The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the hallmarks of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in ~130 days and reached the terminal stage of disease in ~150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease, confirming that these mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.

Changes in prion protein (PrP) folding are associated with fatal neurodegenerative disorders, but the neurotoxic species is unknown. Like other proteins that traffic through the endoplasmic reticulum, misfolded PrP is retrograde transported to the cytosol for degradation by proteasomes. Accumulation of even small amounts of cytosolic PrP was strongly neurotoxic in cultured cells and transgenic mice. Mice developed normally but acquired severe ataxia, with cerebellar degeneration and gliosis. This establishes a mechanism for converting wild-type PrP to a highly neurotoxic species that is distinct from the self-propagating PrP(Sc) isoform and suggests a potential common framework for seemingly diverse PrP neurodegenerative disorders.

The cytoplasm seems to provide an environment that favors conversion of the prion protein (PrP) to a form with the physical characteristics of the PrP Sc conformation, which is associated with transmissible spongiform encephalopathies. However, it is not clear whether PrP would ever exist in the cytoplasm under normal circumstances. We report that PrP accumulates in the cytoplasm when proteasome activity is compromised. The accumulated PrP seems to have been subjected to the normal proteolytic cleavage events associated with N-and C-terminal processing in the endoplasmic reticulum, suggesting that it arrives in the cytoplasm through retrograde transport. In the cytoplasm, PrP forms aggregates, often in association with Hsc70. With prolonged incubation, these aggregates accumulate in an ''aggresome''-like state, surrounding the centrosome. A mutant (D177N), which is associated with a heritable and transmissible form of the spongiform encephalopathies, is less efficiently trafficked to the surface than wild-type PrP and accumulates in the cytoplasm even without proteasome inhibition. These results demonstrate that PrP can accumulate in the cytoplasm and is likely to enter this compartment through normal protein quality-control pathways. Its potential to accumulate in the cytoplasm has implications for pathogenesis.

Prions containing misfolded prion protein (PrP Sc ) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP Sc structure accompanied by a reduction in specific infectivity of >10 5 -fold, to undetectable levels, despite the ability of adapted "protein-only" PrP Sc molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP Sc structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP Sc conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions.phospholipid | bioassay | repertoire | convergence | diversity

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP Sc ) can be produced de novo from a mixture of the normal conformer (PrP C ) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nucleaseresistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP Sc molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP Sc formation in the absence of RNA. PE facilitated the propagation of PrP Sc molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP Sc formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP Sc molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.PrP | scrapie P rions are mechanistically unique infectious agents that contain a misfolded, membrane-bound, glycoprotein (PrP Sc ) formed by the conformational change of a host-encoded conformer (PrP C ) (1). Conversion of PrP C into PrP Sc is the central event in the formation of infectious prions, but the molecular mechanism underlying conformational change remains poorly understood. In particular, the number and identity of endogenous factors other than PrP required for prion formation has not been determined (2).Cell culture and biochemical studies have implicated several classes of macromolecules such as GAGs, nucleic acids, proteins, and lipids as potential cofactors for prion formation (3). Reconstitution experiments with defined substrates (in which purified PrP molecules are mixed with Prnp 0/0 brain homogenate or purified cofactors that facilitate its conversion to PrP Sc ) have suggested that the conversion mechanism may be relatively simple, requiring only a few components (4, 5). Wild-type hamster prions possessing specific infectivity levels similar to those associated with natural scrapie have been formed de novo by using a defined mixture of purified native PrP C , copurified lipid, and RNA molecules (4), and infectious prions have also been formed de novo from bacterially expressed, recombinant PrP substrate in a reaction facilitated by synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA molecules (5, 6). In summary, infectious prions have not yet been produced either with a single cofactor or in the abs...

A rare conformation of the prion protein, PrPSc, is found only in mammals with transmissible prion diseases and represents either the infectious agent itself or a major component of it. The mechanism for initiating PrPSc formation is unknown. We report that PrP retrogradely transported out of the endoplasmic reticulum produced both amorphous aggregates and a PrPSc-like conformation in the cytosol. The distribution between these forms correlated with the rate of appearance in the cytosol. Once conversion to the PrPSc-like conformation occurred, it was sustained. Thus, PrP has an inherent capacity to promote its own conformational conversion in mammalian cells. These observations might explain the origin of PrPSc.

Variants in hepatocyte nuclear factor-4␣ (HNF4␣), a transcription factor that influences the expression of glucose metabolic genes, have been correlated with maturityonset diabetes of the young, a monogenic form of diabetes. Previously, in a genome scan of Ashkenazi Jewish type 2 diabetic families, we observed linkage to the chromosome 20q region encompassing HNF4␣. Here, haplotype-tag single nucleotide polymorphisms (htSNPs) were identified across a 78-kb region around HNF4␣ and evaluated in an association analysis of Ashkenazi Jewish type 2 diabetic (n ‫؍‬ 275) and control (n ‫؍‬ 342) subjects. We found that two of nine htSNPs were associated with type 2 diabetes: a 3 intronic SNP, rs3818247 (29.2% case subjects vs. 21.7% control subjects; P ‫؍‬ 0.0028, odds ratio [OR] 1.49) and a 5 htSNP located ϳ3.9 kb upstream of P2, rs1884614 (26.9% case subjects vs. 20.3% control subjects; P ‫؍‬ 0.0078, OR 1.45). Testing of additional SNPs 5 of rs1884614 revealed a >10-kb haplotype block that was associated with type 2 diabetes. Conditioning on the probands' rs1884614 genotype suggested that the chromosomal region identified by the htSNP accounted for the linkage signal on chromosome 20q in families in which the proband carried at least one risk allele. Notably, the associations and the partitioned linkage profiles near P2 were independently observed in a Finnish sample, suggesting the presence of potential regulatory element(s) that may contribute to the risk for type 2 diabetes.

Quinones permeate our biotic environment, contributing to both homeostasis and cytotoxicity. All quinones generate reactive oxygen species through redox cycling, while partially substituted quinones also undergo arylation (Michael adduct formation) yielding covalent bonds with nucleophiles such as cysteinyl thiols. In contrast to reactive oxygen species, the role of arylation in quinone cytotoxicity is not well understood. We found that the arylating quinones, including unsubstituted 1,4-benzoquinone (1,4-BzQ) and partially substituted vitamin E congener ␥-tocopherol quinone (␥-TQ), were cytotoxic, with ␥-TQ > 1,4-BzQ, whereas the fully substituted nonarylating vitamin E congener ␣-tocopherol quinone was not. In vitro, both arylating quinones formed Michael adducts with the thiol nucleophile N-acetylcysteine (NAC) at rates where 1,4-BzQ > ␥-TQ. In cultured cells, concurrent addition of NAC eliminated 1,4-BzQ caused toxicity, but preincubation was required for the same NAC detoxification effect on ␥-TQ. These data clearly established the role of arylation in quinone toxicity and revealed that arylating quinone structure affects cytotoxicity by governing detoxification through the rate of adduct formation. Furthermore, arylating quinones induced endoplasmic reticulum (ER) stress by activating the pancreatic ER kinase (PERK) signaling pathway including elF2␣, ATF4, and C͞EBP homologous protein (CHOP). Detoxification by NAC greatly attenuates CHOP induction in arylating quinone-treated cells, suggesting that ER stress is a cellular mechanism for arylating quinone cytotoxicity.quinone adduction ͉ thiol nucleophiles ͉ tocopherols ͉ CHOP ͉ cytotoxicity Q uinones and their phenolic precursors are present throughout the biotic environment and include polyphenols and tocopherols in the diet, drugs in medicine, environmental pollutants such as polycyclic aromatic hydrocarbons, and their metabolic products (1-8). They are involved in a wide variety of biological and chemical processes, including electron transport in animals and plants, photosynthesis, posttranslational modification of proteins, metabolism of cellular signaling molecules such as estrogens and catecholamines, metabolism of antioxidant and signaling tocopherol congeners (vitamin E), and the elimination of polycyclic aromatic hydrocarbons introduced by combustion processes associated with our petroleum-based chemical environment.Quinones are a class of highly reactive compounds. Although all quinones are redox cycling agents that generate reactive oxygen species (ROS), partially substituted quinones also function as arylating agents (1-3, 5, 6). The arylating quinones react with cellular nucleophiles such as thiols on cysteine residues of proteins, glutathione (GSH), and detoxifying agents such as N-acetylcysteine (NAC), forming covalently linked quinonethiol Michael adducts (1-3, 5, 6) that retain the ability to function as redox cycling agents (4, 9). In contrast to well studied ROS generation and consequent oxidative stress in living cells (1-3), the role o...