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

Sang-Gyun Kang,Nathalie Daude,Satyabrata Kar,David Westaway,Charles E Mays,Jing Yang

doi:10.1007/s12035-019-1623-1

Sang-Gyun Kang, Nathalie Daude + Show 4 more

Open Access

https://doi.org/10.1007/s12035-019-1623-1

Copy DOI

Journal: Molecular Neurobiology	Publication Date: May 25, 2019
Citations: 3	License type: open-access

Affiliation: University of Alberta

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.

Highlights

Prion infections result in a conformational remodeling of the cellular isoform of the prion protein called PrPC, yielding a beta-sheet enriched and infectivity-associated isoform denoted PrPSc [1]
The fixed cells were blocked with 1% BSA in PBST (PBS with 0.1% Tween 20) for 30 min and probed with mAb or pAb at 4 °C overnight: anti-Shadoo protein (Sho) pAb, 06SH1 and 06SH3 [11]; anti-PrP mAb, SAF83 (Cayman, MI, USA, 189765); anti-microtubule-associated protein 2 (MAP2) pAb (Abcam, ab5392)
The inferred impaired import of Sho into endoplasmic reticulum (ER) under proteasomal inhibition conditions was correlated with notable nuclear translocation of Sho in N2a-Shadoo protein (Sho) gene (Sprn) cells, similar to that observed in the primary Tg.Sprn-mixed neuronal and glial cell cultures (MNGCs)

Summary

Introduction

Prion infections result in a conformational remodeling of the cellular isoform of the prion protein called PrPC, yielding a beta-sheet enriched and infectivity-associated isoform denoted PrPSc [1]. In prion infections where proteinase K–resistant isoforms of PrPSc (resPrPSc) are accumulating and where protein degradation mechanisms have an extra burden, the levels of mature Sho do not increase. Rather, they decrease [11, 17,18,19], with a similar effect being deduced for PrPC [20]. The work in this area encompasses diverse and sometimes conflicting findings

Methods

Results

Conclusion