Showing papers by "Susan Lindquist published in 2007"

A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.

Abstract: In the post-genomic era, academic and biotechnological research is increasingly shifting its attention from single proteins to the analysis of complex protein networks. This change in experimental design requires the use of simple and experimentally tractable organisms, such as the unicellular eukaryote Saccharomyces cerevisiae, and a range of new high-throughput techniques. The Gateway system has emerged as a powerful high-throughput cloning method that allows for the in vitro recombination of DNA with high speed, accuracy and reliability. Two Gateway-based libraries of overexpression plasmids containing the entire complement of yeast open reading frames (ORFs) have recently been completed. In order to make use of these powerful resources, we adapted the widely used pRS series of yeast shuttle vectors for use in Gateway-based cloning. The resulting suite of 288 yeast Gateway vectors is based upon the two commonly used GPD and GAL1 promoter expression systems that enable expression of ORFs, either constitutively or under galactose-inducible conditions. In addition, proteins of interest can be fused to a choice of frequently used N- or C-terminal tags, such as EGFP, ECFP, EYFP, Cerulean, monomeric DsRed, HA or TAP. We have made this yeast Gateway vector kit available to the research community via the non-profit Addgene Plasmid Repository (http://www.addgene.org/yeast_gateway).

A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures

Abstract: The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to “burst-phase” intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was ≈43 Å. This increased with denaturant in a noncooperative fashion to ≈63 Å, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion.

The power of automated high-resolution behavior analysis revealed by its application to mouse models of Huntington's and prion diseases

Abstract: Automated analysis of mouse behavior will be vital for elucidating the genetic determinants of behavior, for comprehensive analysis of human disease models, and for assessing the efficacy of various therapeutic strategies and their unexpected side effects. We describe a video-based behavior-recognition technology to analyze home-cage behaviors and demonstrate its power by discovering previously unrecognized features of two already extensively characterized mouse models of neurodegenerative disease. The severe motor abnormalities in Huntington's disease mice manifested in our analysis by decreased hanging, jumping, stretching, and rearing. Surprisingly, behaviors such as resting and grooming were also affected. Unexpectedly, mice with infectious prion disease showed profound increases in activity at disease onset: rearing increased 2.5-fold, walking 10-fold and jumping 30-fold. Strikingly, distinct behaviors were altered specifically during day or night hours. We devised a systems approach for multiple-parameter phenotypic characterization and applied it to defining disease onset robustly and at early time points.

Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity

Abstract: Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their protein-remodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATP-γS unexpectedly unleash activation, disaggregation and unfolding activities independent of cochaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide-binding domains. However, for some substrates, mixtures of ATP and ATP-γS abolish remodeling, whereas for others, ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide 'holding' (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress and enables Hsp104 to control prion inheritance.

Phenotypic diversity and altered environmental plasticity in Arabidopsis thaliana with reduced Hsp90 levels.

Abstract: The molecular chaperone HSP90 aids the maturation of a diverse but select set of metastable protein clients, many of which are key to a variety of signal transduction pathways. HSP90 function has been best investigated in animal and fungal systems, where inhibition of the chaperone has exceptionally diverse effects, ranging from reversing oncogenic transformation to preventing the acquisition of drug resistance. Inhibition of HSP90 in the model plant Arabidopsis thaliana uncovers novel morphologies dependent on normally cryptic genetic variation and increases stochastic variation inherent to developmental processes. The biochemical activity of HSP90 is strictly conserved between animals and plants. However, the substrates and pathways dependent on HSP90 in plants are poorly understood. Progress has been impeded by the necessity of reliance on light-sensitive HSP90 inhibitors due to redundancy in the A. thaliana HSP90 gene family. Here we present phenotypic and genome-wide expression analyses of A. thaliana with constitutively reduced HSP90 levels achieved by RNAi targeting. HSP90 reduction affects a variety of quantitative life-history traits, including flowering time and total seed set, increases morphological diversity, and decreases the developmental stability of repeated characters. Several morphologies are synergistically affected by HSP90 and growth temperature. Genome-wide expression analyses also suggest a central role for HSP90 in the genesis and maintenance of plastic responses. The expression results are substantiated by examination of the response of HSP90-reduced plants to attack by caterpillars of the generalist herbivore Trichoplusia ni. HSP90 reduction potentiates a more robust herbivore defense response. In sum, we propose that HSP90 exerts global effects on the environmental responsiveness of plants to many different stimuli. The comprehensive set of HSP90-reduced lines described here is a vital instrument to further examine the role of HSP90 as a central interface between organism, development, and environment.

The Prion Protein Knockout Mouse: A Phenotype Under Challenge

Abstract: The key pathogenic event in prion disease involves misfolding and aggregation of the cellular prion protein (PrP). Beyond this fundamental observation, the mechanism by which PrP misfolding in neurons leads to injury and death remains enigmatic. Prion toxicity may come about by perverting the normal function of PrP. If so, understanding the normal function of PrP may help to elucidate the molecular mechansim of prion disease. Ablation of the Prnp gene, which encodes PrP, was instrumental for determining that the continuous production of PrP is essential for replicating prion infectivity. Since the structure of PrP has not provided any hints to its possible function, and there is no obvious phenotype in PrP KO mice, studies of PrP function have often relied on intuition and serendipity. Here, we enumerate the multitude of phenotypes described in PrP deficient mice, many of which manifest themselves only upon physiological challenge. We discuss the pleiotropic phenotypes of PrP deficient mice in relation to the possible normal function of PrP. The critical question remains open: which of these phenotypes are primary effects of PrP deletion and what do they tell us about the function of PrP?

Prion recognition elements govern nucleation, strain specificity and species barriers

Abstract: Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and establish and overcome transmission barriers between species are poorly understood. We exploit surface-bound peptides to overcome complexities of investigating such problems in solution. For the yeast prion Sup35, we find that the switch to the prion state is controlled with exquisite specificity by small elements of primary sequence. Strikingly, these same sequence elements govern the formation of distinct self-perpetuating conformations (prion strains) and determine species-specific seeding activities. A Sup35 chimaera that traverses the transmission barrier between two yeast species possesses the critical sequence elements from both. Using this chimaera, we show that the influence of environment and mutations on the formation of species-specific strains is driven by selective recognition of either sequence element. Thus, critical aspects of prion conversion are enciphered by subtle differences between small, highly specific recognition elements.

Inhibition of alpha-synuclein toxicity

Abstract: Compounds and compositions are provided for treatment or amelioration of one or more symptoms of α-synuclein toxicity, α-synuclein mediated diseases or diseases in which α-synuclein fibrils are a symptom or cause of the disease.

Prion pathogenesis is independent of caspase-12.

Abstract: The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer's disease and many others. The ER stress response and upregulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.

Diminishing apoptosis by deletion of Bax or overexpression of Bcl-2 does not protect against infectious prion toxicity in vivo

Abstract: B-cell lymphoma protein 2 (Bcl-2) and Bcl-2-associated X protein (Bax), key antiapoptotic and proapoptotic proteins, respectively, have important roles in acute and chronic models of neurologic disease. Several studies have implicated Bax and Bcl-2 in mediating neurotoxicity in prion diseases. To determine whether diminishing apoptotic cell death is protective in an infectious prion disease model we inoculated mice that either were null for proapoptotic Bax or overexpressed antiapoptotic Bcl-2. Interestingly, genetic manipulation of apoptosis did not lessen the clinical severity of disease. Moreover, some disease parameters, such as behavioral alterations and death, occurred slightly earlier in mice that are null for Bax or overexpress Bcl-2. These results suggest that Bax and Bcl-2 mediated apoptotic pathways are not the major contributing factor to the clinical or pathological features of infectious prion disease.

Alternative assembly pathways of the amyloidogenic yeast prion determinant Sup35–NM

Abstract: The self-perpetuating conformational change of the translation termination factor Sup35 is associated with a prion phenomenon of Saccharomyces cerevisiae. In vitro, the prion-determining region (NM) of Sup35 assembles into amyloid-like fibres through a mechanism of nucleated conformational conversion. Here, we describe an alternative assembly pathway of NM that produces filaments that are composed of β-strands and random coiled regions with several-fold smaller diameters than the amyloid fibres. NM filaments are not detectable with either thioflavin T or Congo Red and do not show SDS or protease resistance. As filaments do not self-convert into fibres and do not act as seed, they are not intermediates of amyloid fibre formation. Instead, they represent a stable off-pathway form. Similar to mammalian prion proteins, Sup35 contains oligopeptide repeats located in the NM region. We found that the number of repeats determines the partitioning of the protein between filaments and amyloid-like fibres. Low numbers of repeats favour the formation of the filamentous structure, whereas high numbers of repeats favour the formation of amyloid-like fibres.

Illuminating aggregate heterogeneity in neurodegenerative disease.

Abstract: Luminescent conjugated polymers (LCPs) bind to prion aggregates and emit different fluorescent spectra depending on their binding conformation. As such, they are promising tools for investigating the biophysical basis of prion strains.

A Phenotype Under Challenge

Abstract: The key pathogenic event in prion disease involves misfolding and aggregation of the cellular prion protein (PrP). Beyond this fundamental observation, the mechanism by which PrP misfolding in neurons leads to injury and death remains enigmatic. Prion toxicity may come about by perverting the normal function of PrP. If so, understanding the normal function of PrP may help to elucidate the molecular mechansim of prion disease. Ablation of the Prnp gene, which encodes PrP, was instrumental for determining that the continuous production of PrP is essential for replicating prion infectivity. Since the structure of PrP has not provided any hints to its possible function, and there is no obvious phenotype in PrP KO mice, studies of PrP function have often relied on intuition and serendipity. Here, we enumerate the multitude of phenotypes described in PrP deficient mice, many of which manifest themselves only upon physiological challenge. We discuss the pleiotropic phenotypes of PrP deficient mice in relation to the possible normal function of PrP. The critical question remains open: which of these phenotypes are primary effects of PrP deletion and what do they tell us about the function of PrP?

Protein aggregation domains from prions and methods of use thereof

Abstract: Using the Sup35 prion proteins of two distantly related yeast species, it is established that prion replication is initiated by small elements of primary sequence, which can be identified using arrays of short peptides. Subtle differences in replication elements govern the formation of distinct aggregate conformations (prion strains) and also determine their species-specific seeding activities. A Sup35 chimera that promiscuously forms prions in more than one species does so by virtue of carrying the replication element of each species. Mutations or conditions that cause the chimera to assemble into distinct prion strains favor recognition of distinct replication elements. Therefore, subtle differences in small sequences that constitute prion replication elements encode important determinants of prion propagation and transmission. The protein aggregation domains, methods for identification thereof, and polypeptides and higher order aggregates including the protein interaction domains, as well as arrays including peptides derived from an aggregation-prone polypeptide are provided.

Protein Aggregation Domains and Methods of Use Thereof

Abstract: Using the Sup35 prion proteins of two distantly related yeast species, it is established that prion replication is initiated by small elements of primary sequence, which can be identified using arrays of short peptides. Subtle differences in replication elements govern the formation of distinct aggregate conformations (prion strains) and also determine their species-specific seeding activities. A Sup35 chimera that promiscuously forms prions in more than one species does so by virtue of carrying the replication element of each species. Mutations or conditions that cause the chimera to assemble into distinct prion strains favor recognition of distinct replication elements. Therefore, subtle differences in small sequences that constitute prion replication elements encode important determinants of prion propagation and transmission. The protein aggregation domains, methods for identification thereof, and polypeptides and higher order aggregates including the protein interaction domains, as well as arrays including peptides derived from an aggregation-prone polypeptide are provided.