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

Abstract

The Shadoo protein (Sho) exhibits homology to the hydrophobic region of the cellular isoform of prion protein (PrPC). As prion-infected brains gradually accumulate infectivity-associated isoforms of prion protein (PrPSc), 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, NH4Cl, 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 NH4Cl and 3-MA did not affect Sho or PrPC 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 materialThe online version of this article (10.1007/s12035-019-1623-1) contains supplementary material, which is available to authorized users.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin

“…However, Sho lacks a copper-binding region, indicative of a potential for distinct functions from PrP C , harboring instead argininerich tetra-repeat segments towards its N-terminal, conferring the ability to bind RNA and nucleic acids [57,58]. Additionally, this region plays a role in the nuclear localization of Sho as we demonstrated earlier [59] in different cells expressing the protein, and, in line with this, nuclear localization of Sho as a response to proteasome inhibition was recently reported, invoking the role of the arginine-rich repeat region in this process [60].…”

“…In the case of Sho, two bands appear at around the expected weight of Sho-EYFP (~45-49 kDa), recognized by both α-Sho and α-GFP antibodies in Sho-EYFP cells, which are absent in parental N2a cell samples (Figure 2B, lanes 1-2 and 4-5). It should be noted that there are no unequivocally good anti-Shadoo antibodies available and that cross-reactive bands are frequently apparent on blots of various cells by anti-Sho antibodies [47,60,63,64,82]. However, since α-GFP shows similar bands, these might be two forms of Sho.…”

The study of the membrane microdomain localization of the prion-family proteins, prion and Shadoo and their interaction with calnexin

“…Sho as a glycoprotein is synthesised in the secretary pathway and is attached to the external leaflet of the plasma membrane via a GPI-anchor 68 . Sho is also present in nucleus besides ER and GA 69,70 . Recently, it was demonstrated that the ER signal peptide of Sho can mediate an alternative targeting of Sho to the mitochondria, a process that is governed by structural features in its intrinsically disorder elements; and that the GPI anchor signal peptide is sufficient to promote efficient ER import of the protein 71 .…”

“…These cells are well characterized and broadly used as a cellular model system in the study of physiology of cellular and the pathological prion protein 90,156 . N2a cells possess detectable amounts of endogenous PrP but not Sho; at least the endogenous Shadoo expression cannot be detected in these cells with the currently available shadoo antibodies 70,86,96,97,157 . To compare Sho and PrP, we generated N2a cells stably expressing either Sho or PrP in fusion with a fluorescent protein (EYFP for Sho and EGFP for PrP) tag by using the plasmid constructs described in the Materials and methods section for PrP (for the plasmid Map see Appendix-I, Figure A1) and earlier by our group for Sho 69 .…”

“…This indicates that two forms of Sho are present, since these two bands are visualized by α-GFP as well. Since there are no unambiguously excellent α-Sho antibodies available, cross reactive bands were commonly produced on the blots of various other cells by anti-Sho antibodies 64,70,96,97,157 . Since Sho and PrP possess one and two N-glycosylation sites, respectively, they may exist as a mixture of proteins with different states of glycosylation, hence, with different molecular weights.…”