Leonard Guarente - US grants

Genetic methods will be employed to elucidate the mechanism of upstream activation of transcription of the CYCl gene of Saccharomyces cerevisiae, and the coordination of CYCl activation with that of other cytochrome genes. Base substitution mutations of the CYCl upstream activation sites (UASs) will be generated as well as mutations in genes encoding trans-acting proteins which mediate the activation. Regulatory genes will be cloned, their sequence and regulation determined, and their encoded products expressed to high levels in E. coli and yeast. In vitro assays for binding of these proteins to the UASs will be developed and used to purify the proteins. The purified proteins will be studied biochemically and employed in an in vitro transcription system programmed by CYCl DNA. The molecular mechanism of upstream activation will be studied in vivo and in the purified system. The regulatory circuits governing coordinate control of nuclear and mitochondrial cytochrome genes will be deduced by an examination of the pleiotropy of mutations affecting CYCl trans-acting regulators. Whether genes encoding subunits of the mitochondrial RNA polymerase are a part of this circuit will be determined by molecular analysis after these genes are cloned.

The long term objective of the experiments is to gain an understanding at the molecular level of how proteins become localized to mitochondria in eukaryotic cells. In particular a detailed genetic analysis of import of the yeast HEM1 product, Delta-amino levalinate (ALA) synthase, will be conducted. Mutations resulting in single amino acid changes or short substitutions in the ALA synthase signal sequence will be isolated by a combined approach of gene fusion, in vitro mutagenesis and genetic selection. After crossing these mutations into the yeast genome generating Hem- mutants, suppressors that recognize the altered signals will be selected as Hem+ revertants. In a separate selection, mutants with a reduced ability to recognize the wild-type ALA synthase signal will be isolated. A genetic analysis of all mutations isolated will be carried out. Whether import of other mitochondrial proteins is altered in mutant strains will be tested. Effects of mutations on import in vitro will be determined to pinpoint whether a component of the import machinery has been mutated, and, if so, whether that component is cytosolic or mitochondrial. Genes that are believed to encode components of the import machinery will be cloned, sequenced, and fused to lacZ. Antibody raised to the fusions will be used to determine the location of the products of cloned genes in yeast cells. Selected proteins will be overexpressed, purified, and employed in biochemical studies. Finally, the role played by mitochondrial signal sequences in regulating import of two other yeast proteins, citrate synthase and cytochrome oxidase subunit IV, will be analyzed.

In the past granting period, we learned that a fundamental mechanismof aging is conserved from yeast to animals. Thus, yeast is a valuable model for at least some important aspects of aging in higher organisms. A specific gene, SIR2, is conserved in organisms from bacteria to humans. This gene clearly regulates aging by promoting longevity in yeast and C. elegans, suggesting that its effects will prove to be general. The activity of Sir2p is to deacetylate proteins when provided with the co-substrate, NAD (nicotinamide-adenine dinucleotide). Our studies in yeast indicate that the replicative aging of mother cells can be extended by calorie restriction, and that this extension requires Sir2p. Since calorie restriction is the only intervention known to extend life span in mammals, these findingsmay have broad implications. We have further learned that the known effects of Sir2p on genomic silencing are pertinent to yeast aging. In the next period we propose to study the mechanism of Sir2p-mediated silencing in greater detail by analyzingsir2 mutations genetically and biochemically. We further propose to use a combination of yeast genetic and molecular approaches to define the specificmetabolic mechanism by which calorie restriction extends life span and how this might relate to Sir2p. In addition, we will probe the intriguing link between sojourns in stationary phase and yeast replicative aging. This particularstudy might provide a conceptual link between the aging of dividing and post-mitotic cells. Finally, we will begin an analysis of two new genes, SSD1 and MPT5, that affect yeast aging in a SIR2-independent way. These experiments will add to a substantial base of knowledge of aging in the simple budding yeast and will be a platform for studies of aging in higher organisms, includingmammals. 3ERFCRMANCE SITE(S) (organization, city, state) Massachusetts Institute of Technology 77 Massachusetts Avenue, 68-280 Cambridge, MA 02139 KEY PERSONNEL. See instructions on Page 11. Usecontinuationpages as neededto provide the required information in the format shown below. Name Organization Role on Project (/ Leonard Guarente Massachusetts Institute of Technology P.I. PHS CI98 (Rev. 4/98) Page 2 BB Numbisr pages consecutively at the bottom throughout the application. Do QQJ use suffixes such as 3a, 3b. CC Principa^Plstigator/Program Director (Last, first, middle): ^B Type frie name of the principal investigator/program director at the top of each printed page and each continuation instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Face Page Description,

This grant proposal is aimed at the molecular dissection of the process of transcriptional activation from enhancer sites in eukaryotes. Transcription is a fundamental process that controls cell growth and cellular differentiation. Further, the mechanism of activation by many activators is conserved in eukaryotes that range from yeast to mammals. The design of the experiments is to isolate adaptor molecules that connect transcriptional activators bound at enhancers to general transcription factors bound at the TATA box. We have already isolated two candidates for such molecules - - ADA2 and ADA3. Next, we aim to demonstrate the protein- protein contacts that connect the activators to the ADA molecules, the ADA molecules to each other, and the ADA complex to the general transcriptional machinery. Further, we aim to clarify the role of the general transcription factor, TFIIB, in activation. Binding of TFIIB to activators and to RNA polymerase II will be studied. We will also employ in our binding assays TFIIB mutants that display altered activation properties in vivo. We aim to build a molecular picture that displays all of the relevant proteins that connect activators to the general factors and cause activation of transcription. We further hope to delineate those surfaces that participate in these protein-protein interactions.

The human disease Werner's Syndrome causes many symptoms resembling premature aging. The sequence of the gene defective in Werner's individuals encodes a DNA helicase. The yeast homolog of this gene, SGS1, is a DNA helicase concentrated in the yeast nucleolus. Mutations in this gene cause premature aging in yeast and the fragmentation of nucleolar rDNA into circles that accumulate to cause aging. In this grant we will investigate the molecular function of the Werner homolog SGS1 including study of how its loss leads to premature aging. The genesis of yeast rDNA circles will be investigated and the molecular mechanism of their accumulation and killing studied in detail. Finally, the generality of this aging mechanism in mammals will be investigated by screening for DNA circles in tissues of aging mice and humans. The overarching goal is to slow down aging in at least some organs to provide a higher quality of life as people enter their twilight years.

DESCRIPTION (provided by applicant): The work described in this grant application will bring about fundamental knowledge in two important areas of current aging research. The first is calorie restriction (CR), which is a dietary regimen known to provide broad health benefits and longevity. The second is SIR2 and its related proteins (sirtuins), which have emerged in the past five years as important regulators of aging in a broad spectrum of model organisms. Studies in the prior period have begun to identify pathways by which CR operates in C. elegans. The first two aims in this grant will focus in on these pathway and identify hormones and cellular processes that are critical for CR to extend the lifespan of worms. We have pinpointed two neurons in the head that trigger broad physiological changes in response to CR, such as an increase in respiration, and we will figure out what hormonal factor these cells release to bring about the observed changes leading to long lifespan. In the third aim, we will study in detail the four sirtuin genes in C. elegans. Since sirtuins have been linked to CR in other organisms, we will investigate whether this is true in worms, which sirtuins are involved, and how they bring about their effects. The C. elegans system we have set up to study CR offers many advantages, including all of the genetic and molecular tools that have solved many other important biological problems in the nematode model. CR itself has salutary effects against many of the major diseases of aging in rodent models. It is possible that a molecular understanding of CR will allow us to tap genetic pathways to provide broad health benefits for our aging population. This approach is complementary to the current focus on the molecular details of particular diseases to develop specific therapies. Beyond this important practical outcome, this study will provide important new information on the sirtuins and pathways that relate diet to physiology. There is an excellent foundation to build on in the next granting period and important findings are anticipated. Public Health relevance: Dietary restriction is the most robust intervention that extends life span and improves health in mammals. This project details a study of dietary restriction in the roundworm C. elegans that will identify new genes, mechanisms and pathways driving this process. Our findings may lead to new therapeutic approaches to diseases of aging in humans.

DESCRIPTION (provided by applicant): We plan to further the study of the function of SIRT1 in mice. In Aim 1 we study whether SIRT1 (and other sirtuin) activity declines aging. This study will include a detailed analysis of NAD and NADH levels in cellular compartments of muscle, heart and brain. In Aim 2 we will determine the effects of depletion or augmentation of SIRT1 in brown fat, muscle, heart and 2-cells. This aim will employ tissue specific deletion and over- expression of SIRT1 in mice. In Aim 3 we will study the effects of SIRT1 on bone. This study will use mice knocked out for SIRT1 in osteoblasts or osteoclasts to determine mechanisms by which SIRT1 determines bone density. This aim will also study how calorie restriction affects bone. In Aim 4 we will study the role of SIRT1 in T cells. In this aim we will study immune function in T cell-specific SIRT1 knockout mice and determine the mechanism by which SIRT1 functions in these immune cells.

DESCRIPTION (provided by applicant): Sirtuins are NAD-dependent deacetylases that have been implicated in aging and metabolism in organisms ranging from bacteria to mice. In mammals, there are seven sirtuins termed SIRT1-7. Three (SIRT1, 6, and 7) are primarily nuclear, three (SIRT3-5) are mitochondrial and SIRT2 is cytoplasmic. Numerous studies show that mammalian sirtuins mediate effects of diet on protein acetylation, metabolism and aging. At least some of the health-promoting effects of calorie restriction (CR) can be elicited by raising levels of SIRT1 in mice. These include protection against metabolic decline caused by high fat diets or normal aging, and protection against neurodegenerative diseases in mice engineered to model, for example, Alzheimer's disease. Thus a general picture arises that SIRT1 activity increases in CR to help mediate protection against diseases of aging. Our own unpublished findings show that when SIRT1 is deleted from pituitary, the major hormonal axes driving growth and reproduction are reduced, like in dwarf mice. Since these changes are induced by CR in wild type mice, this phenotype suggests that unlike in peripheral tissues, SIRT1 activity in pituitary is repressed by CR, which we confirmed. This remarkable finding expands the role of pituitary SIRT1 in pituitary to promoting growth and reproduction when food is abundant (in addition to its role in promoting protection is other tissues when food is scarce). Aim 1 of this proposal will flesh out the mechanism by which SIRT1 mediates its effects in the pituitary. It will also probe how pituitary SIRT1 is regulated by diet. This aim may also provide insight into why whole-body activation of SIRT1 slows aging but does not extend life span. Finally, it will probe whether increasing levels of SIRT1 in the pituitary extends the reproductive life span of mice. The central role of SIRT1 in driving reproduction begs the question whether this sirtuin also controls gametogenesis in a cell autonomous manner. Preliminary findings say it does. Aims 2 and 3 will delete or over-express SIRT1 in testes (Aim 2) or oocytes (Aim 3) and determine consequences on gametogenesis, reproductive life span, and the quality of progeny produced. These findings may identify new mechanisms to slow aging in germ cells, and lead to new practical strategies for infertility in humans.

? DESCRIPTION (provided by applicant): Dysferlinopathies are a class of muscular dystrophies caused by recessive mutations in dysferlin (DYSF). Dysferlin mediates the repair of plasma membrane wounds resulting from normal muscle contractile forces. In patients and in mice where Dysferlin protein is reduced or absent, there is progressive degeneration of skeletal muscle that leads to loss of ambulation. The orphan nature of dysferlinopathy has hampered drug discovery efforts. There are no therapeutic treatments for dysferlinopathy patients, despite evidence of genetic disease modifiers that could be targeted for drug discovery purposes. Moreover, despite efforts, no in vitro high-throughput DYSF(-) muscle membrane repair assay has been successfully developed to identify therapeutics for in vivo testing. To address this unmet need, and as a proof-of-concept demonstration of the utility of lower model organisms in drug discovery, we have developed a high-throughput screening platform to identify small molecule and genetic suppressors of dysferlinopathy in Caenorhabditis elegans. Dysferlin and C. elegans FER-1 are conserved homologs within the Ferlin protein family. C. elegans fer-1 mutants are infertile due to defective sperm development. The biological mechanism of this infertility is similar in nature to the plasma membrane repair defect found in mammalian dysferlinopathy, namely an inability to fuse vesicles to plasma membrane. Using a conditional fer-1 missense mutant, we performed compound and mutagenic screens and identified novel small molecules and genetic suppressors capable of restoring fertility to otherwise sterile animals. Interestingly, the suppressors do not restore fertility to a recently obtained fer-1 null allele, suggesting that they operate by restoring FER-1 function rather than bypassing the need for FER-1. In this proposal we seek to optimize the current leads from our existing screening platform, and further improve our platform's selectivity and power in identifying therapeutic candidates by: 1) Determining the mechanisms of action of the fer-1misense mutation suppressors in order to evaluate their therapeutic potential for the one-third of patients who have missense mutations. 2) Identifying genetic suppressors of the fer-1 null mutant that restore fertility in the absence of FER-1. Mammalian counterparts of such suppressors may be targets to aid therapeutic discovery for ALL dysferlinopathy patients. 3) Determining if transgenic expression of human Dysferlin in the germ line of fer-1 deficient animals is sufficient to restore fertility. The creation of a humanized C. elegans dysferlinopathy model will have far reaching implications for therapeutic discovery as it could be used to screen for compound suppressors of specific patient mutations or mutational classes. C. elegans fer-1 mutants form the basis of our multimodal screening platform, which seeks to address difference among patient mutation classes in searching for therapies. As such, this approach will likely hasten the preclinical route to in vivo testing of drugs to benefit dysferlinopathy patients with muscular dystrophy.

Project Summary/Abstract Over the past decade, sirtuins, the NAD-dependent deacetylases, have emerged as important regulators of metabolism and aging. Numerous papers have shown that sirtuins, and in particular SIRT1, have protective roles against age-related diseases, including type II diabetes, cancer, and neurodegenerative disorders. Also, several studies showed that SIRT1 mediates the beneficial effects of calorie restriction in many metabolic tissues, providing a molecular link between this regimen and its beneficial effects on age-related diseases. These findings were followed by the development of small molecules that act as specific activators of SIRT1, with the hope that they will mimic calorie restriction and be used in treating diseases of aging. However, recent evidence suggests that the activity of sirtuins is reduced during aging likely because of a decline in NAD availability. Thus, investigation of the molecular mechanisms that underline age-dependent reduction in sirtuin activity and the molecular basis of interactions of sirtuins with NAD will guide the development of drugs that enhance sirtuin interaction with NAD and counteract the decline in NAD availability that occurs with aging. Our preliminary data show that yeast SIR2 mutants in lysine K475, which resides in the conserved Rossmann fold domain, rescue SIR2 activity in an NAD deficient strain (npt1) and prolong its replicative lifespan. Preliminary biochemical analysis suggests that the mutants have higher catalytic activity, a characteristic observed also with the same mutations in mammalian SIRT1. In Aim1, we will perform biochemical analyses to investigate the molecular mechanism that underlines the increased catalytic activity of these sirtuin mutants. To further elucidate the mechanism of action of these sirtuin mutants, we plan to solve the structure of this domain bound to NAD, in the context of a shorter but still functional SIR2 protein (mini- SIR2) in collaboration with Cynthia Wolberger. Importantly, the yeast residue K475 is conserved in C. elegans and mammals. In Aim 2, we will determine the effects of SIR2 ortholog mutants in these species, by generating knockin animals, determining their lifespans, and challenging them with various stresses. In Aim 3, we will perform high throughput screen to identify compounds that allow sirtuins to function under low NAD conditions, with the hope that these compounds or their derivatives, will reach the clinic to battle diseases of aging. The screen will be performed in collaboration with the High Throughput facility at the Koch Institute for Integrative Cancer Research. .