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Prions replicate by a self-templating mechanism. Infidelity in the process can lead to the emergence of new infectious structures, referred to as variants or strains. The question of whether prions are prone to mis-templating is not completely answered. Our previous experiments with 23 variants of the yeast [ PSI + ] prion do not support broad mutability. However, it became clear recently that the heat shock protein Hsp104 can restrict [ PSI + ] strain variation. This raises the possibility that many transmutable variants of the prion may have been mistaken as faithful-propagating simply because the mutant structure was too sturdy or too frail to take root in the wild-type cell. Here, I alter the strength of Hsp104 in yeast, overexpressing wild-type Hsp104 or expressing the hypo-active Hsp104 T160M mutant, and check if the new environments enable the variants to mutate. Two variants hitherto thought of as faithful-propagating are discovered to generate different structures, which are stabilized with the hypo-active chaperone. In contrast, most transmutable variants discovered in cells overexpressing Hsp104 have been correctly identified as such previously in wild-type cells without the overexpression. The majority of transmutable variants only mis-template the structure of VH, VK, or VL, which are the most frequently observed variants and do not spontaneously mutate. There are four additional variants that never give rise to different structures in all cell conditions tested. Therefore, quite a few [ PSI + ] variants are faithful-propagating, and even the transmutable ones do not freely evolve but can only change to limited structural types., Competing Interests: The author declares no conflict of interest.

Gorkovskiy et al. observed that many [PSI + ] prion isolates, obtained in yeast with the mutant Hsp104 T160M chaperone, propagate poorly in wild-type cells and suggested that Hsp104 is part of the cellular anti-prion system, curing many nascent [PSI + ] variants. Here, we argue that the concept may require reassessment. We induced [PSI + ] variants in both the wild-type and the mutant background. Three new variants were isolated in the T160M background. They exhibited lower thermostability, possessed novel structural features, and were inherently mutable, changing to well-characterized VH, VK, and VL variants in wild-type cells. In contrast, VH, VK, and VL of the wild-type background, could not change freely and were lost in the mutant, due to insufficient chaperone activity. Thus, mutant Hsp104 can impose as much restriction against emerging prion variants as the wild-type protein. Such restriction conserved the transmutable variants in the T160M background, since new structures mis-templated from them could not gain a foothold. We further demonstrate excess Hsp104 T160M or Hsp104 ∆2-147 can eliminate nearly all of the [PSI + ] variants in their native background. This finding contradicts the generally held belief that Hsp104-induced [PSI + ] curing requires its N-terminal domain, and may help settling the current contention regarding how excess Hsp104 cures [PSI + ]., (© 2020 John Wiley & Sons Ltd.)

Twenty-three prion variants of the wild-type Sup35 protein are obtained, including 19 novel ones and 4 previously documented, namely, VH, VK, VL, and W8. Their uniqueness and non-composite nature are demonstrated. Specific infectivity is generated de novo for most variants by adding prion particles to solutions of a purified Sup35 N-terminal fragment, thereby supporting the protein-only composition. Sup35 prions isolated by other laboratories are identified within the collection and found to fall into a narrow set of five variant types that are readily inducible in vivo by Sup35 overexpression. The work establishes an unambiguous and extensive collection of prion variants, demonstrating that a protein, by itself, in the absence of genetic and conformational co-factors, could adopt a great number of structures. In light of recent high-resolution structures of other amyloids, we discuss how the diverse folding is achieved in spite of apparent contradiction to the classical paradigm that a protein's structure is uniquely determined by its sequence.

[PSI + ] variants are different infectious conformations of the same Sup35 protein. We show that when [PSI + ] variants VK and VL co-infect a dividing host, only one prevails in the end and the host genetic background is involved in winner selection. In the 5V-H19 background, the VK variant dominates over the VL variant. The order of dominance is reversed in the 74-D694 background, where VL can coexists with VK for a short period, but will eventually take over. Differential interaction of chaperone proteins with distinct prion variant conformations can influence the outcome of competition. Expanding the Glycine/Methionine-rich domain of Sis1, an Hsp40 protein, helps the propagation of VL. Over-expression of the Hsp70 protein Ssa2 lowers the number of prion particles (propagons) in the cell. There is more reduction for VK than VL, causing the latter to dominate in some of the 5V-H19 and all of the 74-D694 cells tested. Consistently, depleting Ssa1 in 74-D694 strengthens VK. Swapping chromosomal alleles of SSA1/2 and SIS1 between 5V-H19 and 74-D694, including cognate promoters, is not sufficient to change the native dominance order of each background, suggesting there exist additional polymorphic factors that modulate [PSI + ] competition., (© 2018 John Wiley & Sons Ltd.)

Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of (14)N- and (15)N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Prion strains are different self-propagating conformers of the same infectious protein. Three strains of the [PSI] prion, infectious forms of the yeast Sup35 protein, have been previously characterized in our laboratory. Here we report the discovery of a new [PSI] strain, named W8. We demonstrate its robust cellular propagation as well as the protein-only transmission. To reveal strain-specific sequence requirement, mutations that interfered with the propagation of W8 were identified by consecutive substitution of residues 5-55 of Sup35 by proline and insertion of glycine at alternate sites in this segment. Interestingly, propagating W8 with single mutations at residues 5-7 and around residue 43 caused the strain to transmute. In contrast to the assertion that [PSI] existed as a dynamic cloud of sub-structures, no random drift in transmission characteristics was detected in mitotically propagated W8 populations. Electron diffraction and mass-per-length measurements indicate that, similar to the 3 previously characterized strains, W8 fibers are composed of about 1 prion molecule per 4.7-Å cross-β repeat period. Thus differently folded single Sup35 molecules, not dimeric and trimeric assemblies, form the basic repeating units to build the 4 [PSI] strains.

Immense diversity of prion strains is observed, but its underlying mechanism is less clear. Three [PSI] prion strains--named VH, VK, and VL--were previously isolated in the wild-type yeast genetic background. Here we report the generation and characterization of eight new [PSI] isolates, obtained by propagating the wild-type strains with Sup35 proteins containing single amino-acid alterations. The VH strain splits into two distinct strains when propagated in each of the three genetic backgrounds, harboring respectively single mutations of N21L, R28P, and Gi47 (i.e. insertion of a glycine residue at position 47) on the Sup35 N-terminal prion-forming segment. The six new strains exhibit complex inter-conversion patterns, and one of them continuously mutates into another. However, when they are introduced back into the wild-type background, all 6 strains revert to the VH strain. We obtain two more [PSI] isolates by propagating VK and VL with the Gi47 and N21L backgrounds, respectively. The two isolates do not transmit to other mutant backgrounds but revert to their parental strains in the wild-type background. Our data indicate that a large number of [PSI] strains can be built on three basic Sup35 amyloid structures. It is proposed that the three basic structures differ by chain folding topologies, and sub-strains with the same topology differ in distinct ways by local structural adjustments. This "large number of variations on a small number of basic themes" may also be operative in generating strain diversities in other prion elements. It thus suggests a possible general scheme to classify a multitude of prion strains., Competing Interests: The authors have declared that no competing interests exist.

Amyloid polymorphism underlies the prion strain phenomenon where a single protein polypeptide adopts different chain-folding patterns to form self-propagating cross-beta structures. Three strains of the yeast prion [PSI], namely [VH], [VK], and [VL], have been previously characterized and are amyloid conformers of the yeast translation termination factor Sup35. Here we define specific sequences of the Sup35 protein that are necessary for in vivo propagation of each of these prion strains. By sequential substitution of residues 5-55 of Sup35 by proline and insertion of glycine at alternate sites in this segment, specific mutations have been identified that interfere selectively with the propagation of each of the three prion strains in yeast: the [VH] strain requires amino acid residues 7-21; [VK] requires residues 9-37; and [VL] requires residues 5 to at least 52. Minimal polypeptide segments capable of encoding prion conformations were defined by assembly of recombinant Sup35 fragments on purified prion nuclei to form amyloid fibers in vitro, whose infectivity was assayed in yeast. For the [VK] and [VL] strains, the minimal fragments approximately coincide with the strain-specific sequences defined by mutations of the N-terminal portion of the intact Sup35 (1-685); and for the [VH] strain, a longer Sup (1-53) fragment is required. Polymorphic structures of other amyloids might similarly involve different stretches of polypeptides to form cross-beta amyloid cores with distinct molecular recognition surfaces.

We present methods to prepare infectious Sup35 protein aggregates and use them for genetic transformation of yeast. The protein aggregates are prepared from bacterially expressed recombinant protein, which is converted to amyloid fibers by extended incubation or by nucleated growth using yeast prion particles as seeds. The aggregates are introduced into yeast by a modified spheroplast transformation protocol. The phenotype of the yeast transformants is further characterized by robust prion strain typing methods. The methodology can be used to introduce different [PSI(+)] particles to many laboratory yeast genetic backgrounds. It can be adapted for applications in other yeast prion systems as well.

Mass per length (mpl) measurements on single amyloid fibrils that specifically propagate the [VH], [VK], and [VL] strains of the yeast prion [PSI] reveal unanticipated differences in their structures. Many fibrils have approximately 1.0 prion molecule per 4.7-A cross-beta repeat period, which is consistent with a self-replicating model built by parallel beta-sheet hydrogen-bonding of like prion peptide segments, but other fibrils are definitely heavier. The predominantly straight fibrils of the dominant [VH] strain have a bimodal mpl distribution, corresponding to components with approximately 1.0 and 1.2 prions per repeat. Fibrils of the weaker [VK] strain, which are almost all wavy, have a monodisperse mpl distribution with a mean of 1.15 prions per repeat. The recessive [VL] strain sample has approximately 1.05 prions per repeat in single fibrils and includes approximately 10% double fibrils, which are rare in the duplicate [VH] and [VK] samples. All of these samples were assembled from purified recombinant Sup35 prion protein by seeded growth on nuclei extracted from yeast bearing the three [PSI] strains. Infectious and noninfectious spontaneously assembled fibrils of the recombinant prion protein also display different heterogeneous morphologies. The strain-specific morphological differences we have observed directly confirm the structural prediction of the protein-only prion theory but do not have an obvious molecular explanation.

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

The [PSI] genetic element, which enhances the nonsense suppression efficiency in the yeast Saccharomyces cerevisiae, is thought to be amyloid-like aggregates of the Sup35 protein, and to self-propagate by a prion-like mechanism. Analogous to strains of the mammalian prion, variants of [PSI], with different nonsense suppression efficiencies and mitotic stabilities, can be isolated from the same yeast genetic background. In the framework of the "protein-only" hypothesis, variants of prion are assumed to be distinct conformers of the same prion polypeptide. This study aims to provide further support for the structural basis of [PSI] variation. Three variants of [PSI] were induced and distinguished by a panel of 11 single point mutations of the Sup35 protein. The variant phenotypes are intrinsically associated with [PSI] elements, presumably structurally different amyloids, rather than produced from variations in the genetic background. Differential incorporation to [PSI] variants of a Sup35 point mutation as well as N and C-terminally truncated Sup35 fragments is further demonstrated in vivo, suggesting that distinct patches of amino acid residues are involved in the assembly of [PSI] variants. These results establish a method for [PSI] variant-typing and indicate that heritable variations of amyloid structures can be derived from the same polypeptide., (Copyright 2001 Academic Press.)

This is a review article of neuroimaging studies in post-traumatic stress disorder (PTSD). Findings from structural, biochemical, and functional studies are summarized. Magnetic resonance imaging (MRI) volumetric studies have consistently reported decreased hippocampal volumes in PTSD. Proton magnetic resonance spectroscopy studies report decreased N-acetyl aspartate (NAA) ratios and absolute concentrations in the medial temporal lobe. Although still controversial, these findings from volumetric and spectroscopic studies are thought to represent decreased neuronal density of the hippocampus. Functional imaging studies document different patterns of limbic and paralimbic structure activation in PTSD compared with controls. Of theoretical importance are findings of failure to activate the anterior cingulate as well as amygdala activation during symptom provocation studies. Also, increased amygdala activation was found with a behavioral task targeted to this structure. A neurobiological model is presented that takes into account findings from neuroimaging studies in PTSD as well as animal studies of fear conditioning. This model proposes that central to symptom mediation is a dysfunction of the anterior cingulate, with a failure to inhibit amygdala activation and/or an intrinsic lower threshold of amygdala response to fearful stimuli. The model further proposes that hippocampal atrophy is a result of the chronic hyperarousal symptoms mediated by amygdala activation., (Copyright 2001 Copyright by W.B. Saunders Company.)

Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY's potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif's cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates.

The high-mobility-group (HMG) box defines a DNA-bending motif conserved among architectural transcription factors. A "hydrophobic wedge" at the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain "cantilever." First described in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the crux of the HMG box (residues 12 and 13). Here, the role of these side chains in DNA recognition is investigated by alanine mutagenesis. F12A and I13A substitutions in the SRY HMG box each permit native folding and thermal stability (as monitored by circular dichroism and 1H-NMR) but eliminate sequence-specific DNA-binding activity (as detected by gel-mobility shift). On binding to the sharp angles of a four-way DNA junction (4WJ), however, the substitutions each promote formation of a high-molecular-weight aggregate, presumably by DNA-dependent oligomerization. The substitutions have opposite effects on initial binding to the 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enhanced by I13A. A foreshortened "alanine cantilever", not observed among specific HMG boxes, occurs in a non-specific domain (HMG-1A) and may enhance architecture-selective DNA recognition.

The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.

A novel 1H nuclear magnetic resonance (NMR) strategy for "footprinting" specific protein-DNA target sites is demonstrated. Relative rates of site-specifc imino-proton exchange in the free and bound DNA duplex are determined by use of laminar-shifted shaped pulses in NOESY spectra. 2D exchange crosspeaks between imino (omega2 dimension) (omega1 dimension) resonances in principle provide site-specific probes of protein binding. Chemical exchange is distinguished from nuclear Overhauser enhancements(NOEs) to bound water by use of ROESY spectroscopy. This strategy is illustrated in 1H-NMR studies of the SRY high-mobility group (HMG) box, the Y-chromosome-encoded "master switch" for testis determination in man. In a specific complex between the protein and a 15-basepair DNA site, imino-proton exchange was observed to be damped selectively within the six basepair subsite 5'-ATTGTT, previously identified by random binding-site selection as an optimal SRY target sequence. The extent of damping correlates with sites of protein-DNA contacts in the minor groove but not with the magnitude of 1H-NMR complexation shifts. SRY binding has recently been shown to introduce significant distortions in DNA structure. The DNA is sharply bent and underwound; the minor groove is widened and major groove compressed. Our results demonstrate that despite such distortions base pairing is stably maintained. Protein binding in the DNA minor groove shields DNA imino protons from exchange with solvent.

SRY, a genetic "master switch" for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (168T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition alpha-helix of the HMG box [Haqq, C.M., King, C.-Y., Ukiyama, E., Haqq, T.N., Falsalfi, S., Donahoe, P.K., & Weiss, M.A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided 1H-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits > 50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.

The pathway of male sexual development in mammals is initiated by SRY, a gene on the short arm of the Y chromosome. Its expression in the differentiating gonadal ridge directs testicular morphogenesis, characterized by elaboration of Müllerian inhibiting substance (MIS) and testosterone. SRY and MIS each belong to conserved gene families that function in the control of growth and differentiation. Structural and biochemical studies of the DNA binding domain of SRY (the HMG box) revealed a protein-DNA interaction consisting of partial side chain intercalation into a widened minor groove. Functional studies of SRY in a cell line from embryonic gonadal ridge demonstrated activation of a gene-regulatory pathway leading to expression of MIS. SRY molecules containing mutations associated with human sex reversal have altered structural interactions with DNA and failed to induce transcription of MIS.

SRY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome, regulates a genetic switch in male development. Impairment of this switch leads to intersex abnormalities of the newborn and is observed in association with mutations in the SRY DNA-binding domain [the high-mobility-group (HMG) box]. Here we show that the SRY HMG box exhibits a novel mechanism of DNA recognition: partial intercalation of a nonpolar side chain in the DNA minor groove. Base stacking (but not base pairing) is interrupted at the site of insertion. Sequence specificity reflects topological requirements of partial intercalation rather than direct readout of base-specific functional groups. Our results predict that the SRY HMG box inserts an alpha-helix into a widened minor groove at the center of a sharp DNA bend. A similar mechanism may underlie binding of SRY and homologous HMG proteins to four-way junctions (Holliday intermediates) and other noncanonical DNA structures.

Formation of male-specific structures and regression of female primordia are regulated in early male embryogenesis by SRY, a single-copy gene on the Y chromosome. Assignment of SRY as the testis-determining factor in eutherian mammals is supported by molecular analysis of cytogenetic sex reversal (i.e., XX males and XY females) and by complementary studies of transgenic murine models. Here we characterize the putative DNA-binding domain of SRY, which contains a conserved sequence motif shared by high-mobility group nuclear proteins and a newly recognized class of transcription factors. The SRY DNA-binding domain specifically recognizes with nanomolar affinity proximal upstream elements (designated SRYe) in the promoters of the sex-specific genes encoding P450 aromatase and Mullerian inhibiting substance (MIS). P450 aromatase catalyzes the conversion of testosterone to estradiol, and in the male embryo its expression is down-regulated. Conversely, MIS is expressed in the male embryo to induce testicular differentiation and regression of female reproductive ducts. SRYe-binding activity is observed in nuclear extracts obtained from embryonic urogenital ridge immediately preceding morphologic testicular differentiation. Our results support the hypothesis that SRY directly controls male development through sequence-specific regulation of target genes.

Vancomycin and cholestyramine have been utilized both alone and in combination for the treatment of antibiotic-associated pseudomembranous colitis. Previous work for the treatment of antibiotic-associated pseudomembranous colitis. Previous work has demonstrated significant binding of vancomycin by the anion-exchange resin. The antibacterial activity of vancomycin was markedly reduced when the suspension was centrifuged and the supernatant was tested for antibacterial activity. This study confirmed these findings but demonstrated that there was no immediate loss of antibacterial activity of bound vancomycin. The degree of inactivation appeared to be dependent upon the duration of incubation of vancomycin and cholestyramine in the testing system.