Center for Protein Folding Machinery
Executive Summary
Naturally occurring cellular nanomachines called chaperones fold many critical cellular proteins in all human and animal cells [2, 3]. Different classes of chaperones work together to form elaborate cooperative networks and also ensure that potentially damaging misfolded polypeptides are cleared from the cell. Such misfolded proteins would otherwise cause a cascade of cellular damage and ultimately lead to globalized cell death. Our center envisions using chaperones as important targets for therapeutic interventions or diagnostic analyses in several human diseases related to protein misfolding. In particular, chaperonins represent a particularly intriguing class of chaperones because they possess a unique ability to fold some proteins that are not folded by more simple chaperones. We are focusing on the type II molecular chaperonins, namely, the eukaryotic chaperonin, TRiC/CCT and the archaebacterial chaperonin, Mm-cpn. We aim to understand the folding properties of a broad array of eukaryotic proteins that will establish an essential foundation for designing and optimizing proteins with novel functions.
Introduction
Human cells contain many structurally and functionally distinct classes of chaperones of varying size and complexity. These range from those that only bind misfolded polypeptides and prevent their aggregation, to those that recognize specific classes of folding intermediates and promote their folding to the proper native state in an energy dependent manner [4]. Hundreds of enzymes depend on fully-functioning chaperones. As our population grows older, an increasing socio-economic burden stems from a class of diseases resulting from protein misfolding and protein aggregation. Millions of Americans suffer from the most common of these: Alzheimer's disease, Parkinson's disease, and Huntington's disease. In addition to such neurodegenerative diseases, folding defects play important roles in stroke, various types of cancer, and cataract formation. Protein chains that are required for healthy cell and organ function can misfold and ultimately aggregate into toxic fibers and large complexes in all these diseases [1].
An intriguing class of oligomeric, double-ring, high molecular weight chaperones, called chaperonins, possess a unique ability to fold some proteins that otherwise could not be folded by simpler chaperone systems. The specific protein folding nanomachine that our NDC has chosen is called the type II molecular chaperonin, which includes the eukaryotic chaperonin, TRiC/CCT, and the archaebacterial chaperonin, Mm-cpn.
The human chaperonin (TRiC) is essential for de novo folding of approximately 10 percent of newly synthesized proteins. TRiC acts on proteins of high biomedical relevance such as cytoskeletal components (actin and tubulin), cell cycle regulators (cyclin E and Cdc20), and many regulatory proteins containing a beta-propeller domain. Notably, many of these TRiC substrates cannot be folded by other prokaryotic or eukaryotic chaperones. TRiC and Mm-cpn differ in complexity. Although each consists of two eight-membered rings with a total molecular mass close to 1 MDa, eight different proteins form the ring in TRiC. In contrast, the archaebacterial chaperonin Mm-cpn contains eight copies of a single protein. These barrel-shaped chaperonins open and close their central chamber in response to ATP-binding and hydrolysis. We believe that re-engineering chaperonins could present a promising and cell-friendly bio-delivery container for drugs and nano-devices for therapeutic, diagnostic or industrial purposes. Describing the conformational dynamics of this process is important for understanding and controlling protein folding inside living cells.
NDC Aims
The overarching goal of our NDC is to develop chaperonin-based nanomachines to manipulate protein folding pathways for therapeutic and biomedical applications. A quantitative description of the motions of the chaperonin in a sequence of biochemical events during the protein folding process will provide the necessary specifications to permit a rational design of new chaperonins or their substrates. This will open up new therapeutic avenues, such as:
- New anti-tumor therapies by designing small molecule adaptors to enable cellular chaperonins to correct folding defects in tumor suppressor proteins
- New anti-amyloid therapies by selecting small molecules which increase the affinity of cellular chaperonins to prevent polyglutamine aggregation in Huntington's and related amyloid diseases
- Modifying the chaperonin machinery to more effectively fold pharmaceutically important target proteins that currently cannot fold into their native state in the laboratory.
Our NDC has 15 established investigators from six institutions. We take an integrated approach to study these chaperonins by combining the most suitable physical, chemical, computational and engineering methods for measurements and analysis, including cryo-electron microscopy and tomography, single-molecule spectroscopy and imaging, restrained protein structure modeling, protein dynamics simulation, in silico protein folding in the chaperonin environment, and protein engineering design. We continue developing these methodologies to suit the studies of this relatively large and structurally dynamic cellular nanomachine both in vitro and in vivo, and we are certain that these technologies will have wide ranging impact for the study of many large cellular nanomachines.
Operationally, we aim to establish a highly inter-dependent working relationship among all the investigators who specialize in different experimental and computational methodologies (Figure 1). All of our research priorities are driven by the goal of producing translational outcomes. We recognize that it is necessary to work together cohesively and to co-develop research strategies in tackling the fundamentally important and technically challenging problem of manipulating chaperonins. We believe that our team has created a novel research pathway that is optimal for studying cellular nanomachines in general and will lead to more rapid development of medical applications. Furthermore, an important aspect of our center is our constant attention to integrating the results from various methodologies to stimulate collaborations among the participating scientists in our NDC. For instance, there is ongoing interaction between the experimental and computational investigators at various stages of the project. In addition, our team is exploring a design engineering approach to describe how the chaperonin works as a nano-device in their folding cycle and how an engineered chaperonin or substrate can be developed. Consequently, our studies will create approaches and strategies with translational outcomes.
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- Figure 1. Approaches of the NDC which is organized with synergistic interactions across disciplines and laboratories in six institutions.
Accomplishments
We have made dramatic progress in developing novel biophysical and computational methodologies to sample the conformational space of the chaperonins and their interactions with substrates. Our efforts place particular emphasis on achieving the integration of biological, physical and computational approaches of our team. Frequently, two or more independent labs collaborate on experiments and discussion sessions, exchange visits, hold web-based seminars and sub-group meetings. These have been instrumental in inter-disciplinary collaboration, training and seeding new ideas for both technological and conceptual advances. Our findings in the past two years have produced several exciting technological breakthroughs. We also discovered previously unexpected biological properties of TRiC that have a clear relevance for the projects that are more medically oriented. The highlights of our advances are summarized as following:
Finding the molecular switch to open and close the TRiC chamber (Chiu, Ludtke, Sali, Levitt and Frydman).
TRiC has a built-in lid that can be open or closed, depending on its folding stage. We have combined biochemical, structural and computational approaches to pinpoint the location of a mechanical switch that underlies the conformational cycle of TRiC. This mechanical switch will become an important performance specification in our design of the chaperonins with new functionality.
Proposing a Two-Stroke allosteric model for the Mm-cpn built-in lid (Frydman, Chiu).
Based on biochemical and cryoEM study of Mm-cpn, we propose that the built-in lid caused a remodeling of the chaperonin allosteric network whereby the apical protrusions communicate conformational changes between subunits. The regulatory functions of the lid may provide a timer for substrate encapsulation within the closed chamber.
Detecting the chemical behavior of ATP bound to chaperonins (Moerner, Frydman).
With the anti-Brownian electrophoretic ABEL trap, a new device developed in the Moerner lab that holds single molecules in aqueous solution, we show its application to single chaperonins for obtaining a detailed description of the distributions of their behavior, rather than only ensemble averages. Such measurements will be useful for assessing the new behaviors of normal chaperonins relative to the redesigned ones.
Determining the specificity of TRiC binding to medically relevant substrates (Frydman)
The Frydman lab has identified the TRiC subunits that interact with two medically relevant substrates: the tumor suppressor protein VHL and the amyloidogenic protein Huntingtin, which underlies Huntington Disease. These findings potentially open the way for therapeutic intervention to restore protein homeostasis caused by pathogenic mutations in these proteins.
Discovering a new type of chaperonin-substrate interaction (Moerner, Frydman).
Moerner and Frydman have used single molecule fluorescence resonance energy transfer (FRET) to probe the interactions of VHL tumor suppressor protein with TRiC. The fluorescence anisotropy results suggest that VHL remains associated with the apical domain of TRiC after lid closure, rather than being released into the cavity. This is clearly an unanticipated type of substrate-chaperonin interaction. We plan to use cryoEM to validate this hypothesis.
Modeling the effect of the chaperonin cavity on folding of bound proteins (Pande, Frydman).
Pande's group has computationally studied the solvent effects on the folding reaction. The simulation findings suggest that one function of chaperonins may be to trap unfolded proteins and subsequently expose them to a micro-environment in which the hydrophobic effect, a crucial thermodynamic driving force for folding, is enhanced. Such predictions are being verified experimentally in the Frydman lab.
Progressing towards the design of a molecular adaptor between chaperonins and substrate interfaces (Kortemme, Frydman).
Kortemme's simulations have predicted that residues in two TRiC subunits are critical for binding VHL tumor suppressor protein. This prediction has been confirmed experimentally in the Frydman lab. This illustrates the successful interplay between modeling and experiment which is the recurring thematic style of our NDC.
Discovering chaperonins as misfolding and aggregation inhibitor for Huntington Disease (Frydman).
An unexpected finding in the Frydman lab is that TRiC plays a major role in preventing the formation of toxic amyloidogenic aggregates. Their experiments indicate that depletion of TRiC in yeast or in mammalian cells enhances polyglutamine aggregation. Importantly, over-expression of a single TRiC subunit, CCT1, is sufficient to remodel Htt aggregate morphology in vivo and in vitro; importantly it also reduces Htt-induced toxicity in neuronal cells. These results have intriguing implication for using chaperonin as a therapeutic agent for this disease.
Archeal chaperonins bound to lens protein (King, Frydman).
King's lab demonstrated that the Mm-cpn purified in the Frydman lab inhibited aggregation of partially folded HγD-Crys. This result implicates the possibility that Mm-cpn and TRiC may be able to actively refold γ-crystallins, offering an alternative pathway to recover from aggregation-inducing stresses. This subproject is high risk and typifies our style of an unconventional approach.
Producing various technological advances and innovations (Ludtke, Sali, Adams, Kortemme, Pande, Levitt, Gossard and Chiu).
Ludtke introduced a novel image reconstruction method that revealed a conformational mixture during a folding reaction inside the chaperonin. Sali developed a new computational method for predicting sequential ordering and molecular interactions among subunits in TRiC. Adams and Sali are integrating small angle X-ray scattering data and modeling for structure determination of cellular nanomachine and substrates. Kortemme is developing new tools to model complex protein interaction interfaces. Pande is modeling the dynamic behavior of cellular nanomachines in a confinement. Levitt is exploring deformable elastic network theory to simulate the conformational transition of the chaperonin with the cryoEM constraints. Gossard and Chiu are producing animations of protein folding event which can aid design of new chaperonins and substrates.
Nanomedicine Teleseminars (Gossard, King).
Weaving together research groups that are geographically separate and in addition represent diverse disciplines requires an ongoing internal educational component. We have established a monthly series of Nanomedicine Teleseminars, which involve real-time participation of the various groups. We have Webex to link together all participating research groups. Generally a site is chosen and graduate students and postdocs gather together to listen to the presentation and ask questions. The first series, this year, many of the PIs gave overviews of their arenas of expertise (http://proteinfoldingcenter.org/nanomedicine/lectures).
Educational Outreach (Chiu, King, Gossard).
Since its inception, our NDC has sponsored various technology workshops and symposia in national meetings (e.g. Biophysics Society and Protein Society), retreats and summer undergraduate research. These have become an integral activity of our NDC which has a broad impact on many students and postdoctoral fellows beyond the laboratories of the NDC key investigators.
Future Applications
Our Center has realized the important mission to translate our basic research to clinical and biotechnology applications. Therefore, two of our key clinical investigators (Eric Jonasch at MD Anderson Cancer Center and Huda Zoghbi at Baylor College of Medicine) were included as participants in our original proposal. Our plan is to engage them in the third year on our research discussions and to advise us on medical applications of our findings.
Eric Jonasch's clinical research interest is in von Hippel Landau Disease (VHL). The VHL suppressor protein has been shown to require TRiC to fold properly. Tumor-causing mutations within the TRiC-binding sites of the VHL tumor suppressor lead to severe misfolding of VHL suppressor in vivo. It thus suggests that disease-causing mutations may inactivate protein function by interfering with chaperonin-mediated folding. Therefore, VHL is a logical target to pursue in our goal to engineer a chaperonin or adaptor to correct for the misfolds of the mutant VHL that leads to VHL disease. Eric Jonasch with Judith Frydman and Tanja Kortemme are developing a set of pilot experiments in testing the approach of designing molecular adaptor between VHL and TRiC. A functional screen is being designed to discover linker molecules that alter the interaction between mutated pVHL and TRiC, with the goal of restabilizing the mutated protein and normalizing its function within the cell.
Huda Zoghbi's clinical interest pertains to polyglutamine neurodegenerative diseases. In these diseases, an expansion of a polyglutamine tract alters the conformation of the host protein causing it to have altered activity that is detrimental to neurons. There are two phases to the disorders, one that causes neuronal dysfunction from aberrant interactions, and another that involves induction of stress response secondary to the aberrant interactions and eventually leads to protein aggregation. We have discussed the possibility of using chaperonins to help fold the protein. The enthusiasm generated between Huda Zoghbi and our participating investigators has been sufficiently high that strategic experiments are being contemplated.
References
Background
- Dobson, C.M., The structural basis of protein folding and its links with human disease. Philos Trans R Soc Lond B Biol Sci, 2001. 356(1406): p. 133-45.
- Hartl, F.U. and M. Hayer-Hartl, Molecular chaperones in the cytosol: from nascent chain to folded protein. Science, 2002. 295(5561): p. 1852-8.
- Frydman, J., Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem, 2001. 70: p. 603-47.
- Dobson, C.M., Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol, 2004. 15(1): p. 3-16.
Publications with acknowledgement to NIH Roadmap grant support
- Spiess, C. Miller, E.J. McClellan, A.J. and Frydman, J. (2006). Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins. Molecular Cell 24:25-37.
- Reissmann, S., C. Parnot, C.R. Booth, W. Chiu, and J. Frydman (2007). Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins. Nat Struct Mol Biol 14: 432-40.
- Chen, D. H., Song, J. L., Chuang, D. T., Chiu, W. & Ludtke, S. J. (2006). An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate. Structure 14: 1711-22.
- Marsh, M. P., J. T. Chang, C. R. Booth, N. Liang, M. F. Schmid, W, Chiu (2007). Modular software platform for low-dose electron microscopy and tomography. J Microscopy, in press.
Contact Information
Administrative Contact
- Lenora Trujillo (lenorat@bcm.edu)
Phone: 713-798-2191, Fax: 713-798-8682
Key Investigators
- Baylor College of Medicine, Houston, Texas
Wah Chiu (wah@bcm.edu)
Steve Ludtke (sludtke@bcm.edu)
Huda Zoghbi (hzoghbi@bcm.edu) - Stanford University, Palo Alto, California
Judith Frydman (jfrydman@leland.stanford.edu)
Vijay Pande (pande@stanford.edu)
W.E. Moerner (wmoerner@stanford.edu)
Michael Levitt (michael.levitt@stanford.edu)
- University of California, San Francisco, California
Andrej Sali (Sali@salilab,org)
Tanja Kortemme (kortemme@cgl.ucsf.edu) - Lawrence Berkeley National Laboratory, Berkeley, California
Paul Adams (PDAdams@lbl.gov) - Massachusetts Institute of Technology, Cambridge, Massachusetts
Jonathan King (jaking@MIT.EDU)
David Gossard (gossard@MIT.EDU) - M.D. Anderson Cancer Center, Houston, Texas
Eric Jonasch (ejonasch@mdandersin.org)
