Glycosylphosphatidylinositol Anchoring Directs the Assembly of Sup35NM Protein into Non-fibrillar, Membrane-bound Aggregates

Marshall, Karen E.; Offerdahl, Danielle K.; Speare, Jonathan O.; Dorward, David W.; Hasenkrug, Aaron M.; Carmody, Aaron B.; Baron, Gerald S.

doi:10.1074/jbc.m114.556639

Cited by 7 publications

(9 citation statements)

References 92 publications

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“…The C-terminal green fluorescent protein (GFP) domain contains an A206K substitution to avoid dimerization of the fluorophore (65), which could promote aggregation of PrP by bringing two molecules into close proximity. GFP was added for imaging purposes; any aggregation of the TM PrP construct would result in clusters of GFP fluorescence, which could be observed in real time as seen with GPI-anchored Sup35NM (48,49). Furthermore, this domain serves to firmly anchor the TM PrP in the membrane such that it could not be removed without either proteolysis or severe compromise of the integrity of the plasma membrane.…”

Section: Resultsmentioning

confidence: 99%

“…The PrP res aggregates in the two inocula have different ultrastructures, with fibrils adopting an amyloid conformation and microsomebound aggregates adopting a nonfibrillar, apparently nonamyloid structure (7,35,44,49,(118)(119)(120)(121). Furthermore, PrP res in the two inocula might be expected to interact differently with distinct membrane regions.…”

Section: Discussionmentioning

confidence: 99%

“…Confocal images were acquired on a Nikon LiveScan confocal microscope described previously (48,49). Huygens software (Scientific Volume Imaging) was used to deconvolve confocal images.…”

Section: Methodsmentioning

confidence: 99%

“…species that lack many defining characteristics of amyloid (49). Collectively, these data point toward GPI anchoring and raft localization as significant facets of prion propagation and TSE pathogenesis.…”

mentioning

confidence: 96%

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PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

Marshall

Hughson

Vascellari

et al. 2017

J Virol

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Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol-and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C . To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion.IMPORTANCE Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C ) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrP C or transmembrane-anchored PrP C , which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrP res . We show that only GPI-anchored PrP C was able to convert to PrP res and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrP C to rafts are crucial to the ability of PrP C to propagate as a prion.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Confocal images were acquired on a Nikon LiveScan confocal microscope described previously (48,49). Huygens software (Scientific Volume Imaging) was used to deconvolve confocal images.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

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PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

Marshall

Hughson

Vascellari

et al. 2017

J Virol

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“…24 Interestingly, the experimental addition of a GPI anchor to the amyloidogenic yeast protein Sup35p appears to have an analogous biochemical influence, promoting the formation of non-fibrillar aggregates with ultrastructural similarity to PrP Sc . 25 Recent work has shown that anchorless PrP Sc more readily crosses a species barrier. 26 Finally, the co-expression of anchorless and wild-type PrP C molecules in recipient animals appears to allow for the detection of infectious activity in PrP amyloid fibril preparations.…”

Section: Influence Of Post-translational Modifications On Protein Folmentioning

confidence: 99%

Dissociation of recombinant prion autocatalysis from infectivity

Noble¹,

Supattapone

2015

Prion

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Heterologous overexpression of Sup35 in Escherichia coli leads to both monomer and complex states

Wang

Cheng

2022

Proteins

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The heterologous overexpression states of prion proteins play a critical role in understanding the mechanisms of prion‐related diseases. We report herein the identification of soluble monomer and complex states for a bakers' yeast prion, Sup35, when expressed in Escherichia coli. Two peaks are apparent with the elution of His‐tagged Sup35 by imidazole from a Ni2+ affinity column. Peak I contains Sup35 in both monomer and aggregated states. Sup35 aggregate is abbreviated as C‐aggregate and includes a non‐fibril complex comprising Sup35 aggregate‐HSP90‐Dna K, ATP synthase β unit (chain D), 30S ribosome subunit, and Omp F. The purified monomer and C‐aggregate can remain stable for an extended period of time. Peak II contains Sup35 also in both monomer and aggregated (abbreviated as S‐aggregate) states, but the aggregated states are caused by the formation of inter‐Sup35 disulfide bonds. This study demonstrates that further assembly of Sup35 non‐fibril C‐aggregate can be interrupted by the chaperone repertoire system in E. coli.

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Glycosylphosphatidylinositol Anchoring Directs the Assembly of Sup35NM Protein into Non-fibrillar, Membrane-bound Aggregates

Cited by 7 publications

References 92 publications

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

Dissociation of recombinant prion autocatalysis from infectivity

Heterologous overexpression of Sup35 in Escherichia coli leads to both monomer and complex states

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