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 in general, increasing evidence suggests that selective protein translation occurs and determines cell fate. Many xenobiotics or disease states cause an increase in oxidative stress. We have found that low to mild doses of oxidants trigger de novo translation of Nrf2 protein, a transcription factor regulating a network of antioxidant and detoxification genes. Deficiency of Nrf2 protein results in an increased sensitivity to a variety of chemical and pathophysiological stresses. It is not known the components in the translation machinery responsible for Nrf2 protein translation. Normally, initiation of protein 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 555 nucleotide 5' Untranslated Region (5'UTR). Genes containing an Internal Ribosomal Entry Site (IRES) in 5'UTR can bypass 5' m7G cap-dependent translation and undergo stress-induced protein translation. Using LC-MS/MS based proteomics, we have identified the La autoantigen as a binding partner of Nrf2 5'UTR. Oxidants induce La protein to translocate from the nucleus to the cytoplasm, where it exhibits increased binding to Nrf2 mRNA and ribosomes, resulting in Nrf2 protein translation. Nrf2 is typically activated when modification of redox-sensitive cysteine residues in Keap1 renders it incapable of mediating Nrf2 ubiquitination. Whether or not de novo Nrf2 protein translation is sufficient for activating the Nrf2 transcription network has not been addressed. We hypothesize that La facilitates the assembly of the 48S Initiation Complex for de novo Nrf2 protein translation, and La-Nrf2 signaling is essential for cytoprotection via controlling gene expression under oxidative stress.
Aim 1 will test whether RNA recognition motifs of La protein form physical contact with a specific region of Nrf2 5'UTR to mediate de novo Nrf2 protein translation. The region of Nrf2 5'UTR for La binding will be mapped using an RNA Electrophoretic Mobility Shift Assay (EMSA) in combination with RNase/Chemical probing. The motif of La protein responsible for binding to Nrf2 5'UTR will be identified by deletion or mutation.
Aim 2 will test whether La coordinates the assembly of the 48S Initiation Complex to drive new protein translation under oxidative stress. The eIF4E, 4G, 4A, 4B, 4H, 1, 1A, 2, 3, 5, 5B, and PABP are essential elements of the 48S Initiation Complex (48S IC) and will be examined 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 revealed using high resolution LC-MS/MS proteomics. The role of these proteins will be tested for assembly of 48S IC and de novo Nrf2 protein translation.
Aim 3 will test whether de novo Nrf2 protein translation mediates cytoprotection and expression of antioxidant and detoxification genes independent of Keap1 during oxidative stress. La will be knocked out using siRNA and CRISPR, and knocked in using Nrf2 5'UTR binding null mutant to investigate its cytoprotective role. We will further define the La-Nrf2 axis of gene expression using microarray and RNAseq. Keap1 null cells and conditional Keap1 knockout mice will be tested for Nrf2 induction via de novo protein translation, and subsequent cytoprotection (in vitro) or cardiac protection against ischemic injury (in vivo). The PI has expertise for contemporary state-of-the-art cell and molecular biology research. This project will advance our understanding on the mechanism and functional impact of de novo Nrf2 protein translation under oxidative stress.
