that Sec has in proteins. We discuss the strategic methods implemented in this field to address these challenges. Though the Sec translation system is complex, a remarkable amount of information has been obtained and specialized tools have been developed. Continued studies in this area will provide a deeper understanding on the role of Sec in the context of proteins, and the necessity that we have for maintaining this complex translation machinery to make selenoproteins.

Keywords: biochemistry; redox; selenium; selenocysteine; selenoprotein; translation.

Figure 1

Natural insertion of selenocysteine (Sec) into proteins. (A) Sec is synthesized on its tRNA (tRNA^Sec) because of the labile nature of the free amino acid (red letters). This requires tRNA^Sec to first be aminoacylated with serine (Ser) by seryl-tRNA synthetase (SerRS). Ser is then converted to Sec in a single step in bacteria by selenocysteine synthase (SelA) or two steps in archaea and eukaryotes by O-phosphoseryl-tRNA^Sec kinase (PSTK) and O-phosphoserine tRNA^Sec: selenocysteine synthase (SepSecS). Selenophosphate (SePO₃³⁻) is the donor molecule required for converting Ser to Sec, but this is also labile (red letters) and therefore is generated by a separate enzyme (SelD or SPS2) from elemental selenium (HSe⁻). (B) The process of bringing Sec-tRNA^Sec to the ribosome for translation requires multiple factors that differ depending on the domain of life. Briefly, Sec-tRNA^Sec is recognized by a specialized elongation factor (SelB, aSelB or EFSec) and is directed to a UGA codon by an mRNA hairpin (SECIS element) required in the translated (bacteria) or untranslated region (archaea and eukaryotes) of the mRNA.

Figure 2

Schematic of experimental requirements required to study various stages of (A) aminoacylation and (B) translation for selenoproteins. Molecules are color-coordinated to follow when they are needed.

Figure 3

Strategies to express selenoproteins that (A) utilize the SECIS element or (B) are SECIS independent. (A) In bacteria, when the Sec to be inserted is near the C-terminal end of the protein, a SECIS element is placed right after the stop codon (UAA) in the 3′UTR. In eukaryotes, a SECIS element from Toxoplasma gondii is inserted downstream of the designated stop codon in the 3′UTR. (B) Removing the SECIS element in Escherichia coli and Saccharomyces cerevisiae requires the use of an engineered tRNA (allo-tRNA or SctRNA^Sec) that is recognized by Aeromonas salmonicida SelA for conversion of Ser to Sec and the endogenous elongation factor (EF-Tu or EF-1α).

Figure 4

Cell-free methods for selenoprotein expression involving (A) in vitro protein expression and (B) chemical synthesis. (A) Selenoproteins can be expressed using in vitro translation systems based on the previously described in vivo methods. Here we show one method that cannot be replicated in vivo, which involves acylating tRNA^Cys with Sec using CysRS. Under the correct reducing conditions (DTT in an anaerobic environment), selenocystine is reduced to Sec and can be used as a substrate for aminoacylation. In the absence of Cys, all codons encoding for Cys will contain Sec. (B) Native chemical ligation involves the fusion of two peptide fragments together. These can be expressed in a host or chemically synthesized. For selenoproteins, Sec is an active amino acid in the ligation reaction and, with chemical synthesis into a peptide, can facilitate the formation of a selenoprotein.