Chemical Biology Approaches to Interrogate the Selenoproteome.

Abstract

Selenoproteins are the family of proteins that contain the amino acid selenocysteine. Many selenoproteins, including glutathione peroxidases and thioredoxin reductases, play a role in maintaining cellular redox homeostasis. There are a number of examples of homologues of selenoproteins that utilize cysteine residues, raising the question of why selenocysteines are utilized. One hypothesis is that incorporation of selenocysteine protects against irreversible overoxidation, typical of cysteine-containing homologues under high oxidative stress. Studies of selenocysteine function are hampered by challenges both in detection and in recombinant expression of selenoproteins. In fact, about half of the 25 known human selenoproteins remain uncharacterized. Historically, selenoproteins were first detected via labeling with radioactive 75Se or by use of inductively coupled plasma-mass spectrometry to monitor nonradioactive selenium. More recently, tandem mass-spectrometry techniques have been developed to detect selenocysteine-containing peptides. For example, the isotopic distribution of selenium has been used as a unique signature to identify selenium-containing peptides from unenriched proteome samples. Additionally, selenocysteine-containing proteins and peptides were selectively enriched using thiol-reactive electrophiles by exploiting the increased reactivity of selenols relative to thiols, especially under low pH conditions. Importantly, the reactivity-based enrichment of selenoproteins can differentiate between oxidized and reduced selenoproteins, providing insight into the activity state. These mass spectrometry-based selenoprotein detection approaches have enabled (1) production of selenoproteome expression atlases, (2) identification of aging-associated changes in selenoprotein expression, (3) characterization of selenocysteine reactivity across the selenoprotein family, and (4) interrogation of selenoprotein targets of small-molecule drugs. Further investigations of selenoprotein function would benefit from recombinant expression of selenoproteins. However, the endogenous mechanism of selenoprotein production makes recombinant expression challenging. Primarily, selenocysteine is biosynthesized on its own tRNA, is dependent on multiple enzymatic steps, and is highly sensitive to selenium concentrations. Furthermore, selenocysteine is encoded by the stop codon UGA, and suppression of that stop codon requires a selenocysteine insertion sequence element in the selenoprotein mRNA. In order to circumvent the low efficiency of the endogenous machinery, selenoproteins have been produced in vitro through native chemical ligation and expressed protein ligation. Attempts have also been made to engineer the endogenous machinery for increased efficiency, including recoding the selenocysteine codon, and engineering the tRNA and the selenocysteine insertion sequence element. Alternatively, genetic code expansion can be used to generate selenoproteins. This approach allows for selenoprotein production directly within its native cellular environment, while bypassing the endogenous selenocysteine incorporation machinery. Furthermore, by incorporating a caged selenocysteine by genetic code expansion, selenoprotein activity can be spatially and temporally controlled. Genetic code expansion has allowed for the expression and uncaging of human selenoproteins in E. coli and more recently in mammalian cells. Together, advances in selenoprotein detection and expression should enable a better understanding of selenoprotein function and provide insight into the necessity for selenocysteine production.