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Aggregates from Sup35-GFP GPI -Agg cells seed formation of GPI anchorless Sup35-mC aggregates. A, stably transfected cells expressing anchorless forms of mC or Sup35-mC were assayed by immunoblot for expression of the respective proteins. Cell lysates were normalized for total protein and probed with anti-HA or anti-Sup35 M domain antibodies. Culture supernatants were assayed for mCherry-tagged protein by immunoprecipitation . High levels of anchorless mC and Sup35-mC were released to culture supernatants. B, in situ seeding by fixed Sup35-GFP GPI aggregates. Cells were chemically fixed prior to incubation with culture supernatants from cells expressing the indicated forms of GPI-anchorless mCherry protein and confocal imaging. Arrows, Sup35-GFP GPI aggregates. Arrowheads, newly induced Sup35-mC aggregates. White in the merge panel (G) indicates areas of co-localization. Images correspond to a single 0.5-m optical z slice from near the middle of the cells. Scale bar, 10 m. C, cell-free seeding assay. Microsome fractions from Sup35-GFP GPI -Agg cells as a source of seeds or GFP GPI control cells (or PBS buffer control) were incubated with the indicated substrates. Reactions were analyzed by filter trap assay to detect either preexisting (anti-GFP panels) or newly induced mCherry-tagged (anti-RFP panels; anti-RFP binds mCherry) SDS-insoluble aggregates. D, in situ seeding by Sup35-GFP GPI aggregates on live cells. Live cells were incubated with culture supernatants from cells expressing Sup35-mC followed by confocal imaging. Arrows, Sup35-GFP GPI aggregates. Arrowhead, newly induced Sup35-mC aggregate. White in the merge panel (C) indicates the area of co-localization. Images correspond to a single 0.5-m optical z slice from near the middle of the cells. Scale bar, 10 m. E, chymotrypsin resistance assay. After imaging, cultures in D were lysed, digested with chymotrypsin, and immunoblotted with anti-HA tag antibody (lanes 5–10). Control samples of Sup35-mC culture supernatant treated in parallel were also immunoblotted with anti-Sup35 N (lanes 1 and 2) and M domain antibodies (lanes 3 and 4). Chymotrypsin-negative lanes contain one-quarter sample equivalents loaded in chymotrypsin-treated lanes. Arrow, full-length Sup35-mC. Open arrowhead, chymotrypsin-truncated Sup35-mC.
Source publication
In prion-infected hosts, PrPSc usually accumulates as non-fibrillar, membrane-bound aggregates. Glycosylphosphatidylinositol
(GPI) anchor-directed membrane association appears to be an important factor controlling the biophysical properties of PrPSc
aggregates. To determine whether GPI anchoring can similarly modulate the assembly of other amyloid-...
Citations
... These findings, together with observations in human genetic prion disease associated with GPI-anchorless PrP [22][23][24], have led to the suggestion that the GPI-anchor modulates fibril assembly of PrP, potentially through steric interference of fibril formation. To this end, a GPIanchored form of the yeast prion, Sup35, expressed in neuronal cells formed membrane-bound, nonfibrillar aggregates, whereas GPI-anchorless Sup35 formed fibrils [36]. Interestingly, Sup35 accumulated in extracellular vesicles piling on the cell surface, which may be relevant to our findings of extensive plaque-like deposits of ME7 and mNS prions piling on neuronal cell membranes. ...
Many aggregation-prone proteins linked to neurodegenerative disease are post-translationally modified during their biogenesis. In vivo pathogenesis studies have suggested that the presence of post-translational modifications can shift the aggregate assembly pathway and profoundly alter the disease phenotype. In prion disease, the N-linked glycans and GPI-anchor on the prion protein (PrP) impair fibril assembly. However, the relevance of the two glycans to aggregate structure and disease progression remains unclear. Here we show that prion-infected knockin mice expressing an additional PrP glycan (tri-glycosylated PrP) develop new plaque-like deposits on neuronal cell membranes, along the subarachnoid space, and periventricularly, suggestive of high prion mobility and transit through the interstitial fluid. The plaque-like deposits were largely non-congophilic and composed of full length, uncleaved PrP, indicating retention of the glycophosphatidylinositol (GPI) anchor. Prion aggregates sedimented in low density fractions following ultracentrifugation, consistent with oligomers, and bound low levels of heparan sulfate similar to other predominantly GPI-anchored prions. These results suggest that highly glycosylated PrP primarily converts as a GPI-anchored glycoform with low involvement of HS co-factors, limiting PrP assembly mainly to oligomers. Thus, these findings may explain the high frequency of diffuse, synaptic, and plaque-like deposits and rapid conversion commonly observed in human and animal prion disease.
... M isfolding and aggregation of the prion protein (PrP C ) are associated with several 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. ...
... 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. ...
... In the present study, two different types of PrP res inoculum were used: membranebound (in the form of microsomal brain membranes containing GPI-anchored PrP res ) and purified GPI-anchorless PrP res fibrils. 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. ...
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 localisation of PrP(C) to rafts is crucial to the ability of PrP(C) to propagate as a prion.
... Except for those direct drug-drug interactions, DDIs also include the interactions between targets and drugs respectively. Glycosylphosphatidylinositol which is also known as GPI anchor has been widely reported to be the only functional and structural connection between proteins and cell membrane especially in microbes (Kinoshita 2014;Marshall et al. 2014). The biosynthesis of glycosylphosphatidylinositol (hsa00563) has been reported to involve several crucial phospholipid associated metabolisms (Butikofer et al. 2010;Kinoshita 2014;Stokes, Murakami, Maeda, Kinoshita & Morita 2014). ...
Drug-drug interaction (DDI) defines a situation in which one drug affects the activity of another when both are administered together. DDI is a common cause of adverse drug reactions and sometimes also leads to improved therapeutic effects. Therefore, it is of great interest to discover novel DDIs according to their molecular properties and mechanisms in a robust and rigorous way. This paper attempts to predict effective DDIs using the following properties: (1) chemical interaction between drugs; (2) protein interactions between the targets of drugs; and (3) target enrichment of KEGG pathways. The data consisted of 7,323 pairs of DDIs collected from the DrugBank and 36,615 pairs of drugs constructed by randomly combining two drugs. Each drug pair was represented by 465 features derived from the aforementioned three categories of properties. The random forest algorithm was adopted to train the prediction model. Some feature selection techniques, including minimum redundancy maximum relevance (mRMR) and incremental feature selection (IFS), were used to extract key features as the optimal input for the prediction model. The extracted key features may help to gain insights into the mechanisms of DDIs and provide some guidelines for the relevant clinical medication developments, and the prediction model can give new clues for identification of novel DDIs.
... 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. ...
Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication - that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain (1) . We identified restricted, C-terminal structural differences between these two conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor (1) . In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.
... The loss of the GPI anchor of PrP (which is concomitant with a significant reduction in PrP C glycosylation in vivo [55]) appears to have only a modest effect on PrP Sc structure [56], but substantially alters the biochemical properties of PrP Sc and promotes the formation of fibrillar aggregates [57,58] and interferes with PrP Sc replication in vitro [49]. Interestingly, the co-expresssion of anchorless and wild-type PrP C molecules in vivo appears to enhance host susceptibility to recombinant PrP amyloid fibrils [59], while the experimental addition of a GPI anchor to the amyloidogenic yeast protein, Sup35p, prevents the formation of fibrillar structures, leading instead to the formation of PrP Sc -like, non-fibrillar aggregates [60]. While the data from the present study specifically point to a role for the GPI anchor of native mouse PrP C in restricting the range of recombinant PrP Sc conformers that possess infectious activity, we cannot exclude the possibility that N-linked glycans also influence the infectivity-associated recombinant PrP Sc conformational space. ...
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.
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 E. 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.
Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼10⁹ lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular β sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.
