The buildup of reactive oxygen species (ROS) and reactive nitrogen and oxygen species (RNS) is known as oxidative stress. Oxidative stress results in a wide variety of modification to biological macromolecules including nucleic acids, lipids, and proteins. For at least 30 years it has been known that high levels of oxidative stress leads to damage to cellular components resulting in cell death via apoptosis and necrosis. In perhaps the last 10 years it has become clear that ROS and RNS also mediate ‘redox signaling’ via specific macromolecule modifications. The formation of the oxidative post-translational modification (Ox-PTM) 3-nitrotyrosine (nitroTyr) in proteins serves as the main biomarker of oxidative stress and is present in over 50 disease pathologies. In the vast majority of these diseases it is not known if the nitroTyr is simply a bystander or is playing an active role driving disease progression. Genetic code expansion (GCE) has emerged as a method to install noncanonical amino acids (ncAAs) into proteins of interest. Here we develop several GCE systems for the incorporation of nitroTyr to address the role of tyrosine nitration in biology.
This dissertation presents studies aimed to both improve and develop GCE systems for encoding nitroTyr in E. coli and eukaryotic cells, and to apply this ability to studying the effect of tyrosine nitration on the calcium (Ca²⁺) regulatory protein calmodulin (CaM). Four chapters of original work are presented in this dissertation and include one review and three primary research reports. Three of the chapters (Chapters 2, 3, and 5) focus on the development and optimization of GCE systems for the incorporation of the nitroTyr, and the other chapter (Chapter 4) focuses on the application of the GCE systems to evaluate the nitroTyr Ox-PTMs in the regulatory hub protein, CaM, as the first report of regulating a protein function with the nitroTyr PTM. Chapters 2 and 3 are published; Chapters 4 and 5 are at the stage of manuscripts in preparation for submission. These central chapters are bookended by a brief introduction to oxidative stress and GCE (Chapter 1) and concluding remarks on my summary of the impact of this work, and perspective on future directions (Chapter 6).
In the first research chapter (Chapter 2), I review the strengths and weaknesses of GCE for the study of Ox-PTMs, provide an overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs. The GCE systems for nitroTyr reviewed in Chapter 2, show clear phenotypic evidence that they cannot match the efficiency of natural translational systems, however the root causes of these efficiencies were unknown. In Chapter 3, I correlate the efficiency of in vivo GCE with kinetic constants derived from the in vitro aminoacylation reactions carried out by several nitrotyrosyl-aminoacyl-tRNA synthetases (nitrotyrosyl-aaRS-tRNA) derived from the Methanocaldococcus janaschii (M. jannaschii) tyrosyl-aaRS-tRNA. On the basis of these measurements I report that certain modifications made to the tRNA sequence thought to be necessary for maintaining translational fidelity are not necessary and negatively impact the efficiency of the system.
With these optimized tools in hand I applied them to addressing a major outstanding question in the oxidative stress field in chapter 4; can the oxidative stress induced nitrotyrosine PTM regulate protein function? As a way to address the question I investigated the effect of tyrosine nitration on the regulatory hub protein CaM. Tyrosine phosphorylation of CaM is known to modulate its regulation of intracellular Ca²⁺ signling. Proteomic studies have also revealed that both tyrosines in CaM are nitrated in vivo, although the impact of this modification is unknown. The effect of tyrosine nitration on the CaM target protein endothelial nitric oxide synthase (eNOS) is of particular interest as this signaling protein can generate the oxidants that result in tyrosine nitration, potentially indicating a feedback loop and signaling microdomain. Using GCE I show that tyrosine nitration alters CaM regulation with regard to eNOS function in vitro and in eukaryotic cell lysate.
To extend this research further, I developed a system to genetically encode tyrosine nitration directly into proteins in eukaryotic cells (Chapter 5). This is an empowering technology for studying the impact of tyrosine nitration on regulation/dysregulation of cellular function in the native context of the modification. This technology is also of particular utility for the study of nitrated transmembrane proteins that need to be studied in eukaryotic cell culture or animal models.