| Budget Amount *help |
¥16,840,000 (Direct Cost: ¥15,100,000、Indirect Cost: ¥1,740,000)
Fiscal Year 2007: ¥7,540,000 (Direct Cost: ¥5,800,000、Indirect Cost: ¥1,740,000)
Fiscal Year 2006: ¥9,300,000 (Direct Cost: ¥9,300,000)
|
|---|
| Research Abstract |
Calpains are a cytosolic Ca^<2+>-regulated cysteine protease. Calpains directly interact with intracellular proteins to modify or transform functions and/or activities of their substrates, and are thus called "modulator protease". Mammals have 15 independent genes for calpains, about half of which are expressed tissue specifically. These tissue-specific calpain are expected to be involved in specific functions developed in the specific tissues, and, therefore, their physiological functions are of great interest. Two of the tissue specific calpains, nCL-2/-2' and nCL-4, are predominantly expressed in digestive tracts, especially, in the epithelial mucus secreting cells. These cells protect the stomach and intestines from acid/proteases and bacteria. Therefore, these calpains' functions are considered to be related with mucus secretion mechanisms.
To elucidate physiological functions of these calpains, we constructed nCL-2: C105S "knock in" mice, which express a protease inactive mutant of nCL-2 instead of wild type nCL-2 under endogenous expression controls. A missense mutation corresponding to the Cys105 to Ser was introduced in the mouse genomic DNA, and neomycin-resistance gene (neoR) flanked by loxP sequences was inserted in the vicinity of the C105S mutation. After homologously targeted mice were obtained, they were crossed with Cre-recombinase expressing transgenic mice to excise neoR, resulting in nCL-2: C105S knock-in mice.
Using these knock-in mice and their tissues, various biochemical, cell-biological, and molecular biological analyses have been performed. As a result, nCL-2 was shown to form not only monomer but also homo-oligomers via C2-domain like domain (domain III). This novel structure suggests unique functions for nCL-2 in digestive tracts.
|
