Fellowship for Tomoko Sengoku: Developing Novel Therapeutic Agents: Calpastatin and Protein Transductions Domains

  • Geddes, James (PI)
  • Sengoku, Tomoko (CoI)

Grants and Contracts Details


The long term goals of this proposal are to develop effective treatments against neurodegeneration using novel calpain inhibitors based on the endogenous calpain inhibitor, calpastatin. Synthetic calpain inhibitors have been shown in previous studies as effective treatments against cell loss following ischemia, however these inhibitors require invasive administration and high doses due to the relatively poor membrane permeability, potency and specificity. Calpastatin, on the other hand is up to 10x more potent and is specific for calpain. By linking calpastatin to a protein transduction domain (either from the HIV protein, tat or the herpes simplex virus protein, VP22) we will develop and characterize novel and potent calpain inhibitors. The specific aims of this proposal are as follows: Specific Aim #1: To develop novel calpain inhibitors based on the endogenous inhibitor calpastatin. By subcloning the calpastatin gene into various vectors we will link the calpastatin gene to either VP22 or TAT. Automated sequencing (Davis Sequencing) will verify the sequences and appropriate orientation of the genes. BL21 (DE3)pLysS bacterial strain will be used to express the fusion proteins. These fusion proteins will then be purified following a protocol developed by Becker-Hapack and colleagues (2001). Specific Aim #2: To determine the cellular localization of the fusion proteins. Primary rat hippocampal cultures isolated from timed pregnant Sprague Dawley rats will be exposed to various concentrations of the fusion proteins. At the appropriate timepoint (1Omin-24h) ICC using monoclonal antibodies against the 6xHis tag or against calpastatin will be performed. Specific Aim #3: To compare the efficacy of these novel inhibitors with synthetic inhibitors. Hippocampal cultures will be exposed to various concentrations of fusion proteins and then assayed for calpain activity using the synthetic substrate Succ-LLVY-AMC (Guttmann and Johnson, 2000). Western blots will also be used to determine both spectrin loss and levels of fusion protein transduction.
Effective start/end date7/1/026/30/04


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