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Transient arrest in proteasomal degradation during inhibition of translation in the unfolded protein response.

ABSTRACT: The UPR (unfolded protein response) activates transcription of genes involved in proteasomal degradation. However, we found that in its early stages the UPR leads to a transient inhibition of proteasomal disposal of cytosolic substrates (p53 and p27kip1) and of those targeted to ER (endoplasmic reticulum)-associated degradation (uncleaved precursor of asialoglycoprotein receptor H2a). Degradation resumed soon after the protein synthesis arrest that occurs in early UPR subsided. Consistent with this, protein synthesis inhibitors blocked ubiquitin/proteasomal degradation. Ubiquitination was inhibited during the translation block, suggesting short-lived E3 ubiquitin ligases as candidate depleted proteins. This was indeed the case for p53 whose E3 ligase, Mdm2 (murine double minute 2), when overexpressed, restored the degradation, whereas a mutant Mdm2 in its acidic domain restored the ubiquitination but did not completely restore the degradation. Inhibition of proteasomal degradation early in UPR may prevent depletion of essential short-lived factors during the translation arrest. Stabilization of p27 through this mechanism may explain the cell cycle arrest in G1 when translation is blocked by inhibitors or by the UPR.

SUBMITTER: Shenkman M

PROVIDER: S-EPMC1896287 | biostudies-other | 2007 Jun

REPOSITORIES: biostudies-other

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Project description:Background & aimsNetrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown.MethodsIsolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice.ResultsHepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice.ConclusionsUPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.

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Transient arrest in proteasomal degradation during inhibition of translation in the unfolded protein response.

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