Proteasome-Cytochrome c Interactions: A Model System for Investigation of Proteasome Host-Guest Interactions
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- 作者：Holly A. Huffman ; Mehrnoosh Sadeghi ; Erika Seemuller ; Wolfgang Baumeister ; and Michael F. Dunn
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：July 29, 2003
- 年：2003
- 卷：42
- 期：29
- 页码：8679 - 8686
- 全文大小：265K
- 年卷期：v.42,no.29(July 29, 2003)
- ISSN：1520-4995
文摘
Owing to its high thermal stability and structural simplicity, the archaebacterium ThermoplasmaAcidophilum 20S proteasome was selected for mechanistic studies in this work. This oligomeric enzymecomplex consists of a barrel-shaped 20S core (~700kDa) comprised of four stacked seven-memberedrings with a 
rs/alpha.gif" BORDER=0>7rs/beta2.gif" BORDER=0 ALIGN="middle">7rs/beta2.gif" BORDER=0 ALIGN="middle">7rs/alpha.gif" BORDER=0>7 subunit structure situated around a 7-fold symmetry axis. The hollow interior ofthe proteasome has three large interconnected chambers with narrow (13 Å diameter) entrances fromsolution located at either end of the barrel. The 14 rs/beta2.gif" BORDER=0 ALIGN="middle">-subunit proteolytic sites are located on the innersurface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome c (Cytc) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditionsof temperature and denaturant which unfold Cyt c but do not alter the thermophilic proteasome, Cyt c isextensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasomedigest. Analysis of the cleavage products established that unfolded Cyt c and its covalently attached hemeprosthetic group are translocated to the central chamber where proteolysis occurs. In the presence ofsite-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome c can be sequesteredinside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis ofthe complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cytc is essentially intact within the inhibited proteasome. High-performance liquid chromatography data showthat (1) complexes with an apparent stoichiometry of approximately one Cyt c per proteasome can beformed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt coccurs.
rs/alpha.gif" BORDER=0>7rs/beta2.gif" BORDER=0 ALIGN="middle">7rs/beta2.gif" BORDER=0 ALIGN="middle">7rs/alpha.gif" BORDER=0>7 subunit structure situated around a 7-fold symmetry axis. The hollow interior ofthe proteasome has three large interconnected chambers with narrow (13 Å diameter) entrances fromsolution located at either end of the barrel. The 14 rs/beta2.gif" BORDER=0 ALIGN="middle">-subunit proteolytic sites are located on the innersurface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome c (Cytc) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditionsof temperature and denaturant which unfold Cyt c but do not alter the thermophilic proteasome, Cyt c isextensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasomedigest. Analysis of the cleavage products established that unfolded Cyt c and its covalently attached hemeprosthetic group are translocated to the central chamber where proteolysis occurs. In the presence ofsite-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome c can be sequesteredinside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis ofthe complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cytc is essentially intact within the inhibited proteasome. High-performance liquid chromatography data showthat (1) complexes with an apparent stoichiometry of approximately one Cyt c per proteasome can beformed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt coccurs.