title: Proteasome-cytochrome c interactions: a model system for investigation of proteasome host-guest interactions.
abstract: Owing to its high thermal stability and structural simplicity, the archaebacterium Thermoplasma Acidophilum 20S proteasome was selected for mechanistic studies in this work. This oligomeric enzyme complex consists of a barrel-shaped 20S core (approximately 700kDa) comprised of four stacked seven-membered rings with a alpha(7)beta(7)beta(7)alpha(7) subunit structure situated around a 7-fold symmetry axis. The hollow interior of the proteasome has three large interconnected chambers with narrow (13 A diameter) entrances from solution located at either end of the barrel. The 14 beta-subunit proteolytic sites are located on the inner surface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome c (Cyt c) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditions of temperature and denaturant which unfold Cyt c but do not alter the thermophilic proteasome, Cyt c is extensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasome digest. Analysis of the cleavage products established that unfolded Cyt c and its covalently attached heme prosthetic group are translocated to the central chamber where proteolysis occurs. In the presence of site-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome c can be sequestered inside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis of the complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cyt c is essentially intact within the inhibited proteasome. High-performance liquid chromatography data show that (1) complexes with an apparent stoichiometry of approximately one Cyt c per proteasome can be formed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt c occurs.
authors.affiliation: Department of Biochemistry, University of California, Riverside, California 92521, USA.
authors.forename: Holly A
authors.initials: HA
authors.lastname: Huffman
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Sadeghi
authors.forename: Erika
authors.initials: E
authors.lastname: Seemuller
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013053:Spectrophotometry; D013696:Temperature; D013822:Thermoplasma / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D003574:Cytochrome c Group; D009097:Multienzyme Complexes; D010455:Peptides; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex