| Project/Area Number |
15590274
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Pathological medical chemistry
|
|---|
| Research Institution | The University of Tokushima |
|---|
Principal Investigator |
OKAMURA Kazuko The University of Tokushima, Institute for Enzyme research, Instructor, 分子酵素学研究センター, 助手 (10108863)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TANIGUCHI Hisaaki The University of Tokushima, Institute for Enzyme research, Professor, 分子酵素学研究センター, 教授 (10257636)
SHIMOZAWA Nobuyuki Gifu University, School of Medicine, Associate Professor, 医学部, 助教授 (00240797)
FUJIWARA Kazuko The University of Tokushima, Institute for Enzyme research, Associate Professor, 分子酵素学研究センター, 助教授 (20108880)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Project Status |
Completed (Fiscal Year 2004)
|
| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥3,000,000 (Direct Cost: ¥3,000,000)
|
|---|
| Keywords | Lon protease / Peroxisome / Chaperon / Serine protease / ペルオキシソーム移行シグナル |
|---|
| Research Abstract |
Human peroxisomal Lon protease (pxLon), recently found from proteomic analysis of rat liver peroxisome, is homologous to the mitochondrial isoform, but distinct from the isoform in that it contains a C-terminal SKL sequence that functions as a peroxisomal import signal (PST1). To investigate the function in the peroxisome biogenesis, we expressed human pxLon with His-tag at the N-terminus in E. coli. Although a large part of recombinant pxLon was found in inclusion body, some was found in soluble fraction. The soluble protein was partially purified by column chromatography with TALON Metal Affinity Resin and subsequent gei filtration. It showed ATPase and ATP-dependent peptidase and protease activities. The substrate specificity was determined using Fluorescence Resonance Energy Transfer Substrates (FRETS-25Xaa series, Peptide Institute Inc., Osaka, Japan) according to the protocol provided by the manufacturer. pxLon showed the elastase-like substrate specificity, which preferably cut
… More
the peptide bond at the C-terminal side of alanine residue, distinct from that of E. coli Lon protease. Some peroxisomal matrix protein has another import signal (PTS2) at the N-terminus and it is removed after the import to matrix. The enzyme that cut off the PTS2 with consensus sequence of Arg/Lys-Leu/Val/Ile-X5-His/Gln-Leu/Ala has not been identified so far, and pxLon may be the candidate of the signal peptidase. Furthermore, the putative hexamer form of pxLon prevented citrate synthaese from hear treatment -induced aggregation. These results indicated that pxLon might play important role for the quality control of the peroxisome by prevention of the denaturing of the proteins or the removal of oxidatively modified proteins by proteolytic digestion, since peroxisomal proteins are highly subjected to oxidative modification and inactivation. The function of the C-terminal SKL sequence as peroxisomal import signal was verified by expressing full-length and ΔSKL mutant pxLon as fusion protein with the N-terminal FLAG-tag and determining their intracellular localization. Less
|
