Showing papers by "Susan Lindquist published in 2013"

Identification and Rescue of α-Synuclein Toxicity in Parkinson Patient–Derived Neurons

Abstract: The induced pluripotent stem (iPS) cell field holds promise for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to humans to discover and reverse phenotypic responses to α-synuclein (αsyn), a key protein involved in Parkinson’s disease (PD). We generated cortical neurons from iPS cells of patients harboring αsyn mutations, who are at high risk of developing PD dementia. Genetic modifiers from unbiased screens in a yeast model of αsyn toxicity led to identification of early pathogenic phenotypes in patient neurons. These included nitrosative stress, accumulation of endoplasmic reticulum (ER)–associated degradation substrates, and ER stress. A small molecule identified in a yeast screen (NAB2), and the ubiquitin ligase Nedd4 it affects, reversed pathologic phenotypes in these neurons.

Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish.

Abstract: In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting.

Widespread regulation of translation by elongation pausing in heat shock

Abstract: Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation. Heat shocked cells globally inhibit p...

Transgenerational epigenetic inheritance: how important is it?

Abstract: Much attention has been given to the idea of transgenerational epigenetic inheritance, but fundamental questions remain regarding how much takes place and the impact that this might have on organisms. We asked five leading researchers in this area--working on a range of model organisms and in human disease--for their views on these topics. Their responses highlight the mixture of excitement and caution that surrounds transgenerational epigenetic inheritance and the wide gulf between species in terms of our knowledge of the mechanisms that may be involved.

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition

Abstract: Changes to protein interactomes as a result of mutations to the bait protein or addition of a pharmacological inhibitor are robustly monitored with affinity purification coupled with data-independent acquisition–based mass spectrometry and an automated data analysis pipeline. Also in this issue, Collins et al. describe a similar method.

Cryptic Variation in Morphological Evolution: HSP90 as a Capacitor for Loss of Eyes in Cavefish

Abstract: In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting.

Tight coordination of protein translation and HSF1 activation supports the anabolic malignant state.

Abstract: A unifying characteristic of aggressive cancers is a profound anabolic shift in metabolism to enable sustained proliferation and biomass expansion. The ribosome is centrally situated to sense metabolic states but whether it impacts systems that promote cellular survival is unknown. Here, through integrated chemical-genetic analyses, we find that a dominant transcriptional effect of blocking protein translation in cancer cells is complete inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for tumorigenesis. Translational flux through the ribosome reshapes the transcriptional landscape and links the fundamental anabolic processes of protein production and energy metabolism with HSF1 activity. Targeting this link deprives cancer cells of their energy and chaperone armamentarium thereby rendering the malignant phenotype unsustainable.

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition

Abstract: Changes to protein interactomes as a result of mutations to the bait protein or addition of a pharmacological inhibitor are robustly monitored with affinity purification coupled with data-independent acquisition–based mass spectrometry and an automated data analysis pipeline. Also in this issue, Collins et al. describe a similar method.

Yeast reveal a "druggable" Rsp5/Nedd4 network that ameliorates α-synuclein toxicity in neurons.

Abstract: α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson’s disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N -aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum–to-Golgi vesicle trafficking.

Fitness trade-offs restrict the evolution of resistance to amphotericin B.

Abstract: The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.

Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B

Abstract: The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.

Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells.

Abstract: The sensitivity of chaperones to the stability of client proteins is used for kinome-wide profiling of kinase inhibitors.

Profoundly different prion diseases in knock-in mice carrying single PrP codon substitutions associated with human diseases

Abstract: In man, mutations in different regions of the prion protein (PrP) are associated with infectious neurodegenerative diseases that have remarkably different clinical signs and neuropathological lesions. To explore the roots of this phenomenon, we created a knock-in mouse model carrying the mutation associated with one of these diseases [Creutzfeldt–Jakob disease (CJD)] that was exactly analogous to a previous knock-in model of a different prion disease [fatal familial insomnia (FFI)]. Together with the WT parent, this created an allelic series of three lines, each expressing the same protein with a single amino acid difference, and with all native regulatory elements intact. The previously described FFI mice develop neuronal loss and intense reactive gliosis in the thalamus, as seen in humans with FFI. In contrast, CJD mice had the hallmark features of CJD, spongiosis and proteinase K-resistant PrP aggregates, initially developing in the hippocampus and cerebellum but absent from the thalamus. A molecular transmission barrier protected the mice from any infectious prion agents that might have been present in our mouse facility and allowed us to conclude that the diseases occurred spontaneously. Importantly, both models created agents that caused a transmissible neurodegenerative disease in WT mice. We conclude that single codon differences in a single gene in an otherwise normal genome can cause remarkably different neurodegenerative diseases and are sufficient to create distinct protein-based infectious elements.

ML212: A small-molecule probe for investigating fluconazole resistance mechanisms in Candida albicans

Abstract: The National Institutes of Health Molecular Libraries and Probe Production Centers Network (NIH-MLPCN) screened >300,000 compounds to evaluate their ability to restore fluconazole susceptibility in resistant Candida albicans isolates. Additional counter screens were incorporated to remove substances inherently toxic to either mammalian or fungal cells. A substituted indazole possessing the desired bioactivity profile was selected for further development, and initial investigation of structure-activity relationships led to the discovery of ML212.

Indazole derivatives and uses thereof

Abstract: The present invention provides novel compounds (e.g., compounds of Formula (I)), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof. Also provided are methods and kits comprising the inventive compounds, or compositions thereof, for treating and/or preventing a fungal or protozoan infection, inhibiting the activity of a fungal or protozoan enzyme, killing a fungus or protozoon, or inhibiting the growth of a fungus or protozoon. The fungus may be a Candida species, Aspergillus species, or other pathogenic fungal species. The compounds of the invention may inhibit the activity of fungal or protozoan cytochrome b and/or fungal or protozoan Hsp90. The present invention also provides synthetic methods of the inventive compounds.

Hsf1 and hsf1 cancer signature set genes and uses relating thereto

Abstract: In some aspects, the invention relates to Heat Shock Protein-1 (HSF1) gene and HSF1 gene products. In some aspects, the invention provides methods of tumor diagnosis, prognosis, treatment-specific prediction, or treatment selection, the methods comprising assessing the level of HSF1 expression or HSF1 activation in a sample obtained from the tumor. In some aspects, the invention relates to the discovery that increased HSF1 expression and increased HSF1 activation correlate with poor outcome in cancer, e.g., breast cancer. In some aspects, the invention relates to the HSF1 cancer program genes, HSF1 cancer signature set genes, subsets thereof, and uses in tumor diagnosis, prognosis, treatment-specific prediction, treatment selection, or drug discovery, among others.

Compounds for treating infectious diseases

Abstract: The present invention provides compounds of Formula I, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds of the present invention are useful for inhibiting fungal or parasitic growth. The compounds are useful as inhibitors of glycosylphosphatidylinositol (GPI)-anchor biosynthesis, in particular, as inhibitors of fungal Gwt1 activity. The present invention further provides methods of using the compounds described herein for treating fungal or parasitic infections. The compounds can also be used as biological probes to study the effects of inhibiting Gwt1 activity.

Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells

Abstract: Determining the specificity of small molecules is important for research scientists, medicinal chemists, clinicians and their patients alike. In the laboratory, small-molecule drugs are commonly used as chemical probes, and the meaningful interpretation of the results of such experiments requires detailed knowledge of a drug’s targets. In the pharmaceutical industry, target profiling can be used to identify candidate targets for compounds discovered in cell-based screens and to guide medicinal chemistry efforts to obtain a favorable target spectrum during lead compound optimization. In the clinic, experimental drugs often show unexpected efficacy or toxicity that can sometimes be explained through more thorough target profiling. Moreover, identification of additional targets of an approved drug with a previously established safety profile can facilitate its rapid repurposing to new diseases. The protein kinase family illustrates the challenges inherent to target profiling. Almost all protein kinases share the same conserved fold, and, consequently, developing inhibitors that are both highly potent and highly selective has proven difficult1,2. Given the pharmaceutical interest in drugging protein kinases, several high-throughput methods have been developed for profiling inhibitor specificities in vitro3–8. However, the correlation between in vitro results and in vivo efficacy has often been disappointing9,10. Chemical proteomicsbased approaches have shown considerable promise for inhibitor profiling11,12. However, they are not well suited to profiling allosteric inhibitors, which are not competitive with the ATP site–directed labeling agents employed by these methods. Currently, no assays combine the benefits of in vitro assays (high throughput) with those of in vivo approaches (relevant cellular context). We recently developed a quantitative protein-protein interaction assay to survey the association of the HSP90 chaperone and its CDC37 co-chaperone with the majority of human kinases in vivo1. We established that one of the main determinants of HSP90’s association with a particular kinase is the thermal stability of the kinase domain. Here, we exploit this finding and use HSP90 and CDC37 as thermodynamic sensors for profiling small molecule–kinase interactions in living cells. We further demonstrate that this assay is not limited to kinases as targets and HSP90 or CDC37 as sensors, suggesting a more general approach for probing small molecule–target interactions.

Prion-based manipulation of yeast fermentation and growth

Abstract: In some aspects, the invention provides methods of modulating yeast fermentation or growth by modulating the [ GAR+ ] prion. In some embodiments, [ GAR+ ] is induced by contacting yeast cells with a bacterium that produces a small molecule inducer of [ GAR+ ] or by contacting yeast cells with the small molecule or conditioned medium comprising the small molecule or a fraction of such medium that comprises the small molecule. In some aspects, the invention provides methods of identifying a modulator of a prion of interest. In some embodiments a methods of identifying a modulator of a prion comprises (a) providing a bacterially conditioned medium; (b) contacting at least one fraction of the bacterially conditioned medium with a non-bacterial cell; and (c) monitoring the cell for alteration of a phenotype assocatied with a prion of interest. In some embodiments the non-bacterial cell is a yeast cell. In some embodiments the prion is [ GAR+ ].

Erratum: Transgenerational epigenetic inheritance: how important is it?

Abstract: Nature Reviews Genetics 14, 228–235 (2013) In the author list and throughout the article, the name of the author 'Bill Kelly' has been changed to 'William G. Kelly'. The article has been corrected online.

Abstract C132: Targeting heat shock factor 1 improves the antitumor efficiency of hyperthermia.

Abstract: The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1] HSF1 exerts its role by activating a unique transcriptional program in the cancer cells [2] Indeed, increased HSF1 levels, as well as activation of its transcriptional signature, are associated with reduced survival in breast, lung and colon cancer patients [3] Cancer cells are exquisitely dependent on HSF1 for survival Exposure to additional stress, such as heat, further increases their dependency on HSF1 Recently we described how translation is linked to HSF1 activation using a derivative of the natural compound rocaglamide [4] We found that this drug-like inhibitor of translation-initiation inhibits HSF1 and leads to tumor regression in hematopoietic malignancies Here we combine this compound, or genetic inhibition of HSF1 expression, with focal heat therapy delivered via gold nano rods We find that inhibiting HSF1 in solid tumors increases the efficiency of hyperthermia as an anticancer treatment Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C132 Citation Format: Ruth Scherz-Shouval, Alexander F Bagley, Luke Whitesell, Sangeeta N Bhatia, Susan Lindquist Targeting heat shock factor 1 improves the antitumor efficiency of hyperthermia [abstract] In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C132

Environmentally Acquired Traits Epigenetics in the Extreme: Prions and the Inheritance of

Abstract: clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): June 17, 2013 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/330/6004/629.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/330/6004/629.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/330/6004/629.full.html#ref-list-1 , 8 of which can be accessed free: cites 30 articles This article http://www.sciencemag.org/content/330/6004/629.full.html#related-urls 10 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/cell_biol Cell Biology subject collections: This article appears in the following