Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrP (Sc), an aberrant form of the membrane protein PrP (C). In the absence of an established form-specific covalent difference, the infectious properties of PrP (Sc) were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27-30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of alphaPrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrP (Sc) are present as methionine sulfoxides this modification could not be found on brain PrP (C) as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrP (Sc) differs both within and between species, adding another diversity factor to the structure of PrP (Sc) molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrP (Sc) and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.

The cellular prion protein PrP C is synthesized as a family of four distinct forms. Of these, Cyt PrP is a minor member that segregates outside of the secretory route and can generate cytotoxic forms. Using signal sequence mutants, we found that Cyt PrP is translated from a downstream AUG (coding for Met-8 in human PrP or Met-15 in Syrian hamster PrP). Shortening of the signal sequence dictated the spillage of this isoform into the cytosol, from where it accessed the nucleus or formed insoluble cytosolic aggregates if the proteasome is inhibited. The PrP isoform isolated from the nuclear fractions of cell and brain homogenates was partially SUMO-1-conjugated. Expression of HaPrP(M15) in cells caused an antiproliferative phenotype due to a cell cycle arrest at the G 0 /G 1 phase. The identification of this PrP isoform and its properties provides novel insight into PrP C physiological and pathological functions.The cellular prion protein (PrP C ) 2 underlies a group of fatal neurodegenerative diseases through its conversion into selfperpetuating and neurotoxic forms (1-4). Despite a large amount of evidence supporting a role in survival/death and growth/differentiation cell decisions, the physiological function of PrP C and its involvement in disease remain elusive (5-8). A crucial limiting factor for PrP C functional determination is its molecular diversity. Although PrP C is mainly thought of as a glycoprotein attached to the cell surface by a glycosylphosphatidylinositol anchor, PrP C is actually synthesized as a family of four members: the membrane anchored glycoprotein ( Sec PrP), two transmembrane forms with opposite topologies ( Ntm PrP and Ctm PrP), and a soluble form ( Cyt PrP) (3, 9 -12).Of these different members, Cyt PrP, accounts for a minor intracellular subset of PrP C that has attracted much attention because its accumulation sensitizes cells to death (13-15). Initially, CytPrP was thought to be formed by misfolded chains that retrotranslocated through the endoplasmic reticulum-associated protein degradation-proteasome pathway (16,17). However, it was later shown that Cyt PrP is constitutively populated by nascent chains that spill into the cytosol due to inefficient N-terminal signaling (15,18). Regarding the role of Cyt PrP, most knowledge has been provided by models consisting of mutant polypeptide chains that are inappropriately expressed and folded in the cytosol. These PrP(23-230) chains exhibit a widespread intracellular distribution (19 -21) and an alleged role that varies from cytotoxic (14, 20) to innocuous or even protective (19,22,23). These contradictions call into question the fidelity with which such models can describe Cyt PrP. The finding that information for Cyt PrP synthesis is contained in its N-terminal signal sequence (15) prompted us to decipher this code and use it as a tool to isolate its synthesis from that of the major forms and inspect its function. We have found that Cyt PrP is indeed a novel PrP isoform that accesses the nucleus and interferes with cell growth. These r...

No abstract