2011 Progress Report – Executive Summary
The ultimate goal of our NDC is to develop therapeutic strategies for protein misfolding diseases by harnessing our understanding of chaperonin mechanism and function. To this end, we aim to engineer chaperonins with new functional properties, to develop chaperonin-inspired therapeutics and/or substrate adaptor molecules, to prevent aggregation and/or refold proteins responsible for misfolded- protein diseases. Our approach includes developing and integrating new 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.
Our group continues to evolve, moving from a more basic science-oriented team that defined design principles and fundamental properties of the chaperonin system, to one that encompasses a spectrum of research capabilities geared towards therapeutic applications, including translational and clinical investigation. We have also refined our focus on two specific diseases; Huntington disease (HD) and von Hippel Lindau disease (VHL). Our choice was based on the importance of these disorders with respect both to disease burden and to the opportunity to exploit their underlying biology to create new insights into the pathogenesis and treatment of related protein misfolding disorders. Thus, the work that we will undertake is expected to impact the future treatment of a number of devastating neurodegenerative diseases and other disorders. To achieve our goals, we are currently supporting clinical investigators from M.D. Anderson Cancer Center, University of California at San Diego and the University of California at Irvine, and translational researchers from the Massachusetts Institute of Technology.
Both HD and VHL Disease arise as a consequence of well-defined genetic lesions which cause misfolding of chaperonin substrates. However, the underlying molecular nature of their pathology is distinct: HD features the aggregation of a mutant protein that triggers a cascade of pathogenic mechanisms that appear to result in both gain of toxic function and loss of function; in contrast, VHL Disease arises from the loss-of-function of a folding-defective mutant protein. The translational approaches to remedy each disease through chaperonin-based intervention thus differ. In the case of HD, we will use the eukaryotic chaperonin and/or engineered chaperonin-derivatives to prevent the cellular toxicity associated with protein aggregation. In the case of VHL Disease, we will exploit our knowledge of chaperonin-mediated action to promote pVHL refolding and restore functionality to tumor-promoting mutants.
Our accomplishments in the past year include:
- We established cellular systems to assay the effects of individual CCT subunit(s) as well as small molecule modulators, on mHtt aggregation and cell toxicity: Htt and VHL levels, cell viability, and subcellular localization are being assessed with expression vectors for all eight human CCT subunits with their C-terminus fused to mCherry, a fluorescent marker.
- A pre-clinical mouse model for testing of CCT-based therapies is being developed using established Htt models, and the adoption of AAV-viral vector delivery systems.
- We developed an in vitro super-resolution optical imaging approach to study Htt fibrils produced in solution phase. This will permit visualization of Htt-fibril formation under the influence of potential therapeutic agents.
- An in silico screen to identify compounds that interact with the ATP binding site of TRiC has been developed. Several candidates identified are being tested in cell-based systems.
- Using off-patent compounds, we identified three candidates that increased levels of mutated VHL using a cell based screen. We characterized the structures of the intact Mm-Cpn and TRiC chaperonins in different nucleotide binding states using cryo-EM and X-ray crystallography; we also determined the structure of the apical domain of the TRiC subunit using NMR.
- Our clinical team established outreach to the VHL and Htt community to develop a clinical trials infrastructure for chaperonin based research.
- We published 27 manuscripts in the past year, including manuscripts in Cell, Nature and Nature Protocols, PNAS. These investigations were funded by this NDC grant.
The accomplishments outlined above form the foundation of our ongoing efforts to accelerate our translational development path. By developing the tools required to modulate and measure activity of TRiC and its impact on TRiC and VHL substrates in ex vivo, cellular and animal model systems, we are well positioned to undertake our proposed investigation for the following years.
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