Abstract
Shadoo (Sho), a member of prion protein family, has been shown to prevent embryonic lethality in Prnp0/0 mice and to be reduced in the brains of rodents with terminal prion diseases. Sho can also affect PrP structural dynamics and can increase the prion conversion into its misfolded isoform (PrPSc), which is amyloidogenic and strictly related to expression, intracellular localization and association of PrPC to lipid rafts. We reasoned that if Sho possesses a natural tendency to convert to amyloid-like forms in vitro, it should be able to exhibit “prion-like” properties, such as PK-resistance and aggregation state, also in live cells. We tested this hypothesis, by different approaches in neuronal cells, finding that Sho shows folding properties partially dependent on lipid rafts integrity whose alteration, as well as proteasomal block, regulated generation of intermediate Sho isoforms and exacerbated its misfolding. Moreover, a 18 kDa isoform of Sho, likely bearing the signal peptide, was targeted to mitochondria by interacting with the molecular chaperone TRAP1 which, in turn controlled Sho dual targeting to ER or mitochondria. Our studies contribute to understand the role of molecular chaperones and of PrP-related folding intermediates in “prion-like” conversion.
Highlights
Prion diseases are a group of rare, infectious, sporadic or inherited neurodegenerative disorders in mammals characterized by a primary pathogenic event consisting of a conversion of the cellular prion protein (PrPC) into a disease-associated misfolded isoform PrPSc1, 2
We have previously shown that alteration of detergent-resistant membranes (DRMs), called lipid rafts, by cholesterol depletion, leads to PrPC misfolding in the endoplasmic reticulum (ER)[13, 14] and that the ER-associated degradation pathway (ERAD) is involved in the processing of the non-translocated form of PrPC in epithelial cells[15]
Based on a previous report showing that the ER signal peptide of Sho can mediate targeting to mitochondria[25], we decided to explore the subcellular localization of endogenous Sho, using indirect immunofluorescence microscopy followed by confocal analysis
Summary
Prion diseases are a group of rare, infectious, sporadic or inherited neurodegenerative disorders in mammals characterized by a primary pathogenic event consisting of a conversion of the cellular prion protein (PrPC) into a disease-associated misfolded isoform PrPSc1, 2. Despite intensive research over years, the exact mechanism by which the conversion occurs is poorly understood[3], as are the exact function of PrPC in the cells and the phenotypic impacts of conformational variations and protein partners of PrPC. In the last years it has emerged that prion protein family includes two other members in addition to PrPC, the so called Doppel and Shadoo (Sho), this latter being discovered in silico[4] and mainly expressed in the central nervous system (CNS)[5]. We have characterized the subcellular localization of endogenously expressed Sho in neuronal (GT1 and SH-SY5Y) and non neuronal (HeLa) cells, its association with DRMs and their role, which has never been tested before, in its folding. We found that Sho interacts with the mitochondrial chaperone TRAP1, whose expression levels control dual ER/ Mitochondrial targeting of Sho, regulating the correct Sho localization at the interface between the two compartments
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