Biodegradation of bovine spongiform encephalopathy prions in compost.

To reduce the transmission risk of bovine spongiform encephalopathy prions (PrPBSE), specified risk materials (SRM) that can harbour PrPBSE are prevented from entering the feed and food chains. As composting is one approach to disposing of SRM, we investigated the inactivation of PrPBSE in lab-scale composters over 28 days and in bin composters over 106-120 days. Lab-scale composting was conducted using 45 kg of feedlot manure with and without chicken feathers. Based on protein misfolding cyclic amplification (PMCA), after 28 days of composting, PrPBSE seeding activity was reduced by 3-4 log10 with feathers and 3 log10 without. Bin composters were constructed using ~ 2200 kg feedlot manure and repeated in 2017 and 2018. PMCA results showed that seeding activity of PrPBSE was reduced by 1-2 log10 in the centre, but only by 1 log10 in the bottom of bin composters. Subsequent assessment by transgenic (Tgbov XV) mouse bioassay confirmed a similar reduction in PrPBSE infectivity. Enrichment for proteolytic microorganisms through the addition of feathers to compost could enhance PrPBSE degradation. In addition to temperature, other factors including varying concentrations of PrPBSE and the nature of proteolytic microbial populations may be responsible for differential degradation of PrPBSE during composting.

Large-scale prion protein genotyping in Canadian caribou populations and potential impact on chronic wasting disease susceptibility.

Polymorphisms within the prion protein gene (Prnp) are an intrinsic factor that can modulate chronic wasting disease (CWD) pathogenesis in cervids. Although wild European reindeer (Rangifer tarandus tarandus) were infected with CWD, as yet there have been no reports of the disease in North American caribou (R. tarandus spp.). Previous Prnp genotyping studies on approximately 200 caribou revealed single nucleotide polymorphisms (SNPs) at codons 2 (V/M), 129 (G/S), 138 (S/N), 146 (N/n) and 169 (V/M). The impact of these polymorphisms on CWD transmission is mostly unknown, except for codon 138. Reindeer carrying at least one allele encoding for asparagine (138NN or 138SN) are less susceptible to clinical CWD upon infection by natural routes, with the majority of prions limited to extraneural tissues. We sequenced the Prnp coding region of two caribou subspecies (n = 986) from British Columbia, Saskatchewan, Yukon, Nunavut and the Northwest Territories, to identify SNPs and their frequencies. Genotype frequencies at codon 138 differed significantly between barren-ground (R. t. groenlandicus) and woodland (R. t. caribou) caribou when we excluded the Chinchaga herd (p < .05). We also found new variants at codons 153 (Y/F) and 242 (P/L). Our findings show that the 138N allele is rare among caribou in areas with higher risk of contact with CWD-infected species. As both subspecies are classified as Threatened and play significant roles in North American Indigenous culture, history, food security and the economy, determining frequencies of Prnp genotypes associated with susceptibility to CWD is important for future wildlife management measures.

Estimating relative CWD susceptibility and disease progression in farmed white-tailed deer with rare PRNP alleles.

Chronic wasting disease is a prion disease affecting both free-ranging and farmed cervids in North America and Scandinavia. A range of cervid species have been found to be susceptible, each with variations in the gene for the normal prion protein, PRNP, reportedly influencing both disease susceptibility and progression in the respective hosts. Despite the finding of several different PRNP alleles in white-tailed deer, the majority of past research has focused on two of the more common alleles identified-the 96G and 96S alleles. In the present study, we evaluate both infection status and disease stage in nearly 2100 farmed deer depopulated in the United States and Canada, including 714 CWD-positive deer and correlate our findings with PRNP genotype, including the more rare 95H, 116G, and 226K alleles. We found significant differences in either likelihood of being found infected or disease stage (and in many cases both) at the time of depopulation in all genotypes present, relative to the most common 96GG genotype. Despite high prevalence in many of the herds examined, infection was not found in several of the reported genotypes. These findings suggest that additional research is necessary to more properly define the role that these genotypes may play in managing CWD in both farmed and free-ranging white-tailed deer, with consideration for factors including relative fitness levels, incubation periods, and the kinetics of shedding in animals with these rare genotypes.

Small Proteins Encoded by Unannotated ORFs are Rising Stars of the Proteome, Confirming Shortcomings in Genome Annotations and Current Vision of an mRNA.

Short ORF-encoded peptides and small proteins in eukaryotes have been hiding in the shadow of large proteins for a long time. Recently, improved identifications in MS-based proteomics and ribosome profiling resulted in the detection of large numbers of small proteins. The variety of functions of small proteins is also emerging. It seems to be the right time to reflect on why small proteins remained invisible. In addition to the obvious technical challenge of detecting small proteins, they were mostly forgotten from annotations and they escaped detection because they were not sought. In this review, we identify conventions that need to be revisited, including the assumption that mature mRNAs carry only one coding sequence. The large-scale discovery of small proteins and of their functions will require changing some paradigms and undertaking the annotation of ORFs that are still largely perceived as irrelevant coding information compared to already annotated coding sequences.

Aggregation and neurotoxicity of recombinant α-synuclein aggregates initiated by dimerization.

BACKGROUND: Aggregation of the α-Synuclein (α-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). However, a detailed mechanism of α-Syn aggregation/fibrillogenesis and the exact nature of toxic oligomeric species produced during amyloid formation process are still unknown. RESULTS: In this study, the rates of α-Syn aggregation were compared for the recombinant wild-type (WT) α-Syn and a structurally relevant chimeric homologous protein containing an inducible Fv dimerizing domain (α-SynFv), capable to form dimers in the presence of a divalent ligand (AP20187). In the presence of AP20187, we report a rapid random coil into ß-sheet conformational transformation of α-SynFv within 24 h, whereas WT α-Syn showed 24 h delay to achieve ß-sheet structure after 48 h. Fluorescence ANS and ThT binding experiments demonstrate an accelerated oligomer/amyloid formation of dimerized α-SynFv, compared to the slower oligomerization and amyloidogenesis of WT α-Syn or α-SynFv without dimerizer AP20187. Both α-SynFv and α-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity when injected into the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by protein misfolding cyclic amplification method, providing the first evidence for the in vitro propagation of synthetic α-Syn aggregates. CONCLUSIONS: Together, we show that dimerization is important for α-Syn conformational transition and aggregation. In addition, α-Syn dimerization can accelerate the formation of neurotoxic aggregates and amyloid fibrils which can be amplified in vitro. A detailed characterization of the mechanism of α-Syn aggregation/amyloidogenesis and toxicity is crucial to comprehend Parkinson's disease pathology at the molecular level.

An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein.

The prion protein gene PRNP directs the synthesis of one of the most intensively studied mammalian proteins, the prion protein (PrP). Yet the physiological function of PrP has remained elusive and has created controversies in the literature. We found a downstream alternative translation initiation AUG codon surrounded by an optimal Kozak sequence in the +3 reading frame of PRNP. The corresponding alternative open reading frame encodes a polypeptide termed alternative prion protein (AltPrP) with a completely different amino acid sequence from PrP. We introduced a hemagglutinin (HA) tag in frame with AltPrP in PrP cDNAs from different species to test the expression of this novel polypeptide using anti-HA antibodies. AltPrP is constitutively coexpressed with human, bovine, sheep, and deer PrP. AltPrP is localized at the mitochondria and is up-regulated by endoplasmic reticulum stress and proteasomal inhibition. Generation of anti-AltPrP antibodies allowed us to test for endogenous expression of AltPrP in wild-type human cells expressing PrP. By transfecting cells with siRNA against PrP mRNA, we repressed expression of both PrP and AltPrP, confirming endogenous expression of AltPrP from PRNP. AltPrP was also detected in human brain homogenate, primary neurons, and peripheral blood mononuclear cells. These results demonstrate an unexpected function for PRNP, which, in addition to plasma membrane-anchored PrP, also encodes a second polypeptide termed AltPrP.