Showing papers by "Susan Lindquist published in 2009"

Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.

Abstract: Susan Lindquist and colleagues report a genetic interaction between α-synuclein and the ortholog of human ATP13A2 (PARK9) in yeast, two genes that when mutated cause Parkinson's disease. They further show that yeast PARK9 protects cells from manganese toxicity, a known environmental risk factor for Parkinson's disease. Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. α-Synuclein (α-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between α-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by α-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances α-syn misfolding. These data provide a direct functional connection between α-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (α-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of α-syn–induced neuron loss in animals, establishing a diverse, highly conserved interaction network for α-syn.

Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease

Abstract: Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans, Candida albicans, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen Aspergillus terreus, Hsp90 is required for basal resistance to echinocandins, which inhibit β(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth Galleria mellonella, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal C. albicans infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold, Aspergillus fumigatus. In a murine model of disseminated candidiasis, genetic compromise of C. albicans HSP90 expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad-spectrum activity against diverse fungal pathogens.

Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity

Abstract: Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinson's disease For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways

Inhibiting the transcription factor HSF1 as an anticancer strategy.

Abstract: Background: In mammals, the cytoprotective heat-shock response is regulated primarily by heat shock factor 1 (HSF1). Unfortunately, the effects of HSF1 also support the ability of cancer cells to accommodate imbalances in signaling and alterations in DNA, protein and energy metabolism associated with oncogenesis. The malignant lifestyle confers dependence on this ‘non-oncogene’, suggesting a therapeutic role for HSF1 inhibitors. Objective/methods: We begin with an overview of how HSF1 affects cancer biology and how its activity is regulated. We then summarize progress in discovery and development of HSF1 inhibitors, their current limitations and potential as anticancer agents with a fundamentally different scope of action from other clinically validated modulators of protein homeostasis. Results/conclusions: It is likely that within the next 5 years usable inhibitors of HSF1 will be identified and in early pre-clinical evaluation.

A heritable switch in carbon source utilization driven by an unusual yeast prion

Abstract: The stable inheritance of biological information and phenotype across generations is a fundamental property of living systems. Prions, self-perpetuating and heritable protein conformations that cause multiple phenotypes, represent an unusual mechanism of information transfer that occurs via protein instead of nucleic acid (Wickner 1994). Prion proteins can assume at least two conformations and each conformation alters protein function, resulting in different phenotypes (Wickner et al. 2004; Shorter and Lindquist 2005). When in the self-templating or prion conformation, prion proteins acquire characteristics normally restricted to nucleic acids. The first prion protein identified, the mammalian protein PrP, can behave as a transmissible pathogen and causes a neurodegenerative disease in its prion form (PrPSc) (Prusiner 1998). Prion proteins in fungi, which are functionally unrelated to PrP and to each other, act as non-Mendelian elements of inheritance by switching to the self-perpetuating, cytoplasmically transmissible prion conformation (Wickner 1994). Four prions have been extensively characterized in fungi: [PSI+], [URE3], [Het-s], and [RNQ+]. [PSI+] (Cox 1965) is the prion form of the translation termination factor Sup35, which causes nonsense suppression (Stansfield et al. 1995; Patino et al. 1996; Paushkin et al. 1996). [URE3] (Lacroute 1971) is an altered form (Wickner 1994) of the nitrogen catabolite repressor Ure2 (Courchesne and Magasanik 1988). [RNQ+] controls the ability of a cell to acquire other prions (Derkatch et al. 2000, 2001; Sondheimer and Lindquist 2000). [Het-s], found in Podospora anserina, causes heterokaryon incompatibility with certain mating partners (Rizet 1952; Coustou et al. 1997). These four fungal prions, as well as several recently identified prions ([SWI+], [MCA], [OCT+], and [MOT+]) (Du et al. 2008; Alberti et al. 2009; Nemecek et al. 2009; Patel et al. 2009), share key genetic and physical characteristics despite their disparate functions (Chien et al. 2004; Shorter and Lindquist 2005). Their phenotypes appear spontaneously at higher frequencies than those caused by genetic mutations. They are dominant, show non-Mendelian segregation following meiosis, and are also transmissible by cytoduction (cytoplasmic transfer). Physically, they form a self-templating amyloid conformation in the [PRION+] state. Furthermore, their inheritance is linked to the activities of chaperones, proteins that mediate conformational changes in other proteins. Transient changes in chaperone levels, particularly Hsp104, are sufficient to eliminate the prions permanently from cells. This occurs because chaperones alter the prion conformations and transmission to daughter cells. Once the prion template is gone, cells are “cured” of the elements (Uptain and Lindquist 2002; Shorter and Lindquist 2005). Another unusual feature is that transient overexpression of the prion protein causes permanent inheritance of the prion phenotype. This is because the protein∷protein interactions involved in prion formation are more likely to occur at higher protein concentrations (Chernoff et al. 1993; Ter-Avanesyan et al. 1993; Wickner 1994; Serio et al. 2000; Sondheimer and Lindquist 2000; Derkatch et al. 2001; Uptain and Lindquist 2002; Shorter and Lindquist 2005). The yeast prions also share a distinctive feature with mammalian prions: a strong transmission barrier across species. Even subtle differences in amino acid sequence can reduce the ability of prion proteins from one species to convert the homolog from other species, even though the homologous protein is itself capable of forming a prion on its own (Aguzzi et al. 2007; Chen et al. 2007). The precise nature of the mammalian prion template is not known, but all of the well-characterized fungal prions, as well as the newly discovered prions and prion domains (Du et al. 2008; Alberti et al. 2009; Nemecek et al. 2009; Patel et al. 2009), are self-templating amyloid amyloids. The simple and robust character of self-templating amyloids provides a compelling framework for protein-based inheritance (Glover et al. 1997; Shorter and Lindquist 2005). Indeed, in many cases the amyloid has been shown to be the sole determinant needed for prion formation: Recombinant amyloid fibers alone are sufficient to convert [prion−] cells to [PRION+] cells (Maddelein et al. 2002; King and Diaz-Avalos 2004; Tanaka et al. 2004; Brachmann et al. 2005; Patel and Liebman 2007; Alberti et al. 2009). Amyloid structure is therefore commonly held to be a critical feature of all naturally occurring systems for protein-based inheritance. Indeed, a recent genome-wide screen for new prion domains in yeast began by examining proteins likely to be amyloidogenic (Alberti et al. 2009). Here, we took a different approach. We searched the literature for Saccharomyces. cerevisiae phenotypes with prion-like inheritance patterns. One was described many years ago in a screen for cells with an alteration in carbon source utilization (Ball et al. 1976). The basis of the screen was the extreme preference of S. cerevisiae for glucose as a carbon source. In glucose media, cells repress genes necessary to process other carbon sources such as glycerol (Santangelo 2006). Glucosamine, a nonmetabolizable glucose mimetic, induces a similar repression. Therefore, yeast cells cannot use glycerol as a carbon source if even small amounts of glucosamine are present (Hockney and Freeman 1980; Nevado and Heredia 1996). Some cells spontaneously acquire the ability to use glycerol in the presence of glucosamine, presumably due to defects in glucose repression. Some of these exhibit dominant, non-Mendelian inheritance (Ball et al. 1976). Further, the phenotype is neither carried by the mitochondrial genome nor by a plasmid (Kunz and Ball 1977). Employing a variety of methods, we show here that this factor, [GAR+] (for “resistant to glucose-associated repression,” with capital letters indicating dominance and brackets indicating its non-Mendelian character), exhibits all of the genetic characteristics of a yeast prion, and we use a broad range of biochemical and genetic methods to identify proteins that play a key role in [GAR+] inheritance.

Rapid selection of cyclic peptides that reduce alpha-synuclein toxicity in yeast and animal models.

Abstract: Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of alpha-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human alpha-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease.

Amyloid Deposits: Protection Against Toxic Protein Species?

Abstract: Neurodegenerative diseases ranging from Alzheimer disease and polyglutamine diseases to transmissible spongiform encephalopathies are associated with the aggregation and accumulation of misfolded proteins. In several cases the intracellular and extracellular protein deposits contain a fibrillar protein species called amyloid. However while amyloid deposits are hallmarks of numerous neurodegenerative diseases, their actual role in disease progression remains unclear. Especially perplexing is the often poor correlation between these deposits and other markers of neurodegeneration. As a result the question remains whether amyloid deposits are the disease-causing species, the consequence of cellular disease pathology or even the result of a protective cellular response to misfolded protein species. Here we highlight studies that suggest that accumulation and sequestration of misfolded protein in amyloid inclusion bodies and plaques can serve a protective function. Furthermore, we discuss how exceeding the cellular capacity for protective deposition of misfolded proteins may contribute to the formation of toxic protein species.

BETASCAN: Probable β-amyloids Identified by Pairwise Probabilistic Analysis

Abstract: Amyloids and prion proteins are clinically and biologically important beta-structures, whose supersecondary structures are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Recent work has indicated the utility of pairwise probabilistic statistics in beta-structure prediction. We develop here a new strategy for beta-structure prediction, emphasizing the determination of beta-strands and pairs of beta-strands as fundamental units of beta-structure. Our program, BETASCAN, calculates likelihood scores for potential beta-strands and strand-pairs based on correlations observed in parallel beta-sheets. The program then determines the strands and pairs with the greatest local likelihood for all of the sequence's potential beta-structures. BETASCAN suggests multiple alternate folding patterns and assigns relative a priori probabilities based solely on amino acid sequence, probability tables, and pre-chosen parameters. The algorithm compares favorably with the results of previous algorithms (BETAPRO, PASTA, SALSA, TANGO, and Zyggregator) in beta-structure prediction and amyloid propensity prediction. Accurate prediction is demonstrated for experimentally determined amyloid beta-structures, for a set of known beta-aggregates, and for the parallel beta-strands of beta-helices, amyloid-like globular proteins. BETASCAN is able both to detect beta-strands with higher sensitivity and to detect the edges of beta-strands in a richly beta-like sequence. For two proteins (Abeta and Het-s), there exist multiple sets of experimental data implying contradictory structures; BETASCAN is able to detect each competing structure as a potential structure variant. The ability to correlate multiple alternate beta-structures to experiment opens the possibility of computational investigation of prion strains and structural heterogeneity of amyloid. BETASCAN is publicly accessible on the Web at http://betascan.csail.mit.edu.

Loss of Hsp70 Exacerbates Pathogenesis But Not Levels of Fibrillar Aggregates in a Mouse Model of Huntington's Disease

Abstract: Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.

Motor Mechanism for Protein Threading through Hsp104

Abstract: Summary The protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 and NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATPγS or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring.

Unraveling Infectious Structures, Strain Variants and Species Barriers for the Yeast Prion [PSI+]

Abstract: Prions are proteins that can access multiple conformations, at least one of which is β-sheet rich, infectious and self-perpetuating in nature. These infectious proteins show several remarkable biological activities, including the ability to form multiple infectious prion conformations, also known as strains or variants, encoding unique biological phenotypes, and to establish and overcome prion species (transmission) barriers. In this Perspective, we highlight recent studies of the yeast prion [PSI+], using various biochemical and structural methods, that have begun to illuminate the molecular mechanisms by which self-perpetuating prions encipher such biological activities. We also discuss several aspects of prion conformational change and structure that remain either unknown or controversial, and we propose approaches to accelerate the understanding of these enigmatic, infectious conformers.

Protein folding sculpting evolutionary change.

Abstract: Our work suggests that the forces that govern protein folding exert a profound effect on how genotypes are translated into phenotypes and that this in turn has strong effects on evolutionary processes. Molecular chaperones, also known as "heat-shock proteins" (Hsps), promote the correct folding and maturation of many other proteins in the cell. Hsp90 is an abundant and highly specialized chaperone that works on a particularly interesting group of client proteins: metastable signal transducers that are key regulators of a broad spectrum of biological processes. Such proteins often have evolved to finish folding only when they have received a specific signal, such as the binding of a ligand or a posttranslational modification. Importantly, the folding of Hsp90 clients is particularly sensitive to changes in the external and internal environment of the cell. Therefore, Hsp90 is uniquely positioned to couple environmental contingencies to the evolution of new traits. Our work has helped to define two mechanisms by which Hsp90 might influence the acquisition of new phenotypes. First, by robustly maintaining signaling pathways, Hsp90 can buffer the effects of mutations in those pathways, allowing the storage of cryptic genetic variation that is released by stress. In this case, when the Hsp90 buffer is compromised by environmental stress, new traits appear. These traits can also be assimilated, so that they become manifest even in the absence of stress, when genetic recombination and selection enrich causative variants in subsequent generations. Second, Hsp90 can potentiate the effects of genetic variation, allowing new mutations to produce immediate phenotypes. In this case, when Hsp90 function is compromised, new traits are lost. These traits can also be assimilated, so that they are maintained under environmental stress, but this is achieved through new mutations. We have discovered these powerful evolutionary mechanisms in fruit flies, mustard plants, and fungi, but expect them to operate in all eukaryotes. Another line of work relating protein folding to the evolution of new traits involves protein-based hereditary elements known as prions. These produce changes in phenotype through heritable, self-perpetuating changes in protein conformation. Because changes in protein homeostasis occur with environmental stress, prions can be cured or induced by stress, creating heritable new phenotypes that depend on the genetic variation present in the organism. Both prions and Hsp90 provide plausible mechanisms for allowing genetic diversity and fluctuating environments to fuel the pace of evolutionary change. The multiple mechanisms by which protein folding can influence the evolution of new traits provide both a new paradigm for understanding rapid, stepwise evolution and a framework for targeted therapeutic interventions.

BETASCAN: Probable beta-amyloids Identified by Pairwise Probabilistic Analysis

Abstract: Amyloids and prion proteins are clinically and biologically important b-structures, whose supersecondary structures are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Recent work has indicated the utility of pairwise probabilistic statistics in b-structure prediction. We develop here a new strategy for b-structure prediction, emphasizing the determination of b-strands and pairs of b-strands as fundamental units of b-structure. Our program, BETASCAN, calculates likelihood scores for potential b-strands and strand-pairs based on correlations observed in parallel b-sheets. The program then determines the strands and pairs with the greatest local likelihood for all of the sequence’s potential b-structures. BETASCAN suggests multiple alternate folding patterns and assigns relative a priori probabilities based solely on amino acid sequence, probability tables, and pre-chosen parameters. The algorithm compares favorably with the results of previous algorithms (BETAPRO, PASTA, SALSA, TANGO, and Zyggregator) in b-structure prediction and amyloid propensity prediction. Accurate prediction is demonstrated for experimentally determined amyloid b-structures, for a set of known b-aggregates, and for the parallel b-strands of b-helices, amyloid-like globular proteins. BETASCAN is able both to detect b-strands with higher sensitivity and to detect the edges of b-strands in a richly b-like sequence. For two proteins (Ab and Het-s), there exist multiple sets of experimental data implying contradictory structures; BETASCAN is able to detect each competing structure as a potential structure variant. The ability to correlate multiple alternate b-structures to experiment opens the possibility of computational investigation of prion strains and structural heterogeneity of amyloid. BETASCAN is publicly accessible on the Web at http://betascan.csail.mit.edu. Citation: Bryan AW Jr, Menke M, Cowen LJ, Lindquist SL, Berger B (2009) BETASCAN: Probable b-amyloids Identified by Pairwise Probabilistic Analysis. PLoS Comput Biol 5(3): e1000333. doi:10.1371/journal.pcbi.1000333 Editor: Roland Dunbrack, Fox Chase Cancer Center, United States of America Received June 24, 2008; Accepted February 12, 2009; Published March 27, 2009 Copyright: 2009 Bryan, Jr. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NSF Award #(ASE+NHS)(dms)0428715 and NIH grants #U54-LM008748, #R01-GM25874, and #R01-GM080330. No financial source had any role in experiment design, conduct, data collection, analysis, or interpretation, or in the preparation, review, or approval of this manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lindquist_admin@wi.mit.edu (SLL); bab@mit.edu (BB)

context dependent neuroprotective properties of prion protein (PrP)

Abstract: Although it has been known for more than twenty years that an aberrant conformation of the prion protein (PrP) is the causative agent in prion diseases, the role of PrP in normal biology is undetermined. Numerous studies have suggested a protective function for PrP, including protection from ischemic and excitotoxic lesions and several apoptotic insults. On the other hand, many observations have suggested the contrary, linking changes in PrP localization or domain structure--independent of infectious prion conformation--to severe neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor for toxic oligomeric species of a-beta, a pathogenic fragment of the amyloid precursor protein, and likely contributes to disease pathogenesis of Alzheimer disease. We sought to access the role of PrP in diverse neurological disorders. First, we confirmed that PrP confers protection against ischemic damage using an acute stroke model, a well characterized association. After ischemic insult, PrP knockouts had dramatically increased infarct volumes and decreased behavioral performance compared to controls. To examine the potential of PrP's neuroprotective or neurotoxic properties in the context of other pathologies, we deleted PrP from several transgenic models of neurodegenerative disease. Deletion of PrP did not substantially alter the disease phenotypes of mouse models of Parkinson disease or tauopathy. Deletion of PrP in one of two Huntington disease models tested, R6/2, modestly slowed motor deterioration as measured on an accelerating rotarod but otherwise did not alter other major features of the disease. Finally, transgenic overexpression of PrP did not exacerbate the Huntington motor phenotype. These results suggest that PrP has a context-dependent neuroprotective function and does not broadly contribute to the disease models tested herein.

The Schizosaccharomyces pombe Hsp104 disaggregase is unable to propagate the [PSI] prion.

Abstract: The molecular chaperone Hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. Under stress conditions, the disaggregase activity of Hsp104 facilitates the reactivation of misfolded proteins. Hsp104 is also involved in the propagation of fungal prions. For instance, the well-characterized [PSI+] prion of Saccharomyces cerevisiae does not propagate in Δhsp104 cells or in cells overexpressing Hsp104. In this study, we characterized the functional homolog of Hsp104 from Schizosaccharomyces pombe (Sp_Hsp104). As its S. cerevisiae counterpart, Sp_hsp104+ is heat-inducible and required for thermotolerance in S. pombe. Sp_Hsp104 displays low disaggregase activity and cannot propagate the [PSI+] prion in S. cerevisiae. When overexpressed in S. cerevisiae, Sp_Hsp104 confers thermotolerance to Δhsp104 cells and reactivates heat-aggregated proteins. However, overexpression of Sp_Hsp104 does not propagate nor eliminate [PSI+]. Strikingly, [PSI+] was cured by overexpression of a chimeric chaperone bearing the C-terminal domain (CTD) of the S. cerevisiae Hsp104 protein. Our study demonstrates that the ability to untangle aggregated proteins is conserved between the S. pombe and S. cerevisiae Hsp104 homologs, and points to a role of the CTD in the propagation of the S. cerevisiae [PSI+] prion.

Withaferin a analogs and uses thereof

Abstract: The present invention provides a novel class of withanolides that have been isolated from W. somnifera under aeroponic conditions or produced semi-synthetically from withanolide natural products. The invention also provides pharmaceutical compositions thereof and methods for using the same in proliferative diseases, neurodegenerative diseases, autoimmune, and inflammatory diseases.

A nucleolar protein allows viability in the absence of the essential ER-residing molecular chaperone calnexin.

Abstract: In fission yeast, the ER-residing molecular chaperone calnexin is normally essential for viability. However, a specific mutant of calnexin that is devoid of chaperone function (Δhcd_Cnx1p) induces an epigenetic state that allows growth of Schizosaccharomyces pombe without calnexin. This calnexin-independent (Cin) state was previously shown to be mediated via a non-chromosomal element exhibiting some prion-like features. Here, we report the identification of a gene whose overexpression induces the appearance of stable Cin cells. This gene, here named cif1+ for calnexin-independence factor 1, encodes an uncharacterized nucleolar protein. The Cin cells arising from cif1+ overexpression (Cincif1 cells) are genetically and phenotypically distinct from the previously characterized CinΔhcd_cnx1 cells, which spontaneously appear in the presence of the Δhcd_Cnx1p mutant. Moreover, cif1+ is not required for the induction or maintenance of the CinΔhcd_cnx1 state. These observations argue for different pathways of induction and/or maintenance of the state of calnexin independence. Nucleolar localization of Cif1p is required to induce the Cincif1 state, thus suggesting an unexpected interaction between the vital cellular role of calnexin and a function of the nucleolus.

Yeast cells expressing tar dna-binding protein 43 and uses therefor

Abstract: Disclosed are yeast cells expressing TAR DNA-binding protein 43 (TDP-43) and methods of screening yeast cells to identify compounds that prevent or suppress TDP-43 -induced toxicity, compounds that inhibit the formation or maintenance of cytoplasmic inclusions of TDP- 43, genetic suppressors or enhancers of TDP-43 -induced toxicity, and genetic suppressors or enhancers of the formation or maintenance of cytoplasmic inclusions of TDP-43. Compounds identified by such screens can be used to treat or prevent TDP-43 proteinopathies such as frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

Abstract: Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alphasynuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. Cells live in a dynamic environment in which they confront various perturbations such as sudden environmental changes, toxins, and mutations. The response to such perturbations is #To whom correspondence should be addressed. E-mail: lindquist_admin@wi.mit.edu (S. L.); fraenkel-admin@mit.edu (E.F.). 7Present Address: Department of Cell and Developmental Biology, The University of Pennsylvania, Philadelphia, PA, USA 8Present Address: Medical College of Georgia, Augusta, GA, USA 9Present Address: Boston Biomedical Research Institute, Watertown, MA, USA. *These authors contributed equally to this work +These authors contributed equally to this work Summary: A novel approach that integrates genetic hits, differentially expressed genes and known molecular interactions reveals a dramatically enhanced view of cellular responses and was used to create the first cellular map of alpha-synuclein toxicity. NIH Public Access Author Manuscript Nat Genet. Author manuscript; available in PMC 2009 September 1. Published in final edited form as: Nat Genet. 2009 March ; 41(3): 316–323. doi:10.1038/ng.337. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript typically complex and comprises signaling and metabolic changes, as well as changes in gene expression. Revealing the cellular mechanisms responding to a specific perturbation may unravel its nature, thus illuminating disease mechanisms1 or a drug’s mode of action2 ,3, and identify points of intervention with potential therapeutic value4. High-throughput experimental techniques including mRNA profiling and genetic screening are commonly used for revealing components of these response pathways because they provide a genomeand proteome-wide view of molecular changes. mRNA profiling experiments rapidly identify genes that are differentially expressed following stimuli. Genetic screening, including deletion, overexpression and RNAi library screens, identify genetic “hits”, genes whose individual manipulation alters the phenotype of stimulated cells. However, each technique has obvious limitations for revealing the full nature of cellular responses. mRNA profiling experiments do not target the series of events that led to the differential expression. Genetic screens provide strong evidence that a gene is functionally related to the response process. Yet, this relationship is often indirect and hard to decipher, especially in highthroughput experiments that typically result in scores of relevant genes with various functions. It has been noted previously in a few specific instances 2,5–9 that genetic screens do not identify the same genes as mRNA assays conducted in the same conditions. By analyzing the relationship between genetic hits and differentially expressed genes across 179 diverse conditions, we found that this discrepancy is, in fact, a general rule. Furthermore, we found a striking bias in each technique that led us to a new, more coherent view of cellular responses. To bridge the gap between the two forms of high throughput analysis we developed an algorithm that exploits these experimental biases and that takes advantage of molecular interactions data. This approach simultaneously reveals (i) the functional context of genetic hits, and (ii) additional proteins that participate in the response yet were not detected by either the genetic or the mRNA profiling assays themselves. Having validated our approach in a wide array of perturbations, we applied it to unravel cellular responses to increased expression of alpha-synuclein. Alpha-synuclein is a small human protein implicated in Parkinson disease whose native function and role in the etiology of the disease remain unclear 10. We screened an established yeast model for alpha-synuclein toxicity 11,12 using an additional set of 3,500 overexpression yeast strains, exposing the multifaceted toxicity of alpha-synuclein. Application of our approach to the high-throughput genetic and transcriptional data of the yeast model illuminated response pathways whose manipulation altered cellular survival, and provided the first cellular map of the proteins and genes responding to alpha-synuclein expression. The relationship between genetic hits and differentially expressed genes In order to derive a comprehensive view of the relationship between genetic hits and differentially expressed genes identified in a particular condition, we analyzed published mRNA profiles and genetic hits for 179 distinct perturbations in yeast (Methods). These data included responses to a wide array of chemical and genetic insults affecting a multitude of cellular processes. For 30 of these perturbations complete genetic screens were reported, typically identifying >100 genetic hits; only partial genetic data are available for the remaining perturbations. The number of genetic hits, differentially expressed genes and genes common to both for each perturbation are given in Table 1 and Supplementary Table 1. Intriguingly, in almost all cases the overlap was astonishingly small and statistically insignificant (p>0.05, Methods). One possible explanation for the poor overlap between genetic hits and differentially expressed genes is that each assay may be biased toward distinct aspects of cellular responses. Analysis Yeger-Lotem et al. Page 2 Nat Genet. Author manuscript; available in PMC 2009 September 1. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript of Gene Ontology (GO) enrichment confirmed this hypothesis (Methods). The combined hits from all 179 genetic screens were highly enriched for the annotations biological regulation (23.3%, p<10−82), transcription (14%, p<10−44) and signal transduction (6.3%, p<10−30). In contrast, the regulated genes from all perturbations were enriched mostly for various metabolic processes (e.g., organic acid metabolic process 7.1%, p<10−18) and oxidoreductase activities 7.2%, p< 10−34). To ensure these patterns of enrichment do not stem from a handful of data sources but reflect a general tendency, we also analyzed the 30 perturbations for which complete data were available. We found the same enrichment trends, regardless of whether these perturbations were analyzed individually (Supplementary Table 2) or whether all 30 datasets were combined (Supplementary Table 3). Complete enrichment analyses appear in Supplementary Text. Thus, we find that genetic assays tend to probe the regulation of cellular responses, while mRNA profiling assays tend to probe the metabolic aspects of cellular responses. The striking differences in annotations between genetic hits and differentially expressed genes imply that each gene set alone often provides a limited and biased view of cellular responses. In fact, this hypothesis was often borne out in cases where the pathways are well-studied by other, more classical methods of genetic and molecular biological research. In the yeast DNA damage response pathway, for example, a genetic screen 4 detected proteins that sense DNA damage (Mec3, Ddc1, Rad17 and Rad24), while mRNA profiling detected repair enzymes such as Rnr4 13. Yet core components of this pathway that had been uncovered by other intense investigations over many years, such as the signal transducers Mec1 and Rad53 and the transcription factor Rfx1, remained undetected by either high-throughput assay. If we are to fully reap the benefits of applying high-throughput methods to new problems and under-explored biological processes, it is essential that we find new routes to connect these data and obtain a true picture of the regulation of cellular responses. Here we provide a novel framework that bridges the gap between genetic and transcriptional data. Based on known pathways such as the response to DNA damage discussed above, we expect that some of the genetic hits, which are enriched for response regulators, will be connected via regulatory pathways to the differentially regulated genes, which are the output of such pathways. Discovering these pathways may uncover additional components of the cellular response to perturbation that are missing from the experimental data (Figure 1). ResponseNet algorithm for identification of response networks The ResponseNet algorithm identifies molecular interaction paths connecting genetic hits and differentially expressed genes that may include hidden components of the cellular response (Figure 1). The yeast Saccharomyces cerevisiae provides a powerful model system for such analysis due to the extensive molecular interactions data now available (Methods and Supplementary Table 4). Taking advantage of these resources we assembled an integrated network model of the yeast interactome that contains protein-prot

Simple Cellular Solutions to Complex Problems

Abstract: We have developed simple cellular models of complex neurodegenerative diseases by over-expressing disease-associated human proteins in yeast. By combining the unique power of yeast genetics with the highly conserved biology of protein homeostasis in all eukaryotes, we use yeast cells as “living test tubes” to investigate the mechanisms of toxicity associated with problems in protein folding, trafficking, and degradation and complement these basic studies with transcriptional analysis and high-throughput screens for toxicity modifiers.