Website: http://weissmanlab.ucsf.edu Email: jonathan.weissman / ucsf, edu
1700 4th Street, MC 2542Byers Hall, Room 403San Francisco, CA 94158-2542
Administrative AssistantJoan Kanter415 514-1681joan.kanter / ucsf, edu
Summary: Jonathan Weissman is looking at how cells ensure that proteins fold into their correct shape, as well as the role of protein misfolding in disease and normal physiology. He is also developing methods for assaying the entire complement of proteins derived from an organism.
To ensure proper folding, cells have evolved a sophisticated and essential machinery of proteins called molecular chaperones that assist the folding of newly made polypeptides. The importance of proper protein folding is underscored by the fact that a number of diseases, including Alzheimer’s and those involving infectious proteins (prions), result from protein-misfolding events. My research focuses on identifying and understanding the machinery necessary for efficient folding, as well as studying the mechanism and consequences of protein misfolding.
Mechanism of GroEL and Its Eukaryotic Homologs Chaperonins, such as Escherichia coli GroEL, are large ring structures that aid folding of a wide variety of proteins in the cytosol of all organisms. The long-term goal of our chaperonin studies is to understand the physical basis by which chaperonins assist folding, as well as how the particular “cocktail” of chaperones present in a given cell helps determine the spectrum of proteins that can fold. My studies with Arthur Horwich (HHMI, Yale University School of Medicine) revealed a remarkable mechanism in which GroEL inhibits aggregation by providing a protected environment where folding can occur while a polypeptide remains sequestered within the GroEL central cavity.
In addition to inhibiting aggregation, however, chaperonins can also actively catalyze folding of some proteins. Intriguingly, different chaperonins vary greatly in their ability to promote folding of a given substrate. To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL and GroES variants that dramatically enhance folding of a single substrate—green fluorescent protein (GFP). These findings suggest a surprising plasticity of GroEL and GroES, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins, as increased GFP folding comes at the expense of the ability of GroEL and GroES to fold their natural substrates. This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems. We are exploring this question by expanding the repertoire of substrate-optimized chaperones.
The ER is a highly specialized folding compartment responsible for the structural maturation of all secreted proteins. The long-term goal of our ER studies is to understand how the ER maintains an environment that supports folding. To support efficient disulfide formation, the ER must closely regulate its redox potential. Although the chemical requirement for an oxidizing environment has been appreciated for over 30 years, the mechanism by which this is accomplished was unknown. Our studies demonstrated that there is a specific cellular redox machinery required for disulfide-linked protein folding in the ER and identified Ero1p, a novel and ubiquitous protein, as an essential component of this machinery. Recently we have succeeded in reconstituting Ero1p-mediated oxidative folding in vitro with pure components. Strikingly, despite the strong parallel between the oxidative-folding machinery in eukaryotic ER and bacterial periplasm, our results establish that the underlying biochemical mechanisms are unrelated.
To obtain a more global view of how efficient folding is achieved in the ER, we have exploited DNA microarray “chips” to examine the transcriptional response to accumulation of unfolded proteins in the ER. Our studies dramatically changed the view of the unfolded protein response (UPR). Rather than primarily up-regulating molecular chaperones, the UPR results in a concerted reorganization of the ER, altering polypeptide flux from the cytosol to ER, retrograde export from the ER, transport of proteins from the ER to the Golgi, and retrieval of ER proteins from the Golgi. We are extending our studies of the regulation of protein folding in the ER to metazoan systems by combining traditional cell biological and biochemical techniques with genomic approaches, including microarrays and comprehensive RNA interference libraries.
In contrast to the more frequently observed disordered aggregates, some proteins form ordered aggregates, termed amyloid fibrils, that accumulate in a number of human diseases. Our goal is to provide a mechanistic understanding of how amyloid fibers propagate, to elucidate how molecular chaperones control amyloid growth in vivo, and to determine the role of amyloid formation in both disease and normal physiology. Efforts to understand amyloid propagation have been hampered by the lack of a facile genetic or biochemical system for studying this process. This situation has improved greatly with the finding that the [URE3] and [PSI+] states of yeast result from the prion-like aggregation of endogenous proteins. My laboratory has taken advantage of the PSI phenomenon to identify and characterize properties of the protein Sup35p that allow it to propagate a ?-sheet–rich prion form. We are now establishing a more general set of tools for studying amyloid formation of disease-related proteins in yeast.
Our studies also suggest that a common mechanism underlies the prion-like propagation of PSI and polyglutamine-mediated pathogenesis of several neurodegenerative diseases. More recently, our research on the physiological role of PSI argues that rather than being pathogenic, the PSI prion is an evolutionarily selected mechanism of inheritance. Finally, we have exploited our conservation and mutational data on Sup35p to develop algorithms for searching genomic databases for novel prion-forming proteins.
The development of microarray technology has revolutionized biology by making it possible to quantitate the abundance of mRNAs. RNA messages, however, are imperfect proxies for the information that is more directly related to function, such as the abundance and activity of the proteome.
In collaboration with Erin O’Shea (HHMI, University of California, San Francisco), we are developing methods that allow us to determine the function, localization, and abundance of the complete complement of proteins in an organism. Our initial efforts have focused on the production of two comprehensive yeast libraries in which we attach a carboxyl-terminal tag to each gene in its native chromosomal context. The collections consist of ~6,000 yeast strains, each expressing a single protein fused to either the tandem affinity purification (TAP) tag, which provides an extremely high affinity epitope tag, or to the GFP, which allows us to monitor protein localization. In collaboration with the O’Shea lab, we have used these collections to determine the localization and abundance of the yeast proteome. Our labs are now focusing on a range of subjects, including following the regulation of protein abundance across the cell cycle, the lifetime of each protein, and the level of expression “noise,” as assessed by the cell-to-cell variations in protein levels. This work is supported by grants from the National Institutes of Health, the David and Lucile Packard Foundation, and the Searle Scholars Program.