T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor-like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation.

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...

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP Sc , a misfolded conformer of the normal prion protein, PrP C . However, protein-only PrP Sc preparations lack significant levels of prion infectivity, leading to the alternative hypothesis that cofactor molecules are required to form infectious prions. Here, we show that prions with parental strain properties and full specific infectivity can be restored from protein-only PrP Sc in vitro . The restoration reaction is rapid, potent, and requires bank vole PrP C substrate, post-translational modifications, and cofactor molecules. To our knowledge, this represents the first report in which the essential properties of an infectious mammalian prion have been restored from pure PrP without adaptation. These findings provide evidence for a unified hypothesis of prion infectivity in which the global structure of protein-only PrP Sc accurately stores latent infectious and strain information, but cofactor molecules control a reversible switch that unmasks biological infectivity.

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.