Oxidative stress is an important component of disease etiology and progression.At the cellular level while inhibition of overall protein synthesis is a measure of stress ingeneral increasing evidence suggests that selective protein translation occurs anddetermines cell fate. Many xenobiotics or disease states cause an increase in oxidativestress. We have found that low to mild doses of oxidants trigger de novo translation ofNrf2 protein a transcription factor regulating a network of antioxidant and detoxificationgenes. Deficiency of Nrf2 protein results in an increased sensitivity to a variety ofchemical and pathophysiological stresses. It is not known the components in thetranslation machinery responsible for Nrf2 protein translation. Normally initiation ofprotein translation requires recognition of 7-methyl Guanine cap at the 5' end (5' m7G)of an mRNA strand by eIF4E in the eIF4F complex and recruitment of the 43S pre-initiation complex. Human Nrf2 gene encodes an mRNA species containing a 555nucleotide 5' Untranslated Region (5'UTR). Genes containing an Internal RibosomalEntry Site (IRES) in 5'UTR can bypass 5' m7G cap-dependent translation and undergostress-induced protein translation. Using LC-MS/MS based proteomics we haveidentified the La autoantigen as a binding partner of Nrf2 5'UTR. Oxidants induce Laprotein to translocate from the nucleus to the cytoplasm where it exhibits increasedbinding to Nrf2 mRNA and ribosomes resulting in Nrf2 protein translation. Nrf2 istypically activated when modification of redox-sensitive cysteine residues in Keap1renders it incapable of mediating Nrf2 ubiquitination. Whether or not de novo Nrf2protein translation is sufficient for activating the Nrf2 transcription network has not beenaddressed. We hypothesize that La facilitates the assembly of the 48S InitiationComplex for de novo Nrf2 protein translation and La-Nrf2 signaling is essentialfor cytoprotection via controlling gene expression under oxidative stress. Aim 1will test whether RNA recognition motifs of La protein form physical contact with aspecific region of Nrf2 5'UTR to mediate de novo Nrf2 protein translation. The region ofNrf2 5'UTR for La binding will be mapped using an RNA Electrophoretic Mobility ShiftAssay (EMSA) in combination with RNase/Chemical probing. The motif of La proteinresponsible for binding to Nrf2 5'UTR will be identified by deletion or mutation. Aim 2will test whether La coordinates the assembly of the 48S Initiation Complex to drive newprotein translation under oxidative stress. The eIF4E 4G 4A 4B 4H 1 1A 2 3 55B and PABP are essential elements of the 48S Initiation Complex (48S IC) and will beexamined for interactions with La and La/Nrf2 5'UTR complex under oxidative stress.Proteins associated with the La or the La binding site of Nrf2 5'UTR will be revealedusing high resolution LC-MS/MS proteomics. The role of these proteins will be tested forassembly of 48S IC and de novo Nrf2 protein translation. Aim 3 will test whether denovo Nrf2 protein translation mediates cytoprotection and expression of antioxidant anddetoxification genes independent of Keap1 during oxidative stress. La will be knockedout using siRNA and CRISPR and knocked in using Nrf2 5'UTR binding null mutant toinvestigate its cytoprotective role. We will further define the La-Nrf2 axis of geneexpression using microarray and RNAseq. Keap1 null cells and conditional Keap1knockout mice will be tested for Nrf2 induction via de novo protein translation andsubsequent cytoprotection (in vitro) or cardiac protection against ischemic injury (invivo). The PI has expertise for contemporary state-of-the-art cell and molecular biologyresearch. This project will advance our understanding on the mechanism and functionalimpact of de novo Nrf2 protein translation under oxidative stress.