Biology subjects

Beauchemin, K. S.

Publications and source records attributed to Beauchemin, K. S..

5 recordsLinked to original sources

bioRxiv2026

Glycogen Synthase Kinase-3β Regulates Cellular Prion Protein Levels

The normal cellular prion protein (PrPC) is an essential substrate in all forms of prion diseases and a receptor for A{beta} oligomers in Alzheimers disease. However, it is not fully understood how cells regulate PrPC levels. Recently, we identified glycogen synthase kinase-3{beta} (GSK-3{beta}) 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-3{beta} 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-3{beta} activity controls PrPC levels in living cells, revealing a novel regulatory mechanism and promising therapeutic target.

cell biology↗

bioRxiv2026

Energy Flux Regulates Cell Death Induced by California Serogroup Orthobunyaviruses

The California serogroup (CSG) of orthobunyaviruses includes neuroinvasive viruses with varying pathogenicity. La Crosse virus (LACV) is a leading cause of pediatric arboviral encephalitis in the USA, while Inkoo virus (INKV) is widespread in Northern Europe but rarely causes disease. The reassortment potential of CSG viruses raises concerns about emerging virulent strains and highlights the need to develop therapies that are broadly effective against multiple CSG viruses. To identify host factors mediating viral neurotoxicity, we performed genome-wide CRISPR-Cas9 knockout screens in human neuroblastoma cells infected with LACV or INKV. Analysis revealed largely overlapping host dependency factors for both viruses. Unexpectedly, the screens identified mitochondrial energy production as a major pathway required for both LACV- and INKV-induced cell death. Reducing host cell energy production with mild hypothermia or sugar source substitution prolonged cell survival during viral infection with additive effects mediated by different mechanisms. Both manipulations also protected neuroblastoma cells from the Bunyamwera virus (BUNV), a non-CSG orthobunyavirus; and mild hypothermia protected mature human neurons from LACV. These results highlight host energy metabolism as a key modulator of CSG virus cytotoxicity and suggest novel avenues for general non-invasive therapeutic intervention against current and future strains of these and other orthobunyaviruses.

microbiology↗

bioRxiv2026

Convergent strategies for nanobody-mediated inhibition of an epoxide hydrolase

Secreted by Pseudomonas aeruginosa, Cif is an epoxide hydrolase that acts as a virulence factor in the context of cystic fibrosis and thus represents a target for therapeutic inhibition. Here, we present the structures of several high-affinity inhibitory nanobodies, each bound to Cif. Comparison reveals two classes of nanobodies with distinct CDR sequences and convergent recognition strategies. Mimicry between CDR3 and CDR2 loops positions an aromatic residue for insertion through the active-site gate, accessing a cryptic epitope, which sterically blocks substrate access and provides an anchor point for high-affinity engagement. Projection of either inhibitory CDR toward the active-site entrance requires a relative 90{degrees} rotation of the core immunoglobulin domain, and yet both classes engage the same set of stereochemical handholds within a highly overlapping shared epitope. The structurally distinct paratopes thus represent fundamentally distinct solutions, reflecting the remarkable capacity of the immune system to solve highly constrained molecular recognition challenges.

biochemistry↗

bioRxiv2025

Genome-Wide Screens Identify Core Regulators of Cell Surface Prion Protein Expression

Expression of the cellular prion protein, PrPC, on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrPC expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrPC expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrPC. We also sought to determine whether the same or different genes regulate cell surface PrPC in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrPC expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrPC expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrPC expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.

genomics↗

bioRxiv2025

Oligosaccharyltransferase (OST) complex inhibition effectively treats rodent and human prions

Prion diseases are invariably fatal neurodegenerative diseases that occur when the prion protein misfolds into a pathogenic form. There are currently no clinical treatments or cures for prion disease. Current challenges in the development of prion therapeutics include prion strain specificity, which can cause the emergence of drug-resistant prions, and lack of efficacy in treating human prions despite promising results in rodent models. Here we identify a novel therapeutic target for prion disease: the oligosaccharyltransferase (OST) complex. The OST complex is responsible for transferring the mature glycan to the acceptor polypeptide during Nglycosylation. We found that inhibiting OST effectively treats rodent prions in various dividing and non-dividing cell types. Importantly, we also demonstrate efficacy in treating human sCJD prions in non-dividing cerebral organoids. Inhibition of OST results in a 50% reduction in cell surface expression of the prion protein, PrPC. In addition, lysates of cells treated with the OST inhibitor NGI-1 were unable to amplify PrPSc seeds in Protein Misfolding Cyclic Amplification (PMCA) reactions. In summary, our results identify OST as a novel therapeutic target that regulates both the abundance of cell surface PrPC as well as its ability to convert into multiple strains of PrPSc, including human prions, in various in vitro systems. Author SummaryPrion diseases, such as Creutzfeldt-Jakob disease, are fatal brain disorders caused when a normal protein (PrPC) misfolds into a harmful form that spreads through the brain. There are no effective treatments, and drug development has been hampered by "strain" differences in prions that can lead to resistance and by therapies that work in rodents but not in humans. This study identifies a new treatment target: the oligosaccharyltransferase (OST) complex, a cellular machine that adds sugar groups to proteins. Blocking OST with a small molecule (NGI1) limited prion growth in multiple types of rodent cells, including non-dividing cells, and-- critically--also worked against human sporadic CJD prions in laboratory-grown human brain organoids. OST inhibition cut the amount of normal prion protein on cell surfaces in half, reducing the raw material available to convert into the disease-causing form. In addition, extracts from NGI-1-treated cells could no longer drive prion formation in a sensitive lab amplification test. These results suggest that targeting OST may offer a new strategy that works across different prion strains, including human ones.

cell biology↗