Prion Elimination Research Articles

Studies of specific yeast prion chimera to test hypotheses about prion propagation and Hsp104‐mediated prion elimination

In Saccharomyces cerevisiae, at least 10 proteins are known to form amyloid‐based prions which propagate in cell populations due to continuous fragmentation by molecular chaperones. Hsp104, Hsp70, and the Hsp40 Sis1 are all universally essential to this process; however, prions differ in their specific requirement for Sis1 activities, and some require additional Hsp40 chaperones. For reasons which are still being explored, only one prion, [PSI+], is eliminated when Hsp104 is overexpressed. We are utilizing prion‐chimeras originally developed by the Lindquist lab at MIT to test various hypotheses regarding prion propagation and Hsp104‐mediated elimination, including the role of amino acid content of the prion‐forming domain in determining prion‐Hsp40 requirements. Additionally, our investigations explore the hypothesis that the specificity for Hsp104‐mediated prion elimination is due to a unique protein region of the [PSI+]‐forming protein Sup35 known only as the “M” domain. Preliminary results indicate that the presence of the M domain is an important, if not essential, component in determining the ability of a prion to be eliminated by Hsp104 overexpression.Support or Funding InformationThis work was supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program ( www.lafayette.edu), the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606, and the Henry Dreyfus Teacher Scholar Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Dreyfus Foundation.

Variant-specific and reciprocal Hsp40 functions in Hsp104-mediated prion elimination.

SummaryThe amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI +] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI +] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI +] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI +] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.

Variant‐specific and reciprocal Hsp40 functions in Hsp104‐mediated prion elimination: A potential role for the ‘Anti‐prion J‐protein 1’ (Apj1)

The prions of Saccharomyces cerevisiae are heritable aggregates of misfolded yeast proteins. Yeast prions are incorporated into daughter cells through the fragmentation of these aggregates by molecular chaperone proteins, including J‐proteins (Hsp40s), Hsp70s, and Hsp104. Previous studies have shown that the overexpression of Hsp104 cures the prion [PSI+], but not other prions, a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases, despite the fact that the mechanism of [PSI+] elimination by Hsp104 overabundance is currently the subject of significant debate. We recently reported that the human homolog of Sis1, Hdj1, was capable of substituting for Sis1 in the propagation of strong but not weak [PSI+] variants and was deficient in substituting for Sis1 in the elimination of [PSI+] by Hsp104 overexpression. The objective of this study was to extend that investigation to the Sis1‐domain requirements for Hsp104‐mediated [PSI+] curing, expanding upon another prior investigation by extending the analysis to multiple [PSI+] variants and genetic backgrounds. We also asked whether any of the other 12 J‐proteins located in the S. cerevisiae cytosol are also necessary for Hsp104‐induced [PSI+] curing and if prion amyloid structure (variant identity) affects experimental outcomes. The primary method used in this study was complementation by plasmid‐shuffling in the budding yeast S. cerevisiae.Summary of ResultsOur investigation revealed that at least two weak [PSI+] variants can be maintained in the absence of any of 12 non‐essential J‐proteins, ruling out essential roles for any of these J‐proteins in weak [PSI+] prion propagation. Likewise, elimination of these weak [PSI+] variants by Hsp104 overexpression was ubiquitous, demonstrating that no J‐protein other than Sis1 is necessary for the elimination of weak [PSI+] variants by overexpression of Hsp104. In sharp contrast however, we found that strong variants of [PSI+] exhibited exceptional resistance to Hsp104‐mediated elimination only in strains lacking the J‐protein Apj1 (originally named: Anti‐Yeast Prion J‐protein 1) and conversely Apj1 overexpression accelerates prion elimination. Apj1 has been implicated in prion biology several times; often acting similarly to Ydj1 in prion biology. Here we find a novel genetic interaction for Apj1 and prions that is distinct from Ydj1 function. Additional mutation and truncation experiments revealed domains required for Apj1 function in Hsp104‐mediated [PSI+] curing and may ultimately help to explain the prion‐specificity of this curing mechanism.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity

abstractYeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as ‘strains’ in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called ‘variants’, dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI+] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1.1 Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.

A bipolar functionality of Q/N‐rich proteins: Lsm4 amyloid causes clearance of yeast prions

Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI+]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI+], [URE3], and [RNQ+]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI+] elimination, the [PSI+] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi−]-like mother cells. We also found that the antiprion activity is a general property of [PSI+]-inducible factors. These data provoked a novel “unified” model that explains both prion induction and elimination by a single scheme.

Clotting factor activity in thawed Octaplas® LG during storage at 2–6 °C for 6 days from a quality assurance point of view

IntroductionOctaplas® LG is a second-generation solvent/detergent-treated plasma that offers an additional safety benefit by prion elimination. The stability of clotting factors of the new S/D plasma after thawing has not been investigated yet. This study intended to measure the time course of fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI through storage at 2–6°C over 6days. Materials and methodsWe investigated 20 plasma bags (five bags per blood group) and measured fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI immediately after thawing and after 2, 4, 6, 24, 48, 72, 96, 120 and 144h storage at 2–6°C. Five separate plasma bags were thawed and stored at 2–6°C for microbiological assessment. After 6days samples were drawn for blood cultures that were incubated for six more days. ResultsAfter 6days FII, FIX and PC showed no significant changes. FV (−16%, p<0.001), FVII (−19%, p<0.001), FVIII (−19%, p<0.001), FXI (−13%, p<0.0001) and fPS (−4%, p<0.0007) decreased significantly. PI levels were stable at 56%. The microbiological investigation showed no bacterial contamination. ConclusionsIn Octaplas® LG plasma clotting factors decreased slightly through storage of 6days. PI levels were remarkably higher and stable over time in the new Octaplas® LG. Stability of stored Octaplas® LG was limited by the decrease of FVIII to 53%, which may warrant storage up to 24h from a quality assurance point of view. This could result in reduced plasma wastage and costs for healthcare givers.

Prion‐associated proteins in yeast: comparative analysis of isogenic [PSI+] and [psi−] strains

A large group of prion-associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Two-dimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI(+)] and [psi(-)] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prion-containing aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion-associated, but we cannot exclude the possibility that several proteins may propagate as prions.

A G-protein γ subunit mimic is a general antagonist of prion propagation in Saccharomyces cerevisiae

The Gpg1 protein is a Ggamma subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [PSI(+)], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [PSI(+)] but also [PIN(+)], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [PSI(+)] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted alpha-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2Delta and gpb1Delta strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway.

Investigations of prion and virus safety of a new liquid IVIG product

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP sc) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10 log 10. Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18 log 10 for enveloped and >7 log 10 for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.

Prion formation in yeast is influenced by alterations of the ubiquitin proteolysis

Prions are infectious aggregated proteins that cause neurodegenerative diseases in mammals and transmit heritable traits in yeast. De novo appearance of a yeast prion is facilitated by an increase in the concentration of the same protein or by the presence of aggregates of a different QN‐rich protein. Since levels of protein in the cell are controlled at least in part by ubiquitin proteasome system (UPS), we investigated the role of UPS in maintenance of a prion isoform of the yeast translation termination factor Sup35. Deletion of the ubiquitin‐conjugating enzyme Ubc4 increased de novo prion formation and antagonized prion elimination by the disaggregating chaperone Hsp104. Although levels of Sup35 protein were not affected by this UPS alteration, levels of the QN‐rich protein Pin3 were increased. We confirmed that overexpression of Pin3 protein stimulates prion formation by Sup35 and demonstrated that Pin3 is present in the cell in the ubiquitinated form. Prevention of Pin3 ubiquitination by mutation has an effect on protein stability, cellular localization and aggregation. Our data suggest that UPS can regulate prion formation via modulation of QN‐rich proteins in the yeast model.(Supported by grant GM30308 from NIH)

Propriétés mécaniques de greffons humains provenant de têtes fémorales et traitées par un procédé d’épuration physico-chimique (Ostéopure™)

Purpose of the study Bone grafts and bone substitutes must be biocompatible osteoconductors with satisfactory mechanical properties similar to native bone. When the bone treatment is conducted under specific conditions, the elasticity module under infra-maximal loading can be optimized to achieve reproducible values. The purpose of this work was to determine the effect of the cleaning and sterilization process using Osteopure™ on the biomechanical properties of trabecular bone harvested from human femoral heads. Material and method Seventy trabecular bone samples were tested: group 1F (fresh samples); group 1N (after application of Osteopure™ cleaning); group 1S (after Osteopure® cleaning and sterilization). Non-destructive and destructive tests (group 1D) were performed. Two fresh femoral heads were used as controls for the destructive test (group 2). The first non-destructive test was applied directly after section (group 1F). Other samples were then purified with Osteopure™ treatment and a second non-destructive test was conducted (group 1N). A third non-destructive test was conducted after sterlization with 25kgray radiation (group 1S). Treatments 1 and 2 were performed by OST Developpement SA (Clermont-Ferrand). Finally a destruction test was applied along the directional axis (group 1D). For the 31 samples in group 2 (control) the destructive test was applied along the directional axis immediately after section. Compression tests were performed at a deformation speed of 3 mm/min for 0.3% deformation. Results The Young module did not exhibit any significant difference between the three steps of the testing in the three orthogonal directions. The Young module was not significantly different between group 1F and group 2 (controls). Maximal force of compression was significantly different (P<0.01). There was a linear relationship between maximal force at rupture and the Young module obtained during destructive tests, for groups 1D and 2 respectively. The compression curves obtained from sterilized samples (group 1D) were not significantly different from those observed for fresh trabecular bone in group 2 (controls). Discussion The Young module values measured from 70 - 673 MPa. For non-destructive tests, the module values were to the order of 64% of those obtained for destructive tests. Decreased maximal force of rupture observed for treated samples in comparison with fresh samples can be explained by the extraction of most of the lipids. Conclusion The Osteopure™ method does not alter stiffness of bone allografts. The elasticity module observed in treated bones is close to that observed in fresh bones. Mechanical resistance to compression is however only half the force of compression observed in the hip joint for daily activities. The linear relationship between the elasticity mode and loading required for rupture is not affected by treatment with Osteopure™. The advantages related to elimination of prions or viral contamination appear by far to be more important than the minor changes observed in the mechanical characteristis of allografts.

The mechanisms of [URE3] prion elimination demonstrate that large aggregates of Ure2p are dead-end products

The yeast prion [URE3] is a self-propagating inactive form (the propagon) of the Ure2 protein. Ure2p is composed of two domains: residues 1-93--the prion-forming domain (PFD)--and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression. Guanidine hydrochloride, and the overproduction of Ure2p 1-65 or Ure2-GFP have been shown to induce the elimination of [URE3]. We demonstrate here, two different curing mechanisms: the inhibition of [URE3] replication by guanidine hydrochloride and its destruction by Ure2p aggregation. Such aggregation is observed if PFD or Ure2-GFP are overproduced and in heterozygous URE2/URE2-GFP, [URE3] diploids. We found that the GFP foci associated with the presence of the prion were dead-end products, the propagons remaining soluble. Surprisingly, [URE3] propagated via the Ure2-GFP fusion protein alone is resistant to these two curing mechanisms and cannot promote the formation of foci. The relationship between aggregation, prion and Hsp104 gives rise to a model in which the propagon is in equilibrium with larger aggregates and functional protein.

Differential Inhibition of Prion Propagation by Enantiomers of Quinacrine

Prion diseases are fatal neurologic disorders caused by accumulation of a pathogenic isoform (PrPSc) of the prion protein (PrP). The recent discovery of the inhibitory action of quinacrine on PrPSc formation in scrapie-infected neuroblastoma (ScN2a) cells raised the possibility of a treatment for patients with prion disease. To investigate the efficacy of quinacrine enantiomers, we measured the inhibitory effect of these isomers on PrPSc formation in ScN2a cells. (S)-quinacrine exhibited superior antiprion activity compared with (R)-quinacrine and two generic quinacrines that appear to be racemates. Treatment with these various forms of quinacrine did not induce adverse changes affecting cell survival and the expression of marker proteins over a range of potentially therapeutic concentrations. Thus, quinacrine enantiomers demonstrated stereoselectivity on prion elimination but not cytotoxicity in ScN2a cells. Our results raise the possibility that in vivo treatment using one enantiomer of quinacrine may be superior to a racemic mixture, which is the form that is generally used when quinacrine is employed to treat parasitic diseases.

Elimination of prions by branched polyamines and implications for therapeutics.

We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able to purge PrP(Sc), the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal of PrP(Sc) by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrP(Sc) to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure-activity analysis revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrP(Sc) effectively from cells. The removal of PrP(Sc) by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with branched polyamines at acidic pH rendered the PrP(Sc) susceptible to protease in vitro, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.