Peter Walter, Ph.D. - US grants

The proposed research aims at a precise molecular understanding of how certain classes of proteins are selectively targeted to and translocated across the endoplasmic reticulum membrane in mammalian cells. We will identify the cellular constituents of the protein targeting and translocation machinery that are essential, and ultimately also those that are modulatory, to gain a detailed understanding of the mechanism of targeting and translocation and the role which individual molecules play in this fundamentally important process. Specifically, i) we will determine the molecular details of signal recognition particle (SRP) - signal sequence interactions and SRP-ribosome interactions and ask how these interactions are regulated. ii) We will determine the individual contribution of each of the three GTPase domains contained in the SRP and the SRP receptor during protein targeting to the endoplasmic reticulum membrane and the assembly of the protein translocation site. We will design assays to identify other molecules that affect the binding and/or hydrolysis of GTP by individual GTPase domains. iii) We will determine the minimal set of ER proteins that is required for nascent chain targeting, nascent chain insertion and nascent chain translocation and try to decipher the mechanistic role that these protein play in the process. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.

The primary objective of the proposed research is to understand the mechanism by which the correct proteins are targeted to and translocated across the endoplasmic reticulum membrane in the simple eucaryote Saccharomyces cerevisiae. Current models suggest that there are at least two parallel targeting and translocation pathways, one SRP-dependent and co-translational, the other SRP-independent and post-translational. Through the combination of genetic and biochemical approaches, we aim to identify the gene products that catalyze the reactions that are common to both pathways or unique to one or the other and to understand their molecular function, regulation and physiological importance. Specifically, i) we will characterize the mechanism(s) by which SRP and SRP receptor coordinate the interactions between the ribosome and translocon components to form the ribosome membrane junction. ii) We will characterize the components that mediate the SRP-independent pathway. We will analyze how these components function to select proteins into this pathway, and how they function to target proteins to the ER membrane. iii) We will determine the molecular basis of "adaptation", the physiological response that allows cells to improve protein translocation when the SRP-dependent pathway is disrupted. We wish to learn which aspects of the process are unique to yeast and which aspects can be generalized to other eucaryotic cells and bacteria. Ultimately, we hope that through a combined genetic and biochemical approach, we will understand, at a mechanistic level, the function of all of the cellular constituents of the protein translocation machinery that are essential for translocation, as well as the function of those that are modulatory.

9453958 Walter: In this Local Systemic Change Through Teacher Enhancement Project, the University of California, San Francisco (UCSF), proposes a project in partnership with the San Francisco Unified School District (SFUSD), whose purpose is to enable all of the city's elementary schools to accomplish effective, site-based science education reform. UCSF's City Science program will provide an intensive hands-on science program for 400 SFUSD teachers who have taught less than seven years and a less intensive program for 600 more experienced teachers in order to broaden the base of teachers with hands-on science in a cooperative learning environment. It will also provide new opportunities for a subset of the 100 teachers who are current City Science participants to expand their capacity as science teachers while serving to further develop in their role as leaders. They will support specific aspects of the district-wide science education reform and mentor new leadership that will sustain the reform process in their schools.

A basic knowledge of cell biology is a requisite for understanding the defects in cell function that cause human diseases, including many cancers, muscular dystrophy, neurodegenerative disorders, blistering skin diseases, and cardiovascular disease. In recent years, cell biologists have played an increasing role in elucidating the mechanisms underlying genetic disorders, and understanding the biology of eukaryotic cells now becomes key in the quest to develop new and improved methods for the prevention, diagnosis and therapy of human disease. The 2001 Gordon Conference on Molecular Cell Biology focuses on recent developments in cell biology that were instrumental in determining genetic bases of diseases and now provide insights into methods for diagnosis and prevention of disease. The key features of this meeting are its diversity and scientific excellence. Emphasis is on cutting-edge science leading to new scientific principles and novel approaches to cell biology. A wide range of topics is represented, including cell cycle, cell polarity and movement, cell adhesion, genetic diseases, cell biology and disease. The organizers are Peter Walter and Ira Herskowitz (both at University of California, San Francisco). The meeting will bring together and foster discussion among world- renowned cell biologists. Participants will present their most recent and exciting results involving a number of model systems and using a variety of novel technical approaches. Nine sessions, each involving four major talks, are planned. Time will be reserved at the end of each session for short oral presentations on breaking developments. Thirty-five speakers, all leaders in their fields, have agreed to attend; approximately 30% of these are women. One more person will be invited. Daily poster sessions will enable all participants to present and discuss their most recent results. There will be abundant opportunities for informal discussion among speakers, postdoctoral fellows, and graduate students. This grant requests partial support for this exciting meeting.

DESCRIPTION (provided by applicant): The Science & Health Education Partnership (SEP) of the UCSF, in strong collaboration with the San Francisco Unified School District (SFUSD), proposes a National Institutes of Health (NIH) SEPA, the Quattro Alliance for Science and Language Integration. Through the Quattro Alliance, SEP will both develop a professional development program in science and language and undertake a comprehensive research, documentation, and dissemination effort. Quattro will establish a professional community of four groups of participants - elementary school teachers, UCSF volunteers, elementary English language learning (ELL) students, and a collaborative of evaluators - to promote the integration of science learning and language development in elementary schools and to increase access to rigorous science learning for ELL students in SFUSD. Science learning in this effort refers to engaging students in the development of both scientific concepts and investigation skills. Language development here refers to student understanding of the forms, functions, and vocabulary of scientific discourse. This effort will be accomplished through four specific aims: 1) to develop strong scientist-teacher partnerships grounded in the research literature on science & language teaching and learning; 2) to provide ELL students with access to rigorous science learning and the development of the skills required for academic discourse in science; 3) to establish a generative community of science educators who will develop a Framework of goals and concrete classroom strategies that can be used to integrate science learning and language development; 4) to create dissemination materials based on the generation ofa Quattro Framework and evaluation data collected during the project to provide strategies for the integration of science and language to school districts and universities nationwide. These specific aims will be addressed through three integrated programmatic structures: 1) Quattro Beginning Coursework; 2) Quattro Science Clubs, and 3) the Quattro Professional Community. These efforts will employ three innovations in the integration of science and language learning. First, the project will engage significant numbers of both teachers and UCSF scientists and clinicians in partnership, a novel approach to the integration of language development and science learning. Second, this community of science educators will develop an evidence-based and accessible framework for integrating science and language teaching and learning in elementary school classrooms. Finally, the proposed effort will occur in a large, polylingual, urban school district. Documentation of the effort, analysis of evaluation data, and the generation of a framework for integrating science and language learning in the classroom will be assembled into dissemination materials designed for use by universities and school districts across the United States.

Abstract Not Provided.

yeasts; cell fusion; biomedical resource;

[unreadable] DESCRIPTION (provided by applicant): GRC on Stress Proteins in Growth, Development and Disease All organisms are exposed to stressful conditions including environmental stresses such as elevated temperatures and irradiation, physiological stress such as oxidative stress due to metabolic reactions, or pathophysiological stresses such as pharmacological agents, infection and inflammation. These stressful conditions lead to protein misfolding, aggregation, cellular dysfunction and cell death. Recent studies strongly suggest that the ability to sense and respond to stress signals, through the activation of signal transduction pathways, transcription factors and gene productions that function in protein homeostasis is critical for normal growth, development and in the protection against diseases that include cancer, cardiovascular disease and protein folding diseases such as Alzheimer's Huntington's and prion-based disease. Furthermore, studies in model systems have established a strong correlation between longevity and the ability to mount robust stress responses. The meeting will highlight the many cutting- edge advances in the field, including the mounting appreciation of the importance of autophagy pathways to remodel cells under stress conditions and the increasing emphasis on using modeling approaches to understand stress regulation at a systems level. This Gordon Research Conference on "Stress Proteins in Growth, Development and Disease" will be held August 19-24, 2007 at Magdelan College in Oxford, UK. The organizers are Peter Walter, Chair (University of California, San Francisco), and Bernd Bukau, Vice-Chair (Universit[unreadable]t Heidelberg, Zentrum f[unreadable]r Molekulare Biologie). We will emphasize vigorous discussions of recent developments in stress sensing, signaling and gene expression, diseases of protein conformation, roles of stress genes in metabolism, growth and development, stress gene modulation of infection, the cell biology of stress and the roles of stress in aging. Eight sessions are planned, most with speaking opportunities reserved for new investigators, trainees and recent breakthroughs. All 33 speakers invited to date, of which 42% are women, have confirmed their intention to attend. There will be abundant opportunities for detailed, informal discussions among speakers, postdoctoral fellows and graduate students at poster sessions and additional times during the meeting to enhance interactions and information transmission. This application requests partial support for this Gordon Research Conference. GRC on Stress Proteins in Growth, Development and Disease This meeting will enhance our understanding of stress signaling, stress protein function and mechanism of action, and the role of stress in human health, disease and in aging. Cells employ various mechanisms that help protect them from stresses to which all organisms are constantly exposed, including environmental stresses such as elevated temperatures and irradiation, physiological stress such as oxidative stress due to metabolic reactions, or pathophysiological stresses such as pharmacological agents, infection and inflammation. The molecular consequences of such stressful conditions are protein misfolding, cellular dysfunction, and cell death, manifesting itself as aging and a multitude of human diseases. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

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). PUBLIC HEALTH RELEVANCE: The proper folding of newly made proteins is very important for every cell. Numerous human diseases, including neurodegenerative diseases, diabetes, and cancer, as well as normal aging are linked to the failure to fold proteins properly. We will determine the structure of cellular machines that recognize misfolded proteins to help them fold or target them for degration. A detailed structural understanding will contribute to the development of new therapies.

? DESCRIPTION (provided by applicant): Approximately one third of the genes in the human genome encode secreted and plasma membrane proteins that are synthesized on membrane-bound ribosomes at the endoplasmic reticulum (ER). These proteins cross or become embedded in the ER membrane as they are being synthesized, i.e., co-translationally. The ribosomes synthesizing this subclass of proteins are brought to the ER membrane by an evolutionarily conserved molecular machine, the signal recognition particle (SRP), and its ER membrane-embedded receptor. The folding of the growing polypeptide chain is assisted co-translationally by the sequential engagement of different chaperones and disulfide isomerases in the ER, where the protein folding status is constantly monitored. In response to a folding imbalance, or ER stress, a network of signaling pathways, collectively called the unfolded protein response (UPR) readjusts the ER's protein folding capacity to accommodate the load of proteins entering its lumen. The UPR sensors in the ER membrane respond to an accumulation of un- or misfolded proteins in the ER and initiate appropriate preemptive (i.e. fewer clients are allowed to enter the ER) or corrective (i.e. the ER processing capacity is enlarged) actions. Of the UPR sensors found in mammalian cells, IRE1 is the most evolutionarily conserved and best understood. It is a bifunctional ER-resident transmembrane kinase/RNase that upon activation excises an intron from the mRNA encoding its principal effector, the transcription factor XBP1. This induces a frameshift in the XBP1 open reading frame, and leads to production of functional XBP1 transcription factor that initiates a genetic program to adjust the ER's protein folding capacity according to need. To date, the sensing of the ER's folding conditions and the resulting IRE1-mediated signal transduction events have been thought of as autonomous events, occurring after and uncoupled from protein synthesis and translocation. This view has changed profoundly with two independent and complementary discoveries supporting the synchronous monitoring of the protein folding status in the ER by IRE1 and co-translational translocation of ER clients into its lumen. Using RNA crosslinking techniques coupled with deep sequencing, we sought to comprehensively catalog mRNA substrates for IRE1. To our surprise, we found that major crosslinks between IRE1 and RNA mapped to the SRP RNA and ribosomal RNA. Moreover, the crosslink sites converge on a functionally well-defined region on the ribosome (to SRP RNA's Alu domain and to rRNA in proximity of where SRP's Alu domain binds). In addition, we have shown directly that the purified cytosolic portion of IRE1 binds with high affinity to ribosomes. The notion that IRE1 may function on translating ribosomes is underscored by the independent discoveries of Plumb et al. (eLife 2015), who identified a contact between IRE1's lumenal domain and the Sec61 translocon. These investigators mapped and probed the functional importance of this interaction by mutational analyses and showed impairment in IRE1 function. Taken together, these results suggest that IRE1 can identify and cleave substrates co-translationally. In this proposal, we will focus on deepening our understanding of the IRE1-ribosome-SRP interactions, aiming to add solid structural and mechanistic understanding to lay the foundations that then can allow us to test the importance of these newly characterized interactions in vivo. To this end, we will (i) determine IRE1's engagement with different stages of the targeting/translocation process, (ii) isolate IRE1-engaged ribosomes from living cells and characterize their composition and translational status, (iii) determine the structure of IRE1 in complex with the co-translational targeting machinery by cryo- EM, and (iv) determine the functional significance of the IRE1-SRP and IRE1-ribosome interactions by complementary approaches employing both biochemical assays and in vivo mutagenesis approaches. In this way, we seek to build a conceptually new toolbox, which we expect will lead us to expand and refine-if not substantially revise-current models of IRE1 engagement in mammalian cells.

ABSTRACT Cognitive disorders pose a major threat to public health, and represent an enormous economic and social burden. Despite the high incidence of these disorders, effective treatments remain severely limited. Thus, the development of novel therapeutic to treat cognitive disorders is an important goal. Here we focus on the integrated stress response (ISR), a conserved signaling network that restores protein homeostasis by regulating protein synthesis, and a main causative pathway underlying the memory deficits associated with a wide range of cognitive disorders. The goal of this competing renewal is to define the precise molecular, cellular, and circuit mechanisms by which activation of the ISR leads to cognitive dysfunction. In Aim 1, we will generate and characterize mice carrying a human mutation in a key component of the ISR, which activates the ISR, and is associated with intellectual disability. In Aim 2, we will generate and use state-of-the-art, novel molecular- genetic approaches to identify the specific cell types in the brain driving the long-term memory deficits upon activation of the ISR. Finally, in Aim 3, we will develop a novel high-throughput screening platform to identify new inhibitors of the ISR. The results of these Aims will provide new fundamental insights into the biological basis of cognitive dysfunction and hold the hope of opening new therapeutic avenues for cognitive disorders resulting from perturbation of protein homeostasis.