Susan L. Lindquist - US grants

This proposal has 4 major objectives, all focused on the heat shock response: 1) When the cells or tissues of Drosophila melanogaster are exposed to elevated temperatures, they respond by inhibiting the synthesis of normal cellular proteins and inducing the synthesis of a small number of heat shock proteins. This transition represents the cleanest and most dramatic example of selective translation yet described. Using a mixture of heat shock and control cell messenger RNAs and an in vitro translation assay system, we will fractionate heat shock cells in an attempt to identify the factors involved in regulating this transition. 2) We wish to investigate the changing RNA metabolism in yeast cells during heat shock. Using standard pulse labeling and nucleic acid hybridization techniques, we hope to obtain an accurate picture of the balance between message synthesis and degradation. 3) We wish to obtain antibodies to heat shock proteins, and use them to investigate the intracellular location and function of these proteins in Drosophila and other eukaryotes through indirect immunofluorescence. 4) We have recently obtained evidence that heat shock proteins are transported to the nucleus by some special mechanism. We hope to elucidate the processes involved in this transport.

Both eukaryotic and prokaryotic cells respond to mild temperature elevation (and many other forms of stress) by inducing the synthesis of a small group of highly conserved proteins called the heat-shock proteins (HSPs). A good deal of indirect evidence suggests that these proteins provide protection from the toxic effects of heat, but little is known about their specific molecular functions. Recent work has shown that some of these proteins are induced during the normal course of development, suggesting they may also play important roles in normal cellular processes. We wish to investigate the heat-shock response of the yeast, Saccharomyces cerevisiae, because this organism offers so many unique advantages for genetic manipulation. We are in the process of creating mutations in several of the yeast heat-shock genes and producing antibodies specific for the proteins. These mutants and antibodies will be used as tools to study the function and regulation of the proteins during normal growth, sporulation and exposure to stress.

The cells of an organisms respond to mild temperature elevation (and many other forms of stress) by synthesizing a small group of highly conserved proteins called the heat-shock proteins (hsps). A good deal of indirect evidence suggests that these proteins provide protection from the toxic effects of heat, but little is known about their specific molecular functions. Recent work has shown that some of these proteins are induced during the normal course of development, a finding that suggests they may play important roles in normal cellular processes, as well as during exposure to stress. The experiments outlined in this proposal focus on the heat- shock response of the fruit fly, Drosophila melanogester. They address two major issues: what are the functions of the hsps, and how is their synthesis regulated? Specifically, we seek answers to the following: What features of the heat shock genes (in addition to the heat shock consensus elements) are required for proper transcriptional regulation? What is the effect of heat and of heat shock proteins on RNA processing? Which heat-shock proteins are involved in regulating the response and in conferring upon the cell resistance to high temperatures? With what molecules do hsps associate, and how do their associations relate to its cellular functions?

Both eukaryotic and prokaryotic cells respond to mild temperature elevation (and many other forms of stress) by inducing the synthesis of a small group of highly conserved proteins called the heat-shock proteins (HSPs). A good deal of indirect evidence suggests that these proteins provide protection from the toxic effects of heat, but little is known about their specific molecular functions. Recent work has shown that some of these proteins are induced during the normal course of development, suggesting they may also play important roles in normal cellular processes. We wish to investigate the heat-shock response of the yeast, Saccharomyces cerevisiae, because this organism offers so many unique advantages for genetic manipulation. We are in the process of creating mutations in several of the yeast heat-shock genes and producing antibodies specific for the proteins. These mutants and antibodies will be used as tools to study the function and regulation of the proteins during normal growth, sporulation and exposure to stress.

Heat-shock proteins are induced by high temperatures and many toxic agents. They play important roles in maintaining cell viability under stressful environmental conditions. Many of the proteins also play vital roles at normal temperatures, helping other proteins to fold, associate into complexes, and arrive at their proper destinations. This proposal focuses on the highly conserved hsp9O family of proteins, which are found in virtually all plant, animal, and microbial cells. A great deal of biochemical analysis in vertebrate systems has established that the proteins in the hsp9O family associate with a diverse array of other proteins, including steroid hormone receptors, a variety of kinases (most notably the ongogenic tyrosine kinases), actin, and tubulin. Genetically, the functions of the proteins are best characterized in the yeast, Saccharomyces cerevisiae. The genome of this organism encodes two closely related proteins in this family. Together, the proteins constitute an essential gene pair. Cells can live without either protein, but die in the absence of both. The goal of this proposal is to employ the powerful methods of genetic analysis in yeast to analyze the molecular functions of hsp9O proteins. A major focus is to.import previously characterized vertebrate proteins into yeast cells and determine how their functions are affected by associations with hsp9O. The proteins of most immediate interest are the steroid hormone receptors and the oncogenic tyrosine kinases. Other aims include identifying natural yeast targets for hsp9O and determining the functional significance of accessory proteins that are characteristically found in hsp9O protein complexes.

molecular chaperones; prions; gene expression; conformation; stress proteins; spongiform encephalopathy; fungal genetics; gene mutation; immunofluorescence technique; molecular cloning; genetic manipulation; yeasts;

DESCRIPTION (provided by applicant) Our limited knowledge of the mechanisms underlying PD demands the clevelopment of innovative avenues of investigation. The extensive characterization of the budding yeast Saccharomyces cerevisiae and the many tools developed for its manipulation makes it a powerful system for studying complex biological problems. Alpha-synuclein (aS), one of the established key players in PD, will be the focus of our Study. Preliminary data suggest that many of the basic features of aS biology in mammalian neurons is recapitulated in yeast. The proposed studies should generate a wealth of pilot information pertaining both to normal aS biology and potential therapeutic strategies to synergize with studies in other systems. We will combine our own expertise in yeast genetics and cell biology with the expertise of a group of investigators, ranging from biochemists, to geneticists and neurobiologists.

Changes in protein conformation and assembly underlie most processes in cell biology; correspondingly, defects in conformation and assembly are responsible for many human illnesses, including neurodegenerative diseases. Among the most common of these diseases is Parkinson's disease (PD) with a prevalence of approximately 2% after age 65. One of the hallmarks of PD is the presence of cytoplasmic inclusions in neuronal cells, which mainly contain the protein alpha-synuclein (aSyn). The presence of these inclusions of misfolded aSyn is associated with cell death, but the mechanism for toxicity and indeed the function of aSyn remain elusive. We have developed a system to investigate the biological function of aSyn and the toxicity related to its misfolding in the yeast (Saccharomyces cerevisiae). The behavior of aSyn in yeast cells correlates remarkably well with its behavior in mammalian cells. Our goal is to take advantage of the many genetic and molecular techniques available in yeast to investigate the normal and toxic functions of aSyn. We will 1) investigate the cellular quality-control mechanisms influencing aSyn toxicity, 2) identify other modifiers of aSyn toxicity by high throughput screening and 3) determine how factors that modify aSyn toxicity affect a panel of cell biological and biochemical assays we have developed to enhance our understanding of normal and abnormal sSyn biology. Throughout these studies we will collaborate closely with our colleagues at the MGH/MIT Morris Udall Center of Excellence in PD Research to ensure that our results are rapidly assessed in mammalian systems, and reciprocally that the power of yeast genetic and cell biological assays can be employed to facilitate our understanding of any candidate proteins found in the mammalian systems. Our ultimate aim is the development of new strategies for the prevention and treatment of Parkinson's disease.

Changes in protein conformation and assembly underlie most processes in cell biology; correspondingly, defects in conformation and assembly are responsible for many human illnesses, including neurodegenerative diseases. Among the most common of these diseases is Parkinson's disease (PD) with a prevalence of approximately 2% after age 65. One of the hallmarks of PD is the presence of cytoplasmic inclusions in neuronal cells, which mainly contain the protein alpha-synuclein (aSyn). The presence of these inclusions of misfolded aSyn is associated with cell death, but the mechanism for toxicity and indeed the function of aSyn remain elusive. We have developed a system to investigate the biological function of aSyn and the toxicity related to its misfolding in the yeast (Saccharomyces cerevisiae). The behavior of aSyn in yeast cells correlates remarkably well with its behavior in mammalian cells. Our goal is to take advantage of the many genetic and molecular techniques available in yeast to investigate the normal and toxic functions of aSyn. We will 1) investigate the cellular quality-control mechanisms influencing aSyn toxicity, 2) identify other modifiers of aSyn toxicity by high throughput screening and 3) determine how factors that modify aSyn toxicity affect a panel of cell biological and biochemical assays we have developed to enhance our understanding of normal and abnormal sSyn biology. Throughout these studies we will collaborate closely with our colleagues at the MGH/MIT Morris Udall Center of Excellence in PD Research to ensure that our results are rapidly assessed in mammalian systems, and reciprocally that the power of yeast genetic and cell biological assays can be employed to facilitate our understanding of any candidate proteins found in the mammalian systems. Our ultimate aim is the development of new strategies for the prevention and treatment of Parkinson's disease.

DESCRIPTION (provided by applicant): Our laboratory has developed models of human disease in yeast that have uncovered specific cellular defects underlying hereditary and sporadic protein misfolding diseases. We wish to make use of these models for the identification of molecules with therapeutic potential. Cyclic peptides are a proven class of cellular effector that is largely unexplored as a source of molecular therapeutics. Recently, we have developed a novel high throughput method of screening libraries of small, genetically encoded cyclic peptides for molecules that prevent cell death in our yeast models. The genetic encoding of the cyclic peptides allows orders of magnitude more compounds to be screened faster, at lower cost, and with less need for mechanization than with existing procedures. We have applied this method to our model of Parkinson's disease, in which overexpressed human alpha-synuclein aggregates and causes toxicity in yeast. Two promising hits from a library of octamer cyclic peptides have resulted. In this proposal, we seek initially to identify more hits by constructing and screening libraries of cyclic peptides of several different ring sizes. We will characterize the molecular mechanisms of the hits that result by examining their effects on alpha-synuclein aggregation and the structure of cellular organelles, and by determining their protein targets. We will also test representatives of each class of hit in worm and rat models of Parkinson's disease, and begin to optimize their activity through SAR analysis. The rapid selection procedure made possible by genetically encoding cyclic peptides, and the inherent advantages of the cyclic peptide scaffold make this a powerful method for identifying new compounds that can lead to therapeutics for protein misfolding diseases. Parkinson's Disease is a devastating neurodegenerative disorder that affects more than 2% of Americans over the age of 65, making the need acute to accelerate ways of finding drugs to treat it. We have recreated much of the disease in yeast, a very small organism that grows much faster and is much more manipulable than any of the model systems now in use. These significant advantages will allow us to search through many more potential drugs and do it much faster than is now possible, as well as to examine types of potentially promising drugs that so far have received little attention.

DESCRIPTION (provided by applicant): Acquired drug resistance by medically relevant microorganisms poses a grave threat to human health and has enormous economic consequences. Fungal pathogens pose a particular challenge because they are more closely related to humans than bacteria and share many of the same mechanisms at the molecular level that support the growth and survival of the cells comprising their human hosts. The number of drug classes that have distinct targets in fungi is very limited and the usefulness of current antifungal drugs is compromised owing to either significant toxicity for the patient receiving them or the frequent emergence of high grade resistance. The objective of this project is to discover new chemical compounds capable of reversing fungal drug resistance, thereby illuminating the mechanisms responsible for drug resistance in disease-causing fungi and making currently available antifungal safer and more effective. To achieve this ambitious goal, a research plan designed to achieve the following specific aims will be pursued in collaboration with a designated center within the National Institutes of Health (NIH) Molecular Libraries Probe Production Center Network (MLPCN): Aim 1: Optimize and then execute a high throughput robotic screen of hundreds of thousands of individual chemicals to find compounds that can reverse the resistance of a fungal strain that was isolated from a patient receiving the very commonly used antifungal drug fluconazole Aim 2: Evaluate the compounds identified in the primary screen by measuring their potency, their spectrum of activity against various types of fungus, their selectivity for human versus fungal cells and determine the general way in which they reverse antifungal drug resistance Aim 3: Select the 10 most promising compounds and synthesize a panel of derivatives for each one to optimize their antifungal potency and specificity. This project will combine our long established expertise in studying the molecular biology of fungi using genetic and biochemical techniques with the outstanding resources of an MLPCN center and its expertise in high throughput screening technology and medicinal chemistry. In collaboration with the Broad Institute which was recently designated an MLPCN center, the essential primary screening assay has already been developed and validated. As a result, the deliverable outcome from this brief one year project is expected to be several highly useful chemical compounds with which to probe fungal biology. These probes will be invaluable to us and others for studying the mechanisms underlying fungal drug resistance. In addition to their basic research applications, their therapeutic relevance can be readily evaluated using established animal models in future work. By virtue of the way in which they will be discovered, many of these compounds are likely to act in previously unknown and unexploited ways that could prove uniquely effective in addressing the ever increasing problem of acquired drug resistance by disease-causing microorganisms that confronts our society. PUBLIC HEALTH RELEVANCE: Acquired drug resistance by disease-causing microorganisms poses an escalating threat to human health and imposes an enormous economic burden on our health care system. This project will use state of the art screening technologies to discover chemical compounds that can reverse drug-resistance in human disease- causing fungi. By virtue of the novel way in which they will be discovered, many of these compounds are likely to act in previously unknown and unexploited ways that could prove uniquely effective in tackling the serious problem of acquired antibiotic resistance that confronts our society.

DESCRIPTION (provided by applicant): The Whitehead Institute requests $235,000 for the purchase of an nCounter" system from NanoString Technologies. The nCounter" system is a new approach to gene expression analysis that offers the data quality of Real-Time PCR with higher multiplexing, better throughput and lower cost. The nCounter" system will fill a niche for high throughput gene expression projects requiring mid-range multiplexing (50-500 hundred genes) in hundreds up to thousands of samples. This throughput range is not well served by traditional gene expression technologies like Real-Time PCR or microarrays. The system's unique detection technology also inspires proposals for novel applications where traditional technologies of RT-PCR and microarrays are inadequate. The nCounter" system will be integrated into the technology portfolio of the Whitehead Institute's Genome Technology Core (GTC). The GTC serves 20 labs at the Whitehead Institute and dozens more around the world and has a long history of successfully supporting shared instrumentation as a fee-for-service genome core facility. The nCounter system will complement existing gene expression analysis platforms based on microarrays and quantitative PCR to provide complete solutions for expression analysis projects of all sizes. PROJECT NARRATIVE: The ability to measure levels of gene expression in cells is a critical component of nearly every biomedical research project today. Traditional technologies for gene expression analysis are expensive, time consuming and suffer from a variety of technical limitations. The nCounter(tm) from NanoString is a revolutionary technology that will enable many research projects by providing a cheaper, faster and technically superior option for high throughput expression analysis.

DESCRIPTION (provided by applicant): Our laboratory has used yeast to model the cellular defects caused by the human proteins implicated in neurodegenerative diseases. Furthermore, we have studied the reliance of fungal pathogens on the protein folding machinery to evolve drug resistance. Recently, we have begun to apply the lessons we learned from these two research areas to the investigation of the malaria pathogen Plasmodium falciparum. The genome of P. falciparum is very AT-rich and consequently encodes an unusual amount of asparagine-rich proteins, predicted to be non-globular and of low complexity and thus likely to impose unique demands on the protein folding machinery. Strikingly, P. falciparum also shows a marked expansion of the heat shock protein 40 (Hsp40) family of co-chaperones. During its life cycle in its human host P. falciparum infects and remodels red blood cells. We propose that during this process the parasite relies on a greatly expanded class of Hsp40 co-chaperones. We have developed assays to assess the function of Pf Hsp40s in yeast and we seek to identify small molecules that inhibit the functions of those chaperones. In particular we focus on a Pf Hsp40 that has been shown to be crucial to parasite proliferation. Compounds inhibiting the function of this Hsp40 in yeast will be tested in established parasite survival and host cell remodeling assays to elucidate the role of this Hsp40 in the parasite life cycle. PUBLIC HEALTH RELEVANCE: Malaria afflicts 500 million people a year. The parasite causing malaria appears to rely on a set of proteins called Hsp40 chaperones during its infection. We propose to identify compounds that can interfere with the function of these proteins in the hope of ultimately developing a new class of anti-malarial drugs.

DESCRIPTION (provided by applicant): Our laboratory has used yeast to model the cellular defects caused by the human proteins implicated in neurodegenerative diseases. We propose a high- throughput chemical screen using a yeast strain expressing human A2, a protein linked to Alzheimer's disease. Data from a pilot screen demonstrate that our yeast A2 assay is robust and reproducible. The proposed screen will enable us to probe a larger library at multiple concentrations and cover a broader chemical space. Hit compounds from this screen will be counter-screened against yeast models of other neurodegenerative diseases to determine selectivity, and will be further screened for toxicity in mammalian cells and efficacy in mammalian neurons. The ideal chemical candidates would have activity in both our primary yeast and secondary neuronal toxicity assays and would have a pharmacokinetic profile suitable for studies in transgenic mouse models of AD. These chemical probes will provide a complementary approach to deciphering the tangled mechanisms of toxicity and protection that we have begun to uncover through our genetic screens. PUBLIC HEALTH RELEVANCE: A2 peptide accumulates in the brains of patients with Alzheimer disease and may cause neurodegeneration, but its mechanism of action is not known. We propose to identify compounds that can alleviate the cellular toxicity of A2 and validate these in neuronal models. These probes will be valuable tools for dissecting A2 pathology and could potentially become the focus for future pre- clinical development.

DESCRIPTION (provided by applicant): This grant concerns unusual proteins that have the capacity to change shape and to selectively template that change in shape to other proteins of the same type, a property shared by prions and other amyloid-forming proteins across the evolutionary spectrum. Amyloids have devastating personal and economic costs in an extraordinary variety of settings, many of which have only become clear in recent years. These range from degenerative diseases of our aging population to the production of infectious bacterial biofilms that resist eradication by antibiotics, bacteriophage, and even bleach. This project takes advantage of the remarkable methodological approaches yeast cells offer for the study of difficult problems in protein folding and assembly. It also capitalizes on technological innovations recently developed in our laboratory and others for structural and mechanical investigations of amyloid proteins. The model protein for much of this work is Sup35, a translation termination factor of yeast. It has provided a wealth of information on nucleation and assembly pathways in vitro. Also strains of this prion with defined phenotypic consequences and patterns of inheritance have been intensely investigated in vivo. In recent years dozens of new yeast prions have been discovered by our lab and others. By focusing on this class of proteins, proteins with completely unrelated amino acid sequences and diverse biological functions, we aim to uncover fundamental insights on the assembly of these proteins that will be applicable to a variety of problems that currently cause great suffering and economic hardship. Our aims include i) Obtaining an atomic-level structural understanding of the amyloid fibers formed the prion domain of model prion Sup35 (called NM), using solid-state NMR; (ii) Characterizing the mechanical properties of Sup35 prion fibers using optical trapping and microfluidic techniques; (iii) Employing fluorescence microscopy techniques to monitor the dynamic processes involved in prion replication and inheritance in living cells. This work will provide fundamental new knowledge with potential applications to both normal biology and disease.

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent a significant and increasing burden on society. In particular, Alzheimer's disease and Parkinson's disease are the most common neurodegenerative disorders in the world and affect 5.4 million and >500,000 people in the USA, respectively. In fact, the global cost of Alzheimer's disease and related dementias was estimated in 2010 to be ~1% of the world's gross domestic product (GDP). Despite the knowledge that virtually all neurodegenerative diseases originate with problems in the folding and trafficking of specific proteins in cells, there are simply no effectiv treatments to halt or cure these diseases. This is at least in part due to the lack of a mechanisti understanding of the disease pathology at the cellular level. The research proposed herein uses the budding yeast, Saccharomyces cerevisiae, as a cellular model system. This system has previously been shown to recapitulate unique features of neuronal toxicity observed in human neurodegenerative diseases. Significantly, all previous analyses in both yeast and neuronal models were limited to steady-state bulk measurements, which average over many cells and mask essentially all intracellular dynamics. These measurements cannot reveal important dynamic properties, which require observation of single cells over time. By contrast, proposed herein is a novel approach to systematically measure the global response of single cells to the expression of disease-relevant proteins, such as ?-synuclein (Parkinson's disease) and the A? peptide (Alzheimer's disease) by following the time-dependent changes of biochemical pathways, protein localization and trafficking, organelles, and metabolites in single cells. Biological circuits function inside cells and hence require a single-cell analysis to uncover all their behaviors. The single-cell resolution will be achieved through the development of an innovative microfluidic analysis platform, which permits the imaging of individual cells over time and allows for the quantification of dynamic responses in single cells. Such systematic single-cell measurements will reveal novel mechanisms relevant to cellular toxicity and will contribute to a systems-level understanding of how A? and ?-synuclein poison cellular physiology. Further, this high-throughput imaging platform will be employed to investigate the cell biological effects of ~100 small- molecule compounds that were previously identified through large-scale small-molecule screening as potential drug candidates and are currently being verified for their efficacy in neurons. Discoveries made with the high- throughput imaging platform described herein will later be validated in mammalian neuronal systems. Significantly, the systems-level, single-cell analysis approach described herein has never before been applied to the investigation of neurodegenerative diseases and has the potential to uncover novel insights about intracellular perturbations on a global scale, which is necessary for identifying effective therapeutics to combat these diseases.