Mutational Analysis of Topological Determinants in Prion Protein (PrP) and Measurement of Transmembrane and Cytosolic PrP during Prion Infection

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...

Section: Haprp(m15) and Huprp(m8) Isoforms Account For De Novo Synthementioning

confidence: 99%

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

Juanes

Elvira

García-Grande

et al. 2009

“…Polyclonal antibody P45-66, raised against a synthetic peptide encompassing residues 45-66 of mouse PrP (25), was used at 1:2,500 for Western blot. An antibody (anti-SP) that selectively recognizes forms of murine PrP containing an uncleaved signal peptide was used at 1:500 for Western blot (26). Anti-giantin (Covance) and anti-trap (Upstate Biotechnologies) antibodies were used, respectively, at 1:1,000 and 1:500 for immunofluorescence staining.…”

Section: Methodsmentioning

confidence: 99%

Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

Fioriti

Dossena

Stewart

et al. 2005

Self Cite

111

117

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.

“…To determine whether L9R-3AV PrP in cerebellar granule neurons also has the same feature, we immunoprecipitated PrP from [ 35 S]methionine-labeled cultures using anti-SP, an antibody that specifically recognizes PrP molecules containing an intact signal peptide (19). Parallel samples were immunoprecipitated with 3F4 or 8H4 antibodies, which recognize PrP molecules regardless of the presence of the signal peptide (see Fig.…”

Section: L9r-3av Prp Retains An Uncleaved Signal Peptide-we Demonstramentioning

confidence: 99%

“…Based on these and other results, it was suggested that Ctm PrP is a key neurotoxic intermediate in both familial and infectious prion diseases and that the amount of this form can be increased directly by pathogenic mutations, or indirectly by accumulation of PrP Sc (8). However, uncertainties remain about the role of Ctm PrP in prion diseases because of recent reports that Ctm PrP levels do not change significantly during scrapie infection (19) or as a result of most pathogenic mutations (17).…”

mentioning

confidence: 99%

“…To this end, we have identified mutations that cause PrP to be synthesized exclu-sively with the Ctm PrP topology, thus facilitating analysis of this form in the absence of the other topological variants. We demonstrated that Ctm PrP has an uncleaved, N-terminal signal peptide (16) and that substitution of charged residues in the hydrophobic core of the signal sequence strongly favors synthesis of Ctm PrP (19). Combining one of these mutations (L9R) with a previously studied mutation in the transmembrane domain (3AV) resulted in a protein that was expressed entirely as Ctm PrP after in vitro translation and transfection of cultured cells (16).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Transmembrane Form of the Prion Protein Is Localized in the Golgi Apparatus of Neurons

Stewart

Harris

2005

Self Cite

Ctm PrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. In previous studies, we found that PrP molecules carrying mutations in the N-terminal signal peptide (L9R) and the transmembrane domain (3AV) were synthesized exclusively in the Ctm PrP form in transfected cell lines. To characterize the properties of Ctm PrP in a neuronal setting, we have utilized cerebellar granule neurons cultured from Tg(L9R-3AV) mice that developed a fatal neurodegenerative illness. We found that about half of the L9R-3AV PrP synthesized in these neurons represents Ctm PrP, with the rest being Sec PrP, the glycolipid anchored form that does not span the membrane. Both forms contained an uncleaved signal peptide, and they are differentially glycosylated.Sec PrP was localized on the surface of neuronal processes. Most surprisingly, Ctm PrP was concentrated in the Golgi apparatus, rather in the endoplasmic reticulum as it is in transfected cell lines. Our study is the first to analyze the properties of Ctm PrP in a neuronal context, and our results suggest new hypotheses about how this form may exert its neurotoxic effects.