Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes

Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc.

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“…As a result, protein export from the ER is blocked (Misumi et al, 1986;Oda et al, 1987). In addition, BFA Vol.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and trafficking of prion proteins in cultured cells.

Taraboulos

Raeber

Borchelt

et al. 1992

MBoC

256

214

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“…To assess whether this mechanism also applies to the derivatives analysed here, 10 g/ml Brefeldin A (BFA) were added to the culture medium of wtN2a. BFA prevents vesicular and proteins transport from the ER along the secretory pathway (Misumi et al, 1986;Oda et al, 1987;Klausner et al, 1992). After 2 hours, cells were exposed to 200 g/ml suramin, NF449 or NF023 in the presence of BFA and incubated at 37°C overnight (Fig.…”

Section: Retention Of Prp C In the Er Or Tgn Precludes Aggregation Bymentioning

confidence: 99%

Charged bipolar suramin derivatives induce aggregation of the prion protein at the cell surface and inhibit PrPSc replication

Nunziante

Kehler

Maas

et al. 2005

Journal of Cell Science

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The conversion of the cellular prion protein (PrPc) into a pathogenic isoform (PrPSc) is one of the underlying events in the pathogenesis of the fatal transmissible spongiform encephalopathies (TSEs). Numerous compounds have been described to inhibit prion replication and PrPSc accumulation in cell culture. Among these, the drug suramin induces aggregation and re-targeting of PrPc to endocytic compartments. Plasma membrane and sites of conversion into PrPSc are thereby bypassed. In the present study, a library of suramin analogues was tested as a potential class of new anti-prion compounds and the molecular mechanisms underlying these effects were analysed. Treatment of prion-infected neuroblastoma cells with compounds containing symmetrical aromatic sulfonic acid substitutions inhibited de novo synthesis of PrPSc and induced aggregation and reduction of the half-life of PrPc without downregulating PrPc cell surface expression. Half-molecule compounds lacking the symmetrical bipolar structure or the anionic groups had no effect on PrPSc synthesis or PrPc solubility. Cell surface expression of PrPc was necessary for the activity of effective compounds. Suramin derivatives did not induce aggregation of PrPc when transport along the secretory pathway was compromised, suggesting that their effects occur at a post trans-Golgi network (TGN) site, possibly close to the compartment of conversion into PrPSc. In vitro studies with recombinant PrP demonstrated that the inhibitory effect correlated with direct binding to PrP and induction of insoluble PrP aggregates. Our data reveal an anti-prion effect that differs from those characterising other sulphated polyanions and is dependent on the presence of the symmetrical anionic structure of these molecules.

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“…An established characteristic of the action of brefeldin A on cells is that the compound prevents the exit of newly synthesized proteins from the endoplasmic reticulum (ER) [3,4]. The Golgi apparatus structure is rapidly disrupted and what has been interpreted as microtubuledependent retrograde transport of Golgi apparatus enzymes back to the ER is induced [6,7,24,25].…”

Section: Discussionmentioning

confidence: 99%

“…Transport of G protein is inhibited in vesicular stomatitis virus-infected baby hamster kidney cells [2], and secretion of plasma proteins, such as albumin and al-protease inhibitor, are blocked in rat hepatocytes [3,4]. Despite earlier indications that brefeldin A resulted in more or less complete disassembly of the Golgi apparatus and its relocation to the endoplasmic reticulum [1][2][3][4][5][6][7], it now appears that, in most instances, a modified Golgi apparatus remains [8,9].…”

Section: Introductionmentioning

confidence: 99%

Inhibition by brefeldin A of NADH oxidation activity of rat liver Golgi apparatus accelerated by GDP

et al. 1994

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Al~traetReduced pyridine nucleotide has been reported to enhance cell-free transfer of membrane material from a radiolabeled Golgi apparatus donor fraction from rat liver to an acceptor fraction consisting of inside-out plasma vesicles immobilized on nitrocellulose [(1992) Bioehim. Biophys. Acta 1107, 131]. As part of a continuing effort to identify NADH-requiring enzymes in the Goigi apparatus which may be important to membrane trafficking, highly purified fractions of Golgi apparatus from rat liver were tested for their ability to oxidize NADH and the inhibition of the oxidation of NADH by brefeldin A. The isolated Golgi apparatus fractions were found to oxidize NADH with a specific activity comparable to that of the plasma membrane of rat liver. The activity was inhibited by brefeldin A and this inhibition was augmented by GDP. At near optimal concentrations of 7/tM brefeldin A and 1/aM GDP, the activity was > 90% inhibited. Brefeldin A inhibition of NADH oxidation by the Golgi apparatus was time-dependent and GDP appeared to accelerate the inhibition by brefeidin A.

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Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes

Cited by 106 publications

References 14 publications

Synthesis and trafficking of prion proteins in cultured cells.

Synthesis and trafficking of prion proteins in cultured cells.

Charged bipolar suramin derivatives induce aggregation of the prion protein at the cell surface and inhibit PrPSc replication

Inhibition by brefeldin A of NADH oxidation activity of rat liver Golgi apparatus accelerated by GDP

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