| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
This project was planned to determine the relationship between structures and functions of ER-60 protease (ER-60) which was isolated from the endoplasmic reticulum (ER) of rat liver and characterized by the head investigator.
Between 1998-1999, the experiments described below were carried out.
1. ER-60 contains seven cysteine residues, four of which constitute two CGHC motifs which were assumed to include an active center cysteine residue (s) of the proteases. From the experiment with the recombinant human ER-60 with site-directed mutations of the C-terminal cysteine residues (Cys-60 and Cys-409) of the CGHC motifs to alanine, these cysteine residues were suggested to be an active center cysteine residue(s). The double-mutated enzyme with Cys-60 and Cys-409 both modified to alanine (C60A/C409A) showed no activity. The single-mutated enzymes, C60A and C409A exhibited activity. These results suggest the C-terminal cysteine residues of the CGHC motifs are responsible for the protease activity.
2. ER-60 has been demonstrated to associate with substrate proteins (lysozyme or ApoB-100) or molecular chaperones (BiP and protein disulfide isomerase) in the ER lumen. The interaction of the recombinant human ER-60 and lysozyme or luminal molecular chaperones was analyzed with a surface plasmon resonance apparatus, BIAcore 2000. A specific, but not strong, interaction was observed between ER-60 and lysozyme, BiP, protein disulfide isomerase or calreticulin. Their kinetic parameters of the association and dissociation were different from each other. It was demonstrated that binding properties of BiP with unfolded polypeptide or ER-60 were different.
3. The truncated ER-60 (K417 ochre), which was misfolded in the ER, was expressed in the COS-1 cell and determined the its fate after de novo synthesis. The overexposed K417 ochre was accumulated as insoluble aggregates. The stably expressed K417 ochre was degraded by a protease (s) inhibited by ALLN in the ER.
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