Showing papers by "Susan Lindquist published in 2002"

Hsp90 as a capacitor of phenotypic variation

Abstract: Heat-shock protein 90 (Hsp90) chaperones the maturation of many regulatory proteins and, in the fruitfly Drosophila melanogaster, buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorily outbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant development is more plastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbred lines, reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation. The strength and breadth of Hsp90's effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normal development from destabilizing effects of stochastic processes. Manipulating Hsp90's buffering capacity offers a tool for harnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events in the determination of phenotype.

Neurotoxicity and Neurodegeneration When PrP Accumulates in the Cytosol

Abstract: Changes in prion protein (PrP) folding are associated with fatal neurodegenerative disorders, but the neurotoxic species is unknown. Like other proteins that traffic through the endoplasmic reticulum, misfolded PrP is retrograde transported to the cytosol for degradation by proteasomes. Accumulation of even small amounts of cytosolic PrP was strongly neurotoxic in cultured cells and transgenic mice. Mice developed normally but acquired severe ataxia, with cerebellar degeneration and gliosis. This establishes a mechanism for converting wild-type PrP to a highly neurotoxic species that is distinct from the self-propagating PrP Sc isoform and suggests a potential common framework for seemingly diverse PrP neurodegenerative disorders.

Conversion of PrP to a self-perpetuating PrPSc-like conformation in the cytosol.

Abstract: A rare conformation of the prion protein, PrPSc, is found only in mammals with transmissible prion diseases and represents either the infectious agent itself or a major component of it. The mechanism for initiating PrPSc formation is unknown. We report that PrP retrogradely transported out of the endoplasmic reticulum produced both amorphous aggregates and a PrPSc-like conformation in the cytosol. The distribution between these forms correlated with the rate of appearance in the cytosol. Once conversion to the PrPSc-like conformation occurred, it was sustained. Thus, PrP has an inherent capacity to promote its own conformational conversion in mammalian cells. These observations might explain the origin of PrPSc.

Prions as protein-based genetic elements.

Abstract: ▪ Abstract Fungal prions are fascinating protein-based genetic elements. They alter cellular phenotypes through self-perpetuating changes in protein conformation and are cytoplasmically partitioned from mother cell to daughter. The four prions of Saccharomyces cerevisiae and Podospora anserina affect diverse biological processes: translational termination, nitrogen regulation, inducibility of other prions, and heterokaryon incompatibility. They share many attributes, including unusual genetic behaviors, that establish criteria to identify new prions. Indeed, other fungal traits that baffled microbiologists meet some of these criteria and might be caused by prions. Recent research has provided notable insight about how prions are induced and propagated and their many biological roles. The ability to become a prion appears to be evolutionarily conserved in two cases. [PSI+] provides a mechanism for genetic variation and phenotypic diversity in response to changing environments. All available evidence sugges...

Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants

Abstract: AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes. AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis. Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state. In vitro, reducing kcat at NBD2 significantly alters the steady-state kinetic behavior of NBD1. Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2. In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function.

Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion [PSI+]

Abstract: The yeast prion [PSI+] provides an epigenetic mechanism for the inheritance of new phenotypes through self-perpetuating changes in protein conformation. [PSI+] is a nonfunctional, ordered aggregate of the translation termination factor Sup35p that influences new Sup35 proteins to adopt the same state. The N-terminal region of Sup35p plays a central role in prion induction and propagation. The C-terminal region provides translation termination activity. The function of the highly charged, conformationally flexible middle region (M) is unknown. An M deletion mutant was capable of existing in either the prion or the nonprion state, but in either case it was mostly insoluble. Substituting a charged synthetic polypeptide for M restored solubility, but the prions formed by this variant were mitotically very unstable. Substituting charged flexible regions from two other proteins for M created variants that acquired prion states (defined as self-perpetuating changes in function transferred to them from wild-type [PSI+] elements), but had profoundly different properties. One was soluble in both the prion and the nonprion form, mitotically stable but meiotically unstable, and cured by guanidine HCl but not by alterations in heat shock protein 104 (Hsp104p). The other could only maintain the prion state in the presence of wild-type protein, producing Mendelian segregation patterns. The unique character of these M variants, all carrying the same N-terminal prion-determining region, demonstrate the importance of M for [PSI+] and suggest that a much wider range of epigenetic phenomena might be based on self-perpetuating, prion-like changes in protein conformation than suggested by our current methods for defining prion states.

Analysis of prion factors in yeast.

Abstract: Publisher Summary This chapter presents an analysis of prion factors in yeast. Prions are unique proteins that can adopt two or more distinctly different conformational states in vivo . The chapter reviews the principal techniques used for genetic, cell biological, and biochemical characterization of yeast prions. It focuses on [ PSI +]; however, [ URE3 ] and composite prions are also discussed for comparison. Many approaches are available to identify and study prion proteins in yeast. These include (1) approaches based on phenotypic changes, such as nonsense suppression or growth, (2) approaches based on genetic criteria, such as non-Mendelian (cytoplasmic) inheritance, curing by certain stress-inducing agents and by chaperone alterations, and induction de novo by the overproduction of the protein determinant, and (3) approaches based on biochemical criteria, such as aggregation and proteinase resistance in vivo and in vitro . Because no approach is sufficient to establish prion-like behavior, a collection of various approaches should be used.

Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding

Abstract: Hsp104 from Saccharomyces cerevisiae is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer. Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2. Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2. To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W). The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2. The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the Kd and Hill coefficient to be determined for each nucleotide. By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2. An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides. In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle.

Yeast screens for treatment of human disease

Abstract: Screening methods for identifying substances that provide therapeutic value for various diseases associated with protein misfolding are provided. Genetic and chemical screening methods are provided using a yeast system. The methods of the invention provide a rapid and cost-effective method to screen for compounds that prevent protein misfolding and/or protein fibril formation and/or protein aggregation which includes numerous neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease as well as non-neuronal diseases such as type 2 diabetes.

Hsp90 as a capacitor of phenotypic

Abstract: Heat-shockprotein90(Hsp90)chaperonesthe maturationof manyregulatory proteinsand,inthe fruitfly Drosophilamelanogaster,buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorilyoutbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant developmentismoreplastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbredlines,reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation.The strength and breadth of Hsp90’s effects on the buffering and release of genetic variation suggests it may have an impact onevolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normaldevelopment from destabilizing effects of stochastic processes. Manipulating Hsp90’s buffering capacity offers a tool forharnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events inthe determination of phenotype.

Self-perpetuating structural states in biology, disease, and genetics.

Abstract: Over the past half-century, the central dogma, in which DNA makes RNA makes protein, has dominated thinking in biology, with continuing refinements in understanding of DNA inheritance, gene expression, and macromolecular interactions. However, we have also witnessed the elucidation of epigenetic phenomena that violate conventional notions of inheritance. Protein-only inheritance involves the transmission of phenotypes by self-perpetuating changes in protein conformation. Proteins that constitute chromatin can also transmit heritable information, for example, via posttranslational modifications of histones. Both the transmission of phenotypes via the formation of protein conformations and the inheritance of chromatin states involve self-perpetuating assemblies of proteins, and there is evidence for some common structural features and conceptual frameworks between them. To foster interactions between researchers in these two fields, the National Academy of Sciences convened an Arthur M. Sackler Colloquium entitled “Self-Perpetuating Structural States in Biology, Disease, and Genetics” in Washington, DC, on March 22–24, 2002. Participants described new phenomenology and provided insights into fundamental mechanisms of protein and chromatin inheritance. Perhaps most surprising to attendees was emerging evidence that these unconventional modes of inheritance may be common. First described in studies of scrapie and other transmissible encephalopathies in mammals, prions were later shown to cause some classical phenotypes in yeast. In each case, an alternative protein conformation leads to formation of structures resembling amyloid fibers seen in human disease. How these are seeded has been elucidated by in vitro studies, leading to a satisfying picture of prion-like protein propagation. Other cases of prion inheritance have been discovered in genetic screens, which suggests that we are seeing only the tip of the iceberg. Indeed, it now appears that amyloid fiber formation is the default state for misfolded proteins, and fibrillar aggregates found in amyloidoses result from defects in the cellular machinery that prevents protein misfolding. Excitement also pervades the chromatin field, with new insights into how nucleosomes specify and maintain distinct chromatin states. Remarkably, a single modification of a histone tail residue underlies the distinction between euchromatin and heterochromatin, and even maintenance of DNA methylation can depend on histone tail modification. From insights such as these, we have begun to realize that the relationship between chromatin conformation and gene expression might have a simple basis. Genetic and biochemical approaches have begun to elucidate how histone-modifying enzymes and nonhistone structural proteins regulate chromatin inheritance. Although these alternate mechanisms of inheritance have shaken our blind faith in the central dogma, they whet our appetite for further revolutionary insights.

Yeast screens for agents affecting protein folding

Abstract: Screening methods for identifying substances that provide therapeutic value for various diseases associated with protein misfolding are provided. Genetic and chemical screening methods are provided using a yeast system. The methods of the invention provide a rapid and cost-effective method to screen for compounds that prevent protein misfolding and/or protein fibril formation and/or protein aggregation which includes numerous neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease as well as non-neuronal diseases such as type 2 diabetes.