2012 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 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.
Our group continues to evolve, moving from a more basic science-oriented team to one geared towards therapeutic applications, specifically translational and clinical investigation. We focus on two specific diseases: Huntington’s 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. We expect to impact the future treatment of a number of diseases that feature protein misfolding. 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:
- Demonstrated that CCTx modulates VHL.
- Showed VHL protein stability varies as a function of location and type of covalent adaptor.
- Identified proteasome inhibition as a potential therapeutic strategy for malfolded VHL/RCC.
- Developed clinical trial testing proteasome inhibitors in patients with VHL-mutated kidney cancer.
- We created and expressed human CCT1-8-mCherry fusion proteins. Expressing them in cultured cells transfected with wt or expanded repeat Htt (mHtt), we discovered that individual CCT subunits; a) increased the cellular level of TRiC; b) caused degradation of cellular mHtt97; and c) suppressed formation of insoluble mHtt103.
- It was possible to partially purify complexes that contained TRiC. They were discovered to harbor HSP90, other HSPs, and a number of TRiC client proteins.
- The structural basis for CCT subunit containing complexes is yet to be defined, but early findings in native gels suggest that complexes are quite large and possibly contain several subunits.
- Building on earlier findings we confirmed that exogenous application of purified ApiCCT1 modulates aggregation and cellular toxicity in vitro. Recent data suggests that this construct may act, in part, by sequestering mHtt in the medium of mHtt expressing cells.
- In preparation for in vivo work, AAV viruses encoding CCT1 and ApiCCT1 were constructed and a first round of injections was performed.
- To test for TRiC-inspired treatments in humans, human fibroblast from HD patients
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