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.

20S Proteasomes Have the Potential to Keep Substrates in Store for Continual Degradation

“…Specifically, it implies that in cases where substrate unfolding or translocation is not the rate-limiting step, as in this model system, storage must take place concurrently in both antechambers while the processive degradation is carried out within the catalytic chamber. Interestingly, accumulation of substrate does not appear to affect the proteolytic activity of the proteasome, because when a reversible inhibitor was employed Cyt c was digested as normal (25). It is possible therefore that the antechambers retain proteins in a partially folded state, allowing them to enter the catalytic chamber as soon as space allows (41).…”

“…Formation of the host-guest proteasome complex was monitored using their spectroscopic signatures, 409 nm (Cyt c) and 395 nm (GFP) as previously described (25). Briefly, 20S proteasomes (1 M) were inhibited with clasto-lactacystin-␤-lactone (70 M) for 30 min at 22°C prior to mixing with substrate.…”

“…To trap substrate molecules within the T. acidophilum proteasome we formed host-guest complexes between the 20S proteasome and either cytochrome c (Cyt c) (25) or green fluorescent protein (GFP). To prevent degradation of the two substrates the proteasome was covalently inhibited.…”

Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex

“…From the GEMMA data, it appears that a distribution of myoglobin molecules is trapped by the inhibited proteasome. Although a previous study suggested that a single protein substrate, 12 kDa cytochrome c, can be sequestered intact in an inhibited proteasome complex, the heme group of cytochrome c was found to form a "knot" that could sterically hinder substrate processing [52]. Apomyoglobin, lacking the heme group, may be less sterically hindered from entering the 20S proteasome even from both ends of the complex.…”

“…As suggested by Dunn and coworkers [52], a potential protein substrate can be trapped within a proteasome core without proteolysis by using an irreversible proteasome inhibitor. Lactacystin binds specifically to Thr-1 of the ␤-subunit in a covalent manner to impart protease inhibition of the 20S proteasome.…”