Proteasomal Inhibition Redirects the PrP-Like Shadoo Protein to the Nucleus

The Shadoo protein (Sho) exhibits homology to the hydrophobic region of the cellular isoform of prion protein (PrP(C)). As prion-infected brains gradually accumulate infectivity-associated isoforms of prion protein (PrP(Sc)), levels of mature endogenous Sho become reduced. To study the regulatory effect of the proteostatic network on Sho expression, we investigated the action of lactacystin, MG132, NH(4)Cl, and 3-methyladenine (3-MA) in two cell culture models. In primary mixed neuronal and glial cell cultures (MNGCs) from transgenic mice expressing wild-type Sho from the PrP gene promoter (Tg.Sprn mice), lactacystin- and MG132-mediated inhibition of proteasomal activity shifted the repertoire of Sho species towards unglycosylated forms appearing in the nuclei; conversely, the autophagic modulators NH(4)Cl and 3-MA did not affect Sho or PrP(C) glycosylation patterns. Mouse N2a neuroblastoma cells expressing Sho under control of a housekeeping gene promoter treated with MG132 or lactacystin also showed increased nuclear localization of unglycosylated Sho. As two proteasomal inhibitors tested in two cell paradigms caused redirection of Sho to nuclei at the expense of processing through the secretory pathway, our findings define a balanced shift in subcellular localization that thereby differs from the decreases in net Sho species seen in prion-infected brains. Our data are indicative of a physiological pathway to access Sho functions in the nucleus under conditions of impaired proteasomal activity. We also infer that these conditions would comprise a context wherein Sho’s N-terminal nucleic acid–binding RGG repeat region is brought into play. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12035-019-1623-1) contains supplementary material, which is available to authorized users.