Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications

Abstract

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described.

Efficient Site-Specific Prokaryotic and Eukaryotic Incorporation of Halotyrosine Amino Acids into Proteins

“…Immune cells produce reactive oxygen, halogen, and nitrogen species that participate in host immune surveillance, resulting in tissue damage and oxidative modifications to proteins, DNA, and lipids. Amino acid residues such as tyrosine, tryptophan, lysine, methionine, and cysteine are common targets for oxidative modifications, including halogenation, hydroxylation, nitration, nitrosylation, carbamylation, oxidative cross-links, and elevated states of sulfur oxidation . In particular, post-translational modifications (PTM) of tyrosine residues have emerged as biomarkers for oxidative damage that convey chemical information about the oxidative process involved in their generation and have been observed in more than 50 human pathologies. − In neutrophils and eosinophils, myeloperoxidase (MPO) and eosinophil peroxidase, respectively, mediate the formation predominantly of hypochlorous and hypobromous acids. − In macrophages, activation of NADPH oxidase and inducible nitric oxide synthase (iNOS) results in the concurrent formation of superoxide and nitric oxide (NO), which together form peroxynitrite (ONOO – ). , These reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent tyrosine oxidants, forming chloroTyr, bromoTyr, and nitroTyr under oxidative stress conditions. ,,− Site-specific tyrosine nitration and chlorination have been shown to affect protein–protein interactions with detrimental downstream effects on cellular function and have been established as important biomarkers of oxidative disease states. − However, the specific effects of tyrosine halogenation on protein function have not been well documented.…”

“…In particular, post-translational modifications (PTM) of tyrosine residues have emerged as biomarkers for oxidative damage that convey chemical information about the oxidative process involved in their generation and have been observed in more than 50 human pathologies. − In neutrophils and eosinophils, myeloperoxidase (MPO) and eosinophil peroxidase, respectively, mediate the formation predominantly of hypochlorous and hypobromous acids. − In macrophages, activation of NADPH oxidase and inducible nitric oxide synthase (iNOS) results in the concurrent formation of superoxide and nitric oxide (NO), which together form peroxynitrite (ONOO – ). , These reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent tyrosine oxidants, forming chloroTyr, bromoTyr, and nitroTyr under oxidative stress conditions. ,,− Site-specific tyrosine nitration and chlorination have been shown to affect protein–protein interactions with detrimental downstream effects on cellular function and have been established as important biomarkers of oxidative disease states. − However, the specific effects of tyrosine halogenation on protein function have not been well documented. It is proposed that these oxidative stress-induced post-translational modifications (ox-PTMs) alter protein function and protein–protein interactions …”

Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis

“…To examine the specific functional consequences of a singlesite oxTrp at position 72 in apoA-I, we used GCE to cotranslationally incorporate a noncanonical amino acid in vivo (33). We used an engineered Trp-RS:suppressor tRNA pair (engineered machinery) from S. cerevisiae, which incorporates 5-OHTrp in E. coli in response to a genetically programmed amber nonsense codon (TAG) (34).…”

“…In this study, we sought to address the absolute effect of oxidation at Trp 72 in the absence of any other modification in apoA-I. Genetic code expansion (GCE) has emerged as a powerful method to site-specifically incorporate noncanonical amino acids (ncAAs) into proteins in living cells (33). Specifically, engineered aminoacyl-tRNA synthetase:tRNA pairs are used to deliver a desired ncAA in response to a nonsense or frameshift codon.…”

Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins

“…Indeed, to our knowledge, no Nb has yet been developed against any oxPTM and only a single example has been published for any type of PTM ( Moeglin et al, 2021 ). An avenue to bypass this barrier for PTMs is genetic code expansion (GCE), which can install a variety of PTMs into proteins to generate large quantities of homogenous, site-specific PTM-containing proteins ( Neumann et al, 2008 ; Franco et al, 2013 ; Cooley et al, 2014a ; Cooley et al, 2014b ; DiDonato et al, 2014 ; Franco et al, 2015 ; Porter and Mehl 2018 ; Porter et al, 2019 ; Randall et al, 2019 ; Beyer et al, 2020 ; Jang et al, 2020 ; Porter et al, 2020 ). In GCE, non-canonical amino acids (ncAAs) are site-specifically incorporated into stop codons via orthogonal amino-acyl tRNA synthetase (aaRS)/tRNA UAG pairs.…”