2010 Progress Report – Executive Summary
The ultimate goal of our NDC is to engineer chaperonins with new functional properties, and/or substrate adaptor molecules, to prevent aggregation and/or refold proteins responsible for misfolded- protein diseases. Our approach is to develop and integrate methods to characterize the biophysical and biochemical properties of chaperonins, as well as to develop engineering strategies for designing chaperonins or substrates with new functionalities.
With the financial supplement and scale-up support, we now support 6 clinical investigators from Baylor College of Medicine, M.D. Anderson Cancer Center, University of California at San Diego, University of California at Irvine and the Stanford Medical School. These investigators have led us down an exciting pathway towards translational medicine. Each of our team members provides clearly defined complementary project niches and has assembled into several sub-groups to tackle our goal- relevant technical and biological fronts.
In the past year, we have made cutting edge progress in several experimental and computational methods for characterizing chaperonins and nanomachines in general. These include single particle cryo-EM at near atomic resolution, single molecule imaging to measure chemical kinetics, modeling based on low and medium resolution structures of highly flexible nanomachine made up of multiple molecular components at different functional states, and search of bifunctional adaptor molecules based on simulation and models. All these advances are critical to characterize the biophysical properties and functional activities of chaperonins and their substrates. All of these accomplishments meet our original milestones. Some of the developments are beyond our original anticipations. Most excitingly, we have undertaken the structure based design of 30 chaperonin variants some of which have novel biochemical and structural properties. Our clinical investigators are progressing steadily and methodologically in collaboration with our basic science investigators. Our major accomplishments include:
- We have obtained atomic models of archaeal Mm-cpn and mammalian TRiC chaperonins in ATP-induced state respectively from the cryo-EM density maps. This is the highest resolution cryo-EM structure determination of any biological nanomachine ever reported without a crystal structure of its components or domains. These provide the architectural description of the 16- mer subunits with which chaperonin variants and adaptors can be rationally designed.
- X-ray crystal structures of Mm-cpn were also obtained and cross-validate the cryo-EM structures. Both structures provide complementary information to be useful for engineering chaperonins and adaptors.
- 30 chaperonin variants have been generated according to the structural architecture of the Mm- cpn. Four of them have yielded interesting and unique functional behaviors different from the wild type chaperonin. This marks the beginning of the engineering phase of the project based on structural design of the wild type chaperonin.
- Single-molecule trapping shows unexpected characteristics of the ATP binding/hydrolysis cooperativity of the chaperonin. Such single-molecule measurements are important to characterize the biochemical behavior of chaperonins where ATP hydrolysis is critical. It also has applicability to many other nanomachines requiring ATP.
- The discovery that TRiC can rescue Huntingtin fibril aggregation has opened up much structural and computational research initiatives. For instance, cryo-ET of the Htt fibril incubated with TRiC suggests an unexpected mechanism of TRiC interacting with the fibril; super resolution optical microscopy has enabled us to see unprecedented details of the in vitro Htt aggregation as modulated by the chaperonins; a novel modeling method has been implemented to predict oligomerization behavior of Htt peptide at increasing length. All of these basic studies will contribute to the formulation of our strategy to prevent the Htt aggregation, a landmark of the Huntington’s Disease.
- A new computational method for modeling and engineering proteins and protein conformational changes, borrowing methods from robotics, is being developed and validated. The method shows sub-Ångstrom accuracy in predicting conformation of flexible region in proteins, which are often critical for function of enzymes and protein machines and for interactions with other cellular components (e.g. for chaperonin - adaptor interactions).
- Various methodology innovations have been made to improve the computational efficiency for computing the near atomic resolution cryo-EM maps for the chaperonins; to build reliable atomic models for the chaperonins with density maps at marginal resolutions; to segment domains and components of cryo-EM maps and to fit cryo-EM maps with PDB models at various resolutions; and to build model of nanomachine with a variety of bioinformatics and biophysical measurements. These technological advances will be deployed in the future biophysical characterization of chaperonins with substrates at various folding stages. All of our software is publicly accessible and will benefit a broad spectrum of scientific communities.
- Several newly initiated projects under the translational medicine have begun with steady progress. These projects are relevant to misfolded or protein aggregation resulting in a variety of diseases: cystic fibrosis, von Hippel Lindau disease, Down syndrome, Alzheimer’s disease, apoptotic cell death and cataracts. We have accomplished multiple milestone experiments that chaperonin has effects or binding affinity with the targeted proteins including cystic fibrosis regulator, Stat3 ?D crystallins and axonal transport. More rigorous experiments are in progress in each of these studies through collaborations among various labs.
- Armed with the computational prediction, adaptor molecules are being proposed and tested with a cellular screening assay for A?-involved disease.
- We have initiated collaboration with Steve Potkin, a clinician scientist at UC-Irvine to investigate whether chaperonin derivatives can be introduced to the mouse brain to reduce Htt aggregation toxicity.
- Experiments are being set up to use C. elegans as a model for testing the feasibility of adaptor- mediated rescue of the A? peptide toxicity.
- Preliminary results have been acquired with scientists in other NDCs (Mike Sheetz, Wendel Lim, and Ehud Isacoff) and the Roadmap on Computational Biology to leverage our complementary expertise to pursue our respective goals.
- We sponsored two workshops successfully on modeling nanomachines with cryo-EM maps at different resolutions and animation of nanomachines and cells. The workshops were held in Houston and San Francisco with attendance of 100 and 22 respectively.
- 5 outstanding undergraduates were recruited to do research in our 3 PIs labs in the summer of 2009.
- We published 12 manuscripts in the past year high profile journals such as Nature, PNAS, Nature Methods, Nature Structural and Molecular Biology, Structure and Journal of Molecular Biology. 6 manuscripts are pending for review. These investigations were funded by this NDC grant. 10 manuscripts were resulted from partial funding or synergistic effects from the NDC.
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