BackgroundTranslation shutdown is a hallmark of late-phase, sepsis-induced kidney injury. Methods for controlling protein synthesis in the kidney are limited. Reversing translation shutdown requires dephosphorylation of the eukaryotic initiation factor 2 (eIF2) subunit eIF2α; this is mediated by a key regulatory molecule, protein phosphatase 1 regulatory subunit 15A (Ppp1r15a), also known as GADD34.MethodsTo study protein synthesis in the kidney in a murine endotoxemia model and investigate the feasibility of translation controlin vivoby boosting the protein expression of Ppp1r15a, we combined multiple tools, including ribosome profiling (Ribo-seq), proteomics, polyribosome profiling, and antisense oligonucleotides, and a newly generated Ppp1r15a knock-in mouse model and multiple mutant cell lines.ResultsWe report that translation shutdown in established sepsis-induced kidney injury is brought about by excessive eIF2αphosphorylation and sustained by blunted expression of the counter-regulatory phosphatase Ppp1r15a. We determined the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic and antisense oligonucleotide approaches enabled the overexpression of Ppp1r15a, which salvaged translation and improved kidney function in an endotoxemia model. Loss of this uORF also had broad effects on the composition and phosphorylation status of the immunopeptidome—peptides associated with the MHC—that extended beyond the eIF2αaxis.ConclusionsWe found Ppp1r15a is translationally repressed during late-phase sepsis because of the existence of an uORF, which is a prime therapeutic candidate for this strategic rescue of translation in late-phase sepsis. The ability to accurately control translation dynamics during sepsis may offer new paths for the development of therapies at codon-level precision.
The eIF2 initiation complex is central to maintaining a functional translation machinery. Extreme stress such as life-threatening sepsis exposes vulnerabilities in this tightly regulated system, resulting in an imbalance between the opposing actions of kinases and phosphatases on the main regulatory subunit eIF2α. Here, we report that translation shutdown is a hallmark of established sepsis-induced kidney injury brought about by excessive eIF2α phosphorylation and sustained by blunted expression of the counterregulatory phosphatase subunit Ppp1r15a. We determined that the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic approaches enabled the derepression of Ppp1r15a, salvaged translation and improved kidney function in an endotoxemia model. We also found that the loss of this uORF has broad effects on the composition and phosphorylation status of the immunopeptidome that extended beyond the eIF2α axis. Collectively, our findings define the breath and potency of the highly conserved Ppp1r15a uORF and provide a paradigm for the design of uORF-based translation rheostat strategies. The ability to accurately control the dynamics of translation during sepsis will open new paths for the development of therapies at codon level precision.
Sepsis‐induced acute kidney injury (AKI) remains a major clinical problem with no effective therapies established to date. We have previously shown that bacterial sepsis causes global translation shutdown via phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Under physiological conditions, eIF2α phosphorylation is tightly counter‐regulated by two eIF2α holophosphatases since excessive phosphorylation of eIF2α is deleterious. Of the two holophosphatases, growth arrest DNA‐inducible gene 34 (Gadd34) is the only stress‐inducible regulatory subunit. Using ribosome profiling, or Ribo‐Seq, we found that Gadd34 is translationally repressed during late phase sepsis even though eIF2α is already heavily phosphorylated. This failure of Gadd34 induction could explain the sustained phosphorylation of eIF2α and translation shutdown that contributes to delayed renal recovery in sepsis. The 5′‐untranslated region (UTR) of Gadd34 has multiple upstream open reading frames (uORFs) that are conserved across mammalian species. Our Ribo‐Seq analysis of kidneys from septic animals revealed high ribosomal occupancy of a specific Gadd34 uORF, but not the main protein coding sequence (CDS), consistent with a model in which the uORF serves as a translational inhibitor of the downstream CDS. With these findings, the objective of the investigation was to analyze the effect of disrupting translation in the uORF of Gadd34 in septic states. To investigate the inhibitory role of the Gadd34 uORF, we designed plasmid constructs, which consisted of a full length Gadd34 5′‐UTR and luciferase reporter CDS in‐frame with a single nucleotide mutation introduced to abolish the uORF start codon. The uORF point mutation led to a two‐fold increase in luciferase signal compared with the wild‐type control, confirming the inhibitory property of the Gadd34 uORF. Next, we designed antisense oligonucleotides (ASOs) complementary to the upstream portion of the uORF. Interestingly, masking the uORF with ASOs resulted in sequence‐specific increases in translation of the downstream CDS, possibly due to enhanced leaky ribosomal scanning. Finally, we tested the applicability of the ASO approach in vivo using a mouse model of endotoxin‐induced kidney injury. Despite late intervention at eight hours post‐endotoxin challenge, intravenous administration of Gadd34 uORF ASOs significantly reduced renal tissue damage as determined by the levels of hepatitis A virus cellular receptor 1/kidney injury molecule 1 (Havcr1/KIM1). Collectively, these findings indicate that translational suppression of Gadd34 in late phase sepsis is a maladaptive response that could be therapeutically modulated by targeting its uORF. Support or Funding Information This work was supported by NIH grants K08‐DK113223, R01‐DK080063 and a Veterans’ Affairs Merit grant 1I01BX002901.
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