Judith Frydman - US grants

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the biochemistry and cell biology of protein folding in eukaryotic cells. The proposed research will focus on folding events as they occur at the ribosome during synthesis of a polypeptide and will examine the role of molecular chaperones in the folding process. The conceptual framework required to understand the folding of proteins as they emerge from the ribosome originates from our previous work, which indicates that folding in the eukaryotic cytosol is mediated by a chaperone network that is physically and functionally linked to translation. The objective of this proposal is to elucidate the mechanism by which chaperones mediate the folding of newly synthesized proteins in eukaryotic cells. To gain insight into this process we identified critical questions that will allow us to understand de novo folding, namely: (i) how and when do chaperones contact the emerging nascent chains?; (ii) what is the relevance of chaperone-binding for de novo folding?; (iii) what is the contribution of different chaperone systems to overall folding in the cell? (iv) how do chaperones interact with the translation machinery? Our general strategy to answer these questions is to combine in vitro and in vivo approaches to obtain mechanistic and functional insights into the role of chaperones in cellular folding. The first two specific aims will analyze the folding of model proteins in vivo and in cell-free translation lysates that faithfully represent the intact cytosol. Since our in vivo analysis indicates that different proteins exhibit distinct chaperone requirements, the third specific aim will examine the contribution of different chaperones to cellular folding and will identify the substrate spectrum of different molecular chaperones. Finally, our fourth specific aim will explore the interaction between chaperones and the translational machinery. [unreadable] [unreadable] [unreadable]

protein protein interaction; tumor suppressor proteins; molecular chaperones; protein folding; biomedical resource;

ABSTRACT NOT PROVIDED

molecular assembly /self assembly; molecular chaperones; protein structure function; protein folding; intermolecular interaction; biomedical resource;

ABSTRACT NOT PROVIDED

molecular chaperones; intermolecular interaction; ribosomes; biomedical resource;

mass spectrometry

DESCRIPTION (provided by applicant): The long term goal of our research is to understand how proteins fold in living cells. Chaperonins are key components of the cellular folding machinery. These large protein complexes, consisting of two stacked seven- to nine-membered rings, bind unfolded substrates in their central cavity and use binding and hydrolysis of ATP to mediate polypeptide folding. The folding of substrate proteins occurs in the central cavity formed by each ring. There are substantial differences between group I chaperonins, found in prokaryotic cells, and the distantly related group II chaperonins in Archaea and Eukarya,. Group I chaperonins require a ring-shaped cofactor, such GroES for GroEL, that upon binding acts as a lid for the cavity, creating a folding chamber that encloses polypeptide substrates. Group II chaperonins are heterooligomeric and lack a GroES-like cofactor, suggesting that their conformational cycle is significantly different from group I chaperonins. The present proposal aims to elucidate the mechanism of the eukaryotic chaperonin TRiC (TCP1-Ring Complex, also called CCT). Despite its essential role in polypeptide folding, little is known about the mechanism and substrate binding properties of TRiC. To understand how TRiC facilitates folding we propose the following specific aims: 1. Characterize of the nucleotide cycle of the chaperonin TRiC; 2. Define the molecular basis of TRiC-substrate interactions; 3. Explore the mechanism of TRiC-assisted folding.

mass spectrometry

ATP Hydrolysis; CRISP; Computational Technique; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Institution; Instrumentation, Other; Investigators; NIH; National Institutes of Health; National Institutes of Health (U.S.); Research; Research Personnel; Research Resources; Researchers; Resolution; Resources; Solutions; Source; Structure; Time; United States National Institutes of Health; improved; instrumentation

DESCRIPTION (provided by applicant): The TCP-1 Ring Complex (TRiC) is an essential, conserved chaperone required for the folding and activation of 10-15% of the proteins encoded by the human genome. Among its substrates are a large number of cell cycle regulators and tumor suppressor proteins. Currently, no specific agonists or antagonists are available for this key regulator of cellular protein folding. Given the early promise of other chaperone-directed inhibitors as cancer therapeutics, we believe that inhibitors of TRiC activity may also selectively target transformed cells. In this project, we plan to: 1) Identify small molecule inhibitors of TRiC with high throughput screening (HTS) using a homogenous time resolved fluorescence (HTRF) assay against the Molecular Libraries Screening Center Network compound collection. 2) Test the inhibitors from HTS for specificity to TRiC and optimize candidate compounds for activation or inhibition of TRiC-mediated actin folding. 3) Characterize the toxicity and bioavailability of molecules that modulate TRiC activity in tissue culture and measure the efficacy of candidates for inhibition of protein folding in vivo. PUBLIC HEALTH RELEVANCE: The chaperonin TRiC/CCT is essential for folding a large array of cellular proteins, including many cell cycle regulators, yet no inhibitors or modulators of its activity are available. Obtaining inhibitors for this chaperonin would be invaluable for understanding its biological function, and could also lead to a novel type of anti-cancer drugs.

? DESCRIPTION (provided by applicant): Protein folding in the cell is critically dependent on the assistance of molecular chaperones. The ring-shaped chaperonins are essential members of the cellular folding machinery. These large protein complexes consist of two stacked seven- to nine-membered rings. Chaperonins bind unfolded substrates in their central cavity and use binding and hydrolysis of ATP to mediate polypeptide folding. Substrate proteins are thought to fold upon encapsulation in the central cavity formed by each ring. The long term goal of this program is to understand how the chaperonin of eukaryotic cells, TRiC, mediates polypeptide folding. TRiC is hetero- oligomeric and uses ATP cycling to open and close a built-in lid over the central chamber. Intriguingly, TRiC has the ability to fold some eukaryotic proteins, such as actin, that cannot be folded by any other chaperone. Despite its essential role in cellular folding little is known about the mechanism and substrate binding properties of TRiC. Our work in the previous funding period provided important mechanistic and structural insights into this chaperonin. First, we established that the conformational cycle of TRiC is significantly different from that of bacterial chaperonins. Secondly, we found that subunit diversity confers dramatic functional asymmetry to this seemingly symmetric chaperonin. To understand how TRiC facilitates folding we propose the following aims: 1. Characterize of the nucleotide cycle of the chaperonin TRiC: Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. We want to understand how the ATPase cycle of TRiC is coordinated among the different TRiC subunits, and how ATP cycling drives conformational changes in the chaperonin and how the built-in lid that opens and closes in response to ATP-binding and hydrolysis. 2. Define the molecular basis of TRiC-substrate interactions: Little is known about the molecular basis of TRiC-substrate interactions. We want to define the substrate recognition code of the binding sites for the different subunits in the chaperonin and define the motifs within substrates that are recognized by TRiC. 3. Investigate the mechanism of TRiC-assisted substrate folding: The exact role that chaperonins play with respect to the substrate is still a mystery. We will explore the effect of TRiC on substrate proteins during the different stages of the folding cycle by combining biochemical approaches together with crosslinking and fluorescence spectroscopy. Importantly, recent observations have highlighted the links between TRiC and several pathological states including cancer, viral infection and neurodegeneration. Thus our project deciphering the mechanism of this chaperonin in cellular folding will help develop therapies to ameliorate these human diseases.

DESCRIPTION (provided by applicant): The ability of organisms to withstand stress is critical for survival, and there is a growing appreciation that cellular stress responses are vital during growth and development. In fact, an increasing number of human diseases-particularly those affecting neurons-arise from defects in stress response pathways or from the inability of stress proteins to protect cells. Stress response pathways have also been linked to ageing, infection, cancer, and metabolic diseases. Not surprisingly, then, significant efforts to modulate the stress response have been taken by biomedical researchers. Therefore, it is critical that a forum is provided to assemble scientists who seek to connect the mechanisms underlying human diseases with the regulation of stress genes and with stress protein function. One of the premier forums for this unique assembly is the Stress Proteins in Growth, Development and Disease Gordon Research Conference (GRC). The next conference, which is the sixth in this series, will be held from July 17th - July 22nd, 2011, at the Il Ciocco Hotel &Resort, Lucca (Barga), Italy. The Chair for this meeting is Lea Sistonen (Ebo Akademi University, Turku, Finland) and the Vice-Chair is Judith Frydman (Stanford University, Stanford, CA). The Il Ciocco Hotel has a long history in supporting GRCs conferences. Thus far, 30 internationally recognized speakers have committed to attending and speaking at the 2011 meeting, including 10 women and one African-American. Nevertheless, significant efforts will be undertaken to increase the diversity of meeting attendees, to bring more scientists into the stress protein field, and to catalyze the formation of new collaborations. To this end, the schedule has been prepared so that 16 speaking slots will be chosen from abstracts submitted for poster presentations, and preference will be given to younger scientists and to those from under- represented groups. The meeting schedule will also be prepared to ensure that new and emerging themes in stress biology and different model systems are equally represented. In addition, meeting attendance will be "capped" at 150 to facilitate effective interactions and discussions. Overall, we seek to enhance the dissemination of cutting-edge advances and the formation of new collaborations in the stress protein field. This, in turn, will lead to advances and broaden our understanding of the roles of stress proteins in human health, aging, and disease. PUBLIC HEALTH RELEVANCE: Cells-especially neurons-live under harsh conditions. There are times when cells are starved for nutrients, times when cells need to exhibit rapid growth, and times when cells are exposed to toxic agents. Cells may also express misshapen proteins, as occurs in Alzheimer's, Huntington's, and Parkinson's disease. However, to offset the dangerous consequences of stress, which can lead not only to disease but also to ageing, cells produce specific proteins to protect themselves. The 2011 Gordon Research Conference, entitled 3Stress Proteins in Growth, Development, and Disease4, brings together scientists who study the link between stress and human disease, and provides a unique forum for researchers in this field to assemble and identify new avenues to prevent stress-related diseases and degenerative processes such as aging.

PROJECT SUMMARY (See instructions): Enteroviruses are characterized by their high replication rates and extreme sequence plasficity, which allow them to rapidly adapt to different environments and hosts. These viruses depend entirely on the host protein homeostasis machinery, composed of molecular chaperones and quality control (QC) components such as the ubiquitin-proteasome system, for viral protein production and function. Enterovirus replication poses several challenges to the cellular protein homeostasis machinery as the need to produce high amounts of protein in a very short time places a big burden for the host protein production and folding machineries. Furthermore, enteroviral proteins tend to be large, complex and multifunctional, and thus likely to require the assistance of molecular chaperones to fold. Indeed, we have shown that the Hsp90 chaperone system is essential for capsid folding and assembly for many, perhaps most, picornaviruses, including the enterovirus polio- and coxsakie-viruses. Since other aspects of enterovirus replication Involve additional large multlprotein complexes, chaperones are likely to be broadly required for other aspects of the viral cycle. An important challenge to protein homeostasis in RNA viruses arises from their very high mutation rates, which pose a big burden to viral protein stability and are likely to produce high levels of non-functional or destabilized proteins. These mutant proteins must be either maintained in a funcfional state or eliminated from the cell to prevent dominant negative effects on viral function. We hypothesize that these functions are carried out by chaperones, which can buffer metastable proteins, as well as by the ubiquitin-proteasome system, which targets misfolded proteins for degradation. To understand the molecular and cellular mechanisms by which cellular chaperone and quality control machineries control viral protein homeostasis, and allow the virus to replicate we propose the following Aims: Aim 1: Define the chaperone components required for enterovirus replication. Aim 2: Define the role of the Quality control (QC) machinery in picornavirus replication Aim 3: Examine the plasticity and interplay of chaperone and QC pathways during viral infection

DESCRIPTION (provided by applicant): The integrity of the cellular proteome is critically dependent on an elaborate network of protein quality control machines that both aid in the folding of newly made proteins and allow for the recognition and disposal of terminally misfolded forms. Many diverse human diseases, including familial protein folding diseases, neurodegenerative diseases, diabetes, and cancer, as well as normal aging have been linked to the failure to maintain proper protein homeostasis. Thus defining the mechanism of action of the protein quality control machinery is a major goal in the quest for understanding of health and pathology in all living cells. A common theme to this machinery is the ability to recognize portions of unfolded polypeptide chains, either to facilitate their subsequent folding/refolding or degradation, or to signal in adaptive responses aimed at restoring the balance between supply and demand of the protein folding capacity. Most molecular events in protein quality control work on many diverse substrates and hence possess considerable plasticity in substrate binding. While much progress has been made in structural and functional analysis of individual components of these machines, there are few examples where substrate-bound structures have been determined or where a substrate recognition code has been defined and validated. As such, we are lacking in our understanding of core principles that govern workings of these protein machines. We propose to bridge this gap by focusing on a core set of physiologically critical systems that cover a range of molecular features but share the common requirement of having to balance specificity and plasticity in molecular recognition events. In particular, we wil focus on cytosolic chaperone substrate recognition (using examples of the hsp70, hsp90, and TRIC families of chaperones) and the recognition of unfolded proteins in the lumen of the endoplasmic reticulum (ER) for degradation via the ER-associated degradation pathway (ERAD) and for signaling via the unfolded protein response (UPR).

DESCRIPTION (provided by applicant): The cellular proteome is constantly exposed to a wide variety of toxic stresses. These include external stresses, such as elevated temperatures, radiation damage and pharmacological agents as well as physiological stresses encountered during cellular proliferation, differentiation, inflammation and aging. In all organisms, inductionof the stress response is essential for the maintenance of protein homeostasis and modulation of the stress response plays a critical role in life-span regulation and aging-related diseases such as Alzheimer's, Parkinson's and Huntington's disease. The 2013 Gordon Conference on Stress Proteins in Growth, Development & Disease is shaping up to be one of the most exciting and important meetings in this research area. It will highlight the many cutting edge advances in the field, emphasizing a broad range of topics, including exciting developments related to stress sensing, signaling, and gene expression, diseases of protein folding and conformation, roles of stress genes in metabolism, growth and development, stress gene modulation of infection and pathophysiological states, the cell biology of stress, and the roles of stress in aging. This conference, which is the seventh in this series, will be held July 7th-12th at the Mount Snow Resort in West Dover, Vermont. The chair of this meeting is Dr. Judith Frydman (Stanford University, Stanford, CA) and the co-chair is Dr. Ursula Jakob (University of Michigan, Ann Arbor, MI). Thus far, 34 internationally recognized speakers have committed to attending and speaking at the 2013 meeting, including 9 women and one African- American. Moreover, significant effort will be undertaken to increase the diversity of meeting attendees, to bring more scientists into the stress protein field, and to catalyze the formation of new collaborations. To this end, the schedule has been prepared so that 10 speaking slots will be chosen from abstracts submitted for poster presentations, and preference will be given to younger scientists and to those from under-represented groups. The meeting schedule will also be prepared to ensure that new and emerging themes in stress biology and different model systems are equally represented. In addition, meeting attendance will be capped at 200 to facilitate effective interactions and discussions. The formal scientific program together with more informal discussions will enhance the dissemination of new information and the formation of new collaborations, which are invaluable for deeper understanding of the versatile roles of stress proteins in human health, aging and disease.

  SUMMARY    The  goal  of  this  project  is  to  understand  how  protein  quality  control  mechanisms  in  adult  stem  cells  and  their progeny are regulated during aging, with the objective to restore the functionality of old cells. Preservation  of  a  pristine  proteome  is  emerging  as  a  critical  mechanism  for  maintaining  cellular  function  throughout  life.  Disruption  in  the  machinery  that  maintains  protein  quality  control  leads  to  protein  aggregation  diseases  and  accelerated  aging  in  invertebrate  models.  However,  how  cell  types  with  different  roles  regulate  protein  homeostasis during long periods of time remains unexplored, particularly in mammals. The adult brain offers a  unique paradigm for understanding protein quality control mechanisms in cell types with different functions. It  contains reservoirs of quiescent neural stem cells (NSCs) that can activate and in turn generate differentiated  cells with specialized function ? neurons, astrocytes, and oligodendrocytes. During aging, the ability of NSCs to  exit quiescence and their ability to produce new neurons both decline dramatically yet this deterioration is not  inexorable and can be reversed by environmental interventions, including diet. However, the mechanisms that  can regulate NSC function are largely unknown.     We  recently  embarked  on  a  systematic  characterization  of  protein  aggregates  and  proteostasis  mechanisms  in  young  NSCs  and  their  progeny.  Excitingly,  we  find  that  quiescent  NSCs  contain  large  protein  aggregates that are present undegraded in large lysosomes. Nutrient deprivation can clear protein aggregates  and  enhance  their  ability  to  activate,  a  process  that  is  dramatically  affected  by  aging.  Interestingly,  our  RNA-­ seq  profiling  from  young  and  also  mice  reveal  that  quiescent  NSCs  from  old  mice  exhibit  a  large  degree  of  transcriptome-­wide  change  with  age.  The  central  hypothesis  of  this  Project  is  that  the  protein  quality  control  mechanisms  differ  in  cell  types  with  distinct  functions,  which  could  underlie  their  different  degree  of  deterioration  with  age  and  could  be  used  for  specifically  ameliorating  old  cells.  To  test  this  idea,  we  propose  the following experiments:    1.  To  understand  how  protein  aggregates  and  protein  quality  control  mechanisms  are  influenced  by  increasing age and by rejuvenating strategies    2.  To  specifically  modulate  proteostasis  mechanisms  to  ameliorate  function  in  old  NSCs  and  their  differentiated progeny    3.  To  determine  the  composition  of  protein  aggregates  and  generate  new  aggregate  reporters  in  NSCs  and their progeny    Completion of these Aims will provide unique mechanistic insights into the regulation of protein aggregates  and  their  alteration  during  aging  in  regenerative  cells  and  their  differentiated  progeny.  This  study  should  also  provide  fundamental  understanding  of  how  protein  quality  control  is  mechanistically  regulated  in  different  cell  types. This knowledge should pave the way for building new methods for ?rejuvenation? of old cells and restore  protein  aggregates,  which  will  be  a  critical  step  for  improving  tissue  function  during  aging  and  age-­related  disease.        

Defining the role of Host Hsp70 Subnetworks in Dengue Virus Replication Abstract: Viral protein folding and homeostasis depends entirely on the machinery of the host cell. Here we define the dependence of Dengue virus (DENV) on the complex network of Hsp70 chaperones and cochaperones which mediate distinct steps of the virus life cycle. We find that several cytosolic Hsp70 isoforms are required at multiple steps of the viral life cycle to prevent viral protein degradation, promote virion assembly and support viral enzyme function. At each step, Hsp70 function is specified by distinct subnetworks of cofactors, called DnaJs and NEFs, that modulate Hsp70 action and localization at each step. We hypothesize that combinations of DnaJs and NEFs dictate the specific cellular locations, substrate specificities and downstream effectors of Hsp70 in viral replication. We propose to define the mechanism and function of Hsp70 subnetworks in DENV replication through the integration of genetic and proteomic analyses with biochemical and cell biological experiments. Specifically we propose to: (1) Define the mechanism and function of DnaJs in DENV replication; (2) Dissect the role and mechanism of Hsp70 NEFs in DENV replication and (3) Define the mechanism of restriction by the Bag6-centered network. Defining the chaperone subnetworks required for distinct steps of DENV replication will provide new insights into key aspects of the cell biology and molecular mechanism of viral infection. Importantly, Hsp70 provides a susceptible node for antiviral drugs, since compounds inhibiting the Hsp70 cycle blocks DENV infection with negligible toxicity to the host. Our work will thus identify novel targets for pharmacological antiviral intervention and uncover unanticipated cellular mechanisms for viral restriction.

Core B ? Frydman/Finkbeiner - Sensors and Tools for Proteostasis Analyses Summary: This Core will develop tools to define the proteostasis network (PN) during aging and in the context of disease-associated aggregation-prone proteins and will provide technical support to Projects 1?4. The resources will also be used to test and validate compounds from Core D. Core B will have two sites in close geographical proximity. The Frydman group at Stanford will build molecular sensors and assays to measure proteostasis changes during aging, starting with reporters developed for yeast chaperone machinery and the ubiquitin-proteasome system (UPS). The Finkbeiner group at Gladstone/UCSF will build on mammalian expression and analysis tools to assess protein aggregation and autophagy and help PLs address specific questions for which their multiplexed longitudinal single-cell imaging platform is critical. By working hand-in- hand and testing the ability of the sensors to report on different proteostasis events in experiments used by all Projects, we will produce a set of assays that provide a comprehensive view of the PN of any cell. Tools will be available to the research community. A primary task of Core B will be to develop molecular sensors that robustly and quantitatively report on each branch of the PN and that change in aging and in the context of disease-associated misfolded proteins. Imaging sensors will also allow us to examine how proteostasis mechanisms are communicated from one cell to another. Answers to these questions will provide key insights into mechanisms of proteostasis dysfunction and protein aggregation during aging and will open new avenues for maintaining health and treating disease. We Propose to: (1) Develop and validate a panel of proteostasis sensors and reporters and (2) Assist project leaders (PLs) in the analysis of the PN sensors: towards a comprehensive exam of the PN status of any cell. Our ultimate goal is to build a comprehensive set of sensors compatible with 384-well plates that could be used as an integrated panel to interrogate all aspects of the PN. By synthesizing the collective body of knowledge of the proteostasis network into a set of fluorescence-based sensors and reporters, Core B will produce an invaluable toolbox for the whole proteostasis field that will facilitate progress, not only in the labs associated with this PPG but also in other labs interested in linking proteostasis dysfunction with aging and disease.

Project 1 ? Frydman - Dissecting the aging-associated decline in cellular proteostasis Project Summary: The ability to maintain a functional proteome by preserving protein homeostasis, or proteostasis, is essential for cell viability. Yet, this ability declines during the process of aging. Such a collapse in proteostasis results in the accumulation of misfolded and aggregated proteins that are a hallmark of late-onset diseases including, most notably, a wide range of neurodegenerative diseases including Alzheimer?s, Parkinson?s and Huntington?s Diseases. However, it remains largely unknown what cellular changes are responsible for the loss of protein homeostasis and the accumulation of damaged proteins during aging. We propose to define the mechanisms and consequences of this proteostasis decline in order to better understand what cellular interventions could improve the aging process and ameliorate age-related diseases. Proteostasis is maintained through the interplay of molecular chaperones, which are essential for protein folding and function, and quality control factors, including the ubiquitin-proteasome system and autophagy, which target misfolded proteins for elimination. Accumulating evidence suggests that the proteostasis balance is disrupted during aging. Yet, our understanding of how aging alters the interplay of proteostasis regulators is far from complete. This Project will examine several phases that regulate the life cycle of a protein, including translational fidelity, chaperone function, and misfolded protein management, to determine what cellular changes dictate the widespread proteostasis collapse associated with aging. Importantly, we will examine how the presence of aggregation-prone disease-linked proteins such as A-beta, tau and polyQ-expanded Huntingtin exon1 affect the interplay between proteostasis disfunction and aging. This will provide substantial insight into how age-dependent modulation of these processes might contribute to the decline in proteostasis, and associated decline in cell viability, that is a primary hallmark of aging and several late-onset human neurodegenerative diseases. In order to elucidate at a molecular level how aging affects the folding of newly translated proteins and the management of misfolded and stress-denatured proteins, we plan to exploit our collective expertise across a variety of models of aging to: (i) Examine how aging affects biogenesis and folding of newly made proteins and (ii) Determine how aging affects the management of misfolded and aggregation-prone proteins

Project Summary The parent grant proposal grant aims to dissect how proteins fold in eukaryotic cells. Access to cutting-edge microscopy has become critical to study the problem of protein folding in the cell. The ability to pinpoint the location of chaperones and substrates with respect to each other and to cellular organelles in the cell is increasingly dependent on resolving imaging targets in greater detail ? necessitating super-resolution microscopy. The experiments in this grant (as well as all experiments in my other RO1 grant GM074074) will benefit enormously from acquiring a microscope capable of carrying out super-resolution microscopy approaches. We request an administrative supplement to purchase a benchtop super-resolution fluorescence microscope.