Cell division modulates prion accumulation in cultured cells

Ghaemmaghami, Sina; Phuan, Puay‐Wah; Perkins, Beth; Ullman, Julie C.; May, Barnaby C. H.; Cohen, Fred E.; Prusiner, Stanley B.

doi:10.1073/pnas.0708372104

Cited by 80 publications

(75 citation statements)

References 43 publications

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“…On the other hand, the loss of HaPrP(M15) nuclear functionality might favor either cell transformation on depletion (8) or cell death on cytosolic accumulation (15). HaPrP(M15) can regulate the efficiency of prion accumulation, which decreases with cell division (40). The isolation of the synthesis of this isoform from that of other members of the PrP C family suggests that each member of this family might have different physiological roles and that their aberrant crosstalk could also constitute a pathogenic mechanism.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

Juanes

Elvira

García-Grande

et al. 2009

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

Juanes

Elvira

García-Grande

et al. 2009

Journal of Biological Chemistry

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“…2) and expressed equal amounts of PrP C in the time range from 1 to 6 dpp (data not shown). These results matched recently reported PrP amounts in dividing N2a and ScN2a cells [25].…”

Section: Fibrillar Network Form On Top Of Murine Neuroblastoma Cellssupporting

confidence: 82%

Observing fibrillar assemblies on scrapie-infected cells

Wegmann

Miesbauer

Winklhofer

et al. 2008

Pflugers Arch - Eur J Physiol

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The infectious agent in prion diseases is an aberrant-folded isoform of the cellular prion protein (PrP C ). This scrapie-related prion protein (PrP Sc ) has an increased β-sheet content, is detergent insoluble and proteinase K resistant, and accumulates in prion-infected organisms and cells. In vitro, PrP Sc self-aggregates into amyloid fibrils. However, there is no direct experimental proof for the occurrence of PrP Sc -containing fibrils in vivo or in cell cultures. Applying atomic force microscopy (AFM) to scrapie-infected mouse neuroblastoma (ScN2a) cells, we discovered growing patch-like assemblies of amyloid-like fibrillar structures on the cell surfaces. Immunofluorescence and AFM images showed heterogeneous accumulation and aggregation of PrP Sc in ScN2a cell cultures. The percentage of cells having characteristic fibrils on their surface increased with time after scrapie infection. These endogeneous fibrils had lengths from 0.5 to 3 μm and protruded from the cell surface by 108±30 nm, and thus resembled the heterogeneous shapes and networks of in vitro prepared amyloid fibrils.

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“…While these findings are suggestive, the potential effectiveness of these drugs and drug-combinations yet remains to be confirmed in other prion models as well as in cell conditions that may better reflect the in vivo prion biology. In this respect, an interesting study in prion-infected N2a cells showed that cell division strongly influences the accumulation of PrP res and suggested that, since the process of cell division may intensify the kinetics of PrP Sc clearance, inhibitory activity in neuronal cell lines may not reflect real effectiveness in the neurons [38]. Moreover, our previous studies on the role of cholesterol metabolism during cell proliferation indicated an association between the cell capability to synthesize and retain cholesterol esters and the cell growth rate potential [20,24,25].…”

Section: Discussionmentioning

confidence: 99%

In vitro synergistic anti-prion effect of cholesterol ester modulators in combination with chlorpromazine and quinacrine

et al. 2010

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In vitro synergistic anti-prion effect of cholesterol ester modulators in combination with chlorpromazine and quinacrine * E-mail: pania@unica.it°These authors contributed equally to this work. Received 06 October 2009; Accepted 16 December 2009Abstract: Our studies on the role of cholesterol in prion infection/replication showed that brains and peripheral cells of sheep susceptible-to or suffering-from Scrapie were characterized by an altered cholesterol homeostasis, and that drugs affecting cholesterol ester pool were endowed with selective anti-prion activity in N2a cell lines infected with the 22L and RML prion strains. In these prion-infected N2a cell lines, we now report increased anti-prion activity of dual-drug combinations consisting of cholesterol ester modulators associated with prion inhibitors. Synergism was obtained with the cholesterol ester modulators everolimus, pioglitazone, progesterone, and verapamil associated with the anti-prion chlorpromazine, and with everolimus and pioglitazone associated with the anti-prion quinacrine. In addition, comparative lipid analyses in prion-infected vs. uninfected N2a cells, demonstrated a derangement of type and distribution of cholesterol ester, free cholesterol, and triglyceride pools in the infected cells. Single-drug treatments differently affected synthesis of the various lipid forms, whereas combined drug treatments appeared to restore a lipid profile similar to that of the untreated-uninfected cells. We conclude that the anti-prion synergistic effects of cholesterol ester modulators associated with the cholesterol-interfering anti-prion drugs chlorpromazine and quinacrine may arise from the ability of combined drugs to re-establish lipid homeostasis in the prion-infected cells. Overall, these data suggest that inhibition of prion replication can be readily potentiated by combinatorial drug treatments and that steps of cholesterol/cholesterol ester metabolism may represent suitable targets.© Versita Sp. z o.o.

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Cell division modulates prion accumulation in cultured cells

Cited by 80 publications

References 43 publications

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

Observing fibrillar assemblies on scrapie-infected cells

In vitro synergistic anti-prion effect of cholesterol ester modulators in combination with chlorpromazine and quinacrine

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