Aug 2026· PLoS Pathogens· Vol 22, pp. e1014536· 0 citations· 118 references
Medicine
TL;DR
It is shown that stable knock-down of the protein disulfide isomerase P4HB in prion-susceptible CAD5 cells reduces PrPC levels and hinders the generation of protease-resistant PrP (PrPres) following infection with two different prion strains.
Abstract
Prions are misfolded, self-propagating versions of cellular proteins. In humans and animals, misfolding of the cellular prion protein (PrPC) causes invariably fatal transmissible neurodegenerative diseases. Little is known about how mammalian prions replicate in the brain, including whether other proteins participate in prion replication in vivo. Several members of the protein disulfide isomerase family have been shown to reside in close spatial proximity to PrPC in cells and mice, implying that they could be involved in prion biogenesis. Here, we show that stable knock-down of the protein disulfide isomerase P4HB (also called PDIA1) in prion-susceptible CAD5 cells reduces PrPC levels and hinders the generation of protease-resistant PrP (PrPres) following infection with two different prion strains. Moreover, transient knock-down of P4HB decreases PrPres levels in cells with established prion infection. Partial reduction of P4HB activity using the P4HB-selective inhibitor KSC-34 also decreases PrPC levels in uninfected CAD5 cells whereas treatment of prion-infected CAD5 cells with KSC-34 results in higher levels of PrPres. A proportion of P4HB reaches the cell surface where PrPC is located, and a secreted P4HB variant increases PrPres levels in cells. Collectively, these results suggest that P4HB influences PrPC homeostasis and modulates the conversion of PrPC into misfolded species. Thus, targeting P4HB during prion disease may have therapeutic benefit.
Prion diseases are transmissible, neurodegenerative diseases caused by misfolded, protease-resistant, and infectious aggregates of the mammalian prion protein (PrPSc) that replicate by converting properly folded prion protein (PrPC) into PrPSc. Spongiform change and cellular loss in the brain are hallmarks of prion disease, but our understanding of how prions alter cellular fitness remains incomplete. Here we characterized changes in mitochondrial redox state and respiration in neural cells following uptake of two different PrPSc strains, 22L and 87V. Only 22L PrPSc induced changes in cellular respiration and mitochondrial redox state, even in cells that did not produce PrPC. These effects were disrupted by detergent and dependent upon endo-lysosomal acidification, suggesting that both PrPSc membrane association and lysosomal degradation are involved. Interestingly, cells chronically infected with 22L appeared to adapt to infection, showing no signs of mitochondrial dysfunction, but were more susceptible to oxidative stress even though mitochondrial respiration was normal. Thus, during initial prion infection, PrPSc drives mitochondrial dysfunction in a manner that is both strain dependent and independent of PrPC expression, while persistent prion infection increases mitochondrial sensitivity to cellular stress.
Daniel Shoup, L. Varner, B. Race et al.· Journal of Cell Science· 0 citations
Borna disease virus 1 (BoDV-1) is a noncytolytic, neurotrophic virus that causes neurological disorders, where astrocytes contribute to neuropathology. Cellular prion protein (PrPC) is involved in pro-survival signaling and antioxidative defense. Using rat astroglial C6 cells as an in vitro model, we investigated whether persistent BoDV-1 infection alters N-glycan maturation of PrPC. Western blotting showed that PrPC shifted toward lower-molecular-weight glycoforms as cell density increased. This tendency was more evident in BoDV-1-infected C6 cells without detectable changes in total surface PrPC expression. Two-dimensional polyacrylamide gel electrophoresis showed a shift of PrPC toward lower molecular weight and more basic isoelectric points. Conventional RT-PCR detected reduced signals of MGAT5, which encodes N-acetylglucosaminyltransferase V (GnTV), whereas MGAT1–4 signals were not markedly altered. In addition, GnTV overexpression induced a higher-molecular-weight shift of PrPC. PHA-L4 precipitation followed by anti-PrP immunoblotting showed reduced β1-6-branched complex N-glycan signals associated with PrPC. BoDV-1 infection also decreased the RT-PCR signal of E26 transformation-specific 1 (ETS1) as well as reduced ETS-dependent promoter activity and superoxide dismutase activity. These findings suggest that persistent BoDV-1 infection may impair PrPC N-glycan maturation through suppression of the ETS1–MGAT5/GnTV pathway activity, thereby potentially affecting astroglial antioxidative capacity.
The pathogenic conversion of the cellular prion protein (PrPC) into the β-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive noncanonical amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substrate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substrate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and noninfectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new strategy for studying conformational dynamics in amyloid-forming proteins.
Jessica de Alcantara Ferreira, Daniel J. Walsh, Evelyn M. Turnbaugh et al.· Biochemistry· 0 citations
The normal cellular prion protein (PrPC) is an essential substrate in all forms of prion diseases and a receptor for Aβ oligomers in Alzheimer’s disease. However, it is not fully understood how cells regulate PrPC levels. Recently, we identified glycogen synthase kinase-3β (GSK-3β) as a potential regulator of PrPC levels in a whole genome knockout screen. Here, we show that both cell surface and total PrPC levels can be reduced either by siRNA-mediated Gsk3b (but not Gsk3a) knockdown or by CRISPR-mediated Gs3b knockout. Whole cell mass spectrometric analysis showed that PrPC was the 60th most significantly reduced protein (out of 7227 total proteins detected) in Gsk3b knockout cells, compared to wild-type cells. Two different GSK-3 inhibitors, laduviglusib (CHIR-99021) and AZD-1080, reduced PrPC levels in mouse CAD5 and human BE(2)-C cells, both in undifferentiated and differentiated states. PrPC levels were similarly reduced by cycloheximide treatment in both Gsk3b knockout and WT cells, indicating that GSK-3β regulates PrPC levels through a post-translational mechanism. Finally, treatment with either laduviglusib or AZD-1080 reduced PrPSc levels in CAD5 cells infected with three different rodent prion strains. Overall, the results reveal that GSK-3β activity controls PrPC levels in living cells, revealing a novel regulatory mechanism and promising therapeutic target.
Kathryn S. Beauchemin, Anna M. Schmoker, J. Watts et al.· bioRxiv· 0 citations
This study enhances the understanding of the E. coli-genome nature and suggests the existence of specific and experimentally testable novel prions in this organism, and moves a step forward towards the identification of new prion proteins in bacteria.
Katherine Shreeve, Jinoh Jang, Mr. S Srivathsan et al.· Proteins: Structure, Functio...· 0 citations
Protein misfolding cyclic amplification (PMCA) is an ultrasensitive, cell-free technique that mimics the conversion of the host cellular prion protein (PrPC) to its pathogenic version (PrPSc) in an accelerated manner. PMCA can amplify minimal quantities of disease-associated prion particles contained in various sample types, including biological and environmental specimens. This is especially relevant for chronic wasting disease (CWD), a prion disease of cervids that currently manifests at epidemic proportions in North America. Considering the ecology of CWD prions, it is imperative to identify their presence in environmental components. Prion replication by PMCA is achieved through multiple incubation/sonication cycles, and PMCA products contain large prion quantities that can be easily visualized by conventional techniques such as dot blots or western blots. Here, we describe an updated protocol to replicate CWD prions by means of the PMCA technology in animal-derived samples and environmental fomites. We also describe newly optimized PMCA protocols to identify CWD-prions in non-cervid, sympatric hosts. Finally, we also explain modifications of this technique to increase its throughput, focused in the screening of small molecule libraries to identify anti-prion molecules. Multiple reports demonstrate that PMCA is able to detect as little as a single prion polymer in specific tissues (e.g. brain). Considering this, this technique has been proposed as a promising method for the diagnosis of prion diseases. In addition, PMCA faithfully replicates the biological information present in the original prion “seeds”, fact that has greatly helped in our understanding of prion pathogenic mechanisms. Importantly, prion diseases remain without cure, and the optimization of PMCA for drug discovery may importantly advance efforts in this direction.
F. Bravo-Risi, Rebeca Benavente, Rodrigo Morales· BMC Methods· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
