Prep aration of Proteasomes
The proteasome is a protein-destroying machine capable of degrading a variety of proteins involved in the regulation of diverse processes, such as the cell cycle, immune response, signaling cascades, and developmental programs in various eukaryotes (Hershko and Ciechanover, 1998; Kloetzel, 2001; Rock et al., 2002). Early studies identified the proteasome as a latent protease complex with a sedimentation coefficient of 20S, and it was accordingly named the 20S proteasome (Coux et al., 1996). The 20S proteasome is a barrel-like particle formed by the axial stacking of four rings made up of two outer α rings and two innerβ rings, being associated in the order of αββα. The active sites reside in a chamber formed by the centers of the abutting β rings (Bochtler et al., 1999; Unno et al., 2002). The latency of the proteasome may be explained by the tertiary structure, as the center of the α ring is almost closed, preventing penetration of proteins into the inner aspect of the β ring on which the proteolytically active sites are located (Fig. 1). The 20S proteasome possesses a variety of catalytic centers that presumably contribute to the hydrolysis of multiple peptide bonds in single polypeptide substrates by a coordinated mechanism.
Subsequently, the latent 20S proteasome was demonstrated to act as a catalytic core of a large multisubunit proteolytic complex. To date, three protein factors that can stimulate 20S proteasome activity have been described. One is PA700 (also known as the 19S regulatory particle), which can be divided into lid and base complexes. The lid consists of multiple Rpn (non- ATPase) subunits, while the base consists of six proteasomal ATPases, Rptl-Rpt6, and a few additional Rpn subunits (Finley et al., 1998). PA700 can associate with the 20S proteasome in an ATP-dependent manner to form the 26S proteasome with a molecular mass of ~2500 kDa, a eukaryotic ATP-dependent protease (Fig. 2), capable of degrading mainly proteins tagged with a polyubiquitin (Ub) chain, which functions as a degradation signal (Baumeister et al., 1998; Pickart, 2001).
Another activator of the 20S proteasome is PA28 (equivalent to the 11S regulator) (DeMartino and Slaughter, 1993; Rechsteiner et al., 2000). PA28 is attached independently to both ends of the central 20S proteasome ATP to form a football-like proteasome and markedly stimulates the activities of various peptidase of the 20S proteasome in vitro. However, unlike the 26S proteasome, PA28 fails to enhance the hydrolysis of large protein substrates with native or denatured structures, even when they are polyubiquitinated. PA28 is composed of three related proteins, named PA28α, PA28β, and PA28γ, with an overall identity of approximately 50% (Tanaka, 1998). Of these, PA28α and PA28β are induced greatly by a major immunomodulatory cytokine, interferon-γ (Tanaka and Kasahara, 1998). Intriguingly, further studies identified that the "hybrid proteasome," which comprises the 20S proteasome flanked by PA28 on one side and PA700 on the other, functions as a new ATPdependent protease (Tanahashi et al., 2000).
The third proteasome activator is PA200, which is localized in the nucleus and is involved in DNA repair (Ustrell et al., 2002).
II. MATERIALS AND INSTRUMENTATION
Q-Sepharose (Cat. No. 17-1014-03), Q-Sepharose fast flow (Cat. No. 17-0510-10), Superdex 200 pg (Cat. No. 17-1043-01), Mono Q (Cat. No. 17-5166-01), and heparin-Sepharose CL-6B (Cat. No. 17-0467-09) can be purchased from Amersham. BiG-Gel A-1.5m (Cat. No. 151-0440) and hydroxylapatite BiG-Gel HTP (Cat. No. 130-0420) are from Bio-Rad. Polyethylene glycol 6000 (Cat. No. P-2139), ubiquitin (Ub, Cat. No. U-6253), succinyl- Leu-Leu-Val-Tyr-4-methyl-courmaryl-7-amide (Suc-LLVY-MCA, Cat. No. S-6510), and Supelco TSKDEAE 650M is from Sigma. Amicon PM-10 and PM-30 membranes (Cat. No. 13132 and 13232) can be obtained from Millipore. Complete protease inhibitor (Cat. No. 1 697 498) is from Roche.
A. Preparation of 20S Proteasomes
B. Preparation of 26S Proteasomes
Preparation of PA700
Steps for homogenization, ultracentrifugation, and Bio-Gel A-1.5m gel filtration are similar to those used for the preparation of 26S proteasomes. Apply the pooled fractions of the 26S proteasome from the Bio- Gel A-1.5m column directly to a hydroxylapatite column with a 50-ml bed volume that has been equilibrated with buffer C. Wash the column with the same buffer and elute the adsorbed materials with 300ml of a linear gradient of 10-300mM phosphate. Collect 3.0- ml fractions of eluate. Note that the 26S proteasome can be adsorbed in the hydroxylapatite column under a low concentration of ATP and that the 20S proteasome and PA700 regulatory complex can be eluted separately at different phosphate concentrations of approximately 150 and 50mM, respectively. The 20S proteasome is detected by measuring the Suc-LLVYMCA- degrading activity with 0.05% SDS as described earlier, whereas the PA700 complex is monitored by immunoblotting with antibodies against their subunits. Collect protein in fractions containing the PA700 complex from hydroxylapatite chromatography, concentrate it to 2.0mg/ml by ultrafiltration with an Amicon PM-30 membrane, and subject samples of 1.0-2.0mg of protein to 10-30% glycerol density gradient centrifugation, similar to step 6 used in preparation of the 26S proteasome. Collect 1-ml fractions from the bottom of the centrifuge tube. The PA700 complex can be monitored by ATPase activity and/or the aforementioned immunoblotting analysis. Pool fractions 14-18 containing PA700 complex and store at -80°C.
C. Preparation of Football Proteasomes
Preparation of PA28
Apply the PA28-20S proteasome complex from the glycerol density gradient centrifugation, the final material for preparation of football proteasomes, directly to a Q-Sepharose column that has been equilibrated in buffer A and wash extensively with the same buffer. Elute the adsorbed material with a linear gradient of 0-0.8M NaCl in the same buffer because the 20S proteasome and PA28 are separated by this column operation. Eluate the PA28 activator with about 0.3 M NaCl (for details, see step 2). Pool fractions containing PA28 and store at -80°C.
Preparation of Hybrid Proteasomes
Hybrid proteasomes can be reconstituted by purified PA28 (or recombinant PA2800 and 26S proteasomes. Incubate 26S proteasomes (single- and double-capped PA700-20S proteasome complexes) with PA28 or PA280α at 37°C for 15-30min in 20mM Tris-HCl (pH 7.5) buffer containing 2 mM ATP, 5 mM MgCl2, and 1 mM EDTA (Kopp et al., 2001; Cascio et al., 2001).
D. Preparation of PA200
Various fluorogenic peptides are suitable for the measurement of 20S proteasomal activity because proteasomes show broad substrate specificity. However, Suc-LLVY-MCA is recommended as a sensitive substrate. Latent 20S proteasomes can be activated in various ways (Coux et al., 1996). We recommend the use of SDS at low concentrations of 0.02-0.08% for the activation of Suc-LLVY-MCA breakdown; the optimal concentration depends on the enzyme source and the protein concentration used. The fluorogenic peptide (Suc-LLVY-MCA) can be used for assay of PA700-, PA28-, PA200-20S proteasome complexes, but these are active without any treatment, unlike latent 20S proteasomes. For a specific assay, ATP-dependent degradation of polyubiquitinated 125I-labeled lysozyme should be measured, although such an assay is not easy because three kinds of enzymes, E1 (Ub-activating), E2 (Ub-conjugating), and E3 (Ub-ligating) must be purified for the in vitro preparation of ubiquitinated substrate (for the procedures, see Tamura et al., 1991). There are various E2 and E3 enzymes. Most of them have not yet been characterized for use in in vitro-reconstituted proteolytic systems, so it is difficult to prepare large amounts of ubiquitinated proteins for use as substrates for 26S proteasomes. Therefore, for quantitative and sensitive measurement of ATP-dependent proteolysis activity in vitro in mammalian cells, ornithine decarboxylase (ODC) is a useful substrate. ODC is the only known natural substrate independent of ubiquitination for recognition and degradation by 26S proteasomes and hybrid proteasomes. Antizyme (AZ), an ODC inhibitory protein, however, is needed for the process instead of Ub, but both ODC and AZ, required for this in vitro degradation assay, are available as recombinant proteins (Murakami et al., 1999; Tanahashi et al., 2000). Note that AZ is not present in lower organisms, such as yeasts, and thus this assay is not fit for these cells. It is also possible that their purification is monitored by measuring ATPase activity at later steps of their purification, since the 26S proteasome and PA700 regulator complex have intrinsic ATPase activity.
Proteasomes have been purified from a variety of eukaryotic cells by many investigators. Many purification methods have been reported, but no special techniques are necessary because 20S proteasomes are very stable and abundant in cells, constituting 0.5-1.0% of the total cellular proteins. The procedures used for the purification of 20S proteasomes obviously differ, depending on whether they are small or large operations. For their isolation from small amounts of biological materials, such as cultured cells, 10-40% glycerol density gradient centrifugation analysis is very effective. 20S proteasomes are present in a latent form in cells and can be isolated in this form in the presence of 20% glycerol. For their isolation in high yield, a key point is to keep them in their latent form because their activation results in autolytic loss of a certain subunit(s) and marked reduction of enzymatic activities, particularly their hydrolyses of various proteins. Accordingly, all buffers used contain 10-20% glycerol as a stabilizer. Furthermore, a reducing agent is required because 20S proteasomes precipitate in its absence. All purification procedures are performed at 4°C, but operations in a high-performance liquid chromatography (HPLC) apparatus can be carried out within a few hours at room temperature.
For purification of the 26S proteasome, ATP (0.5 or 2mM), together with 20% glycerol and 1 mM DTT, should be added to all solutions used because they strongly stabilize the 26S proteasome complex; the purified enzyme is stable during storage at -70°C for at least 6 months in the presence of 2 mM ATP and 20% glycerol. Other drastic chromatographs should be avoided because these operations may result in dissociation of the 26S complex into its constituents. Alternative methods of purification of the PA28 and PA700 regulatory complexes have been reviewed by DeMartino and Slaughter (1993).
It should be noted that the hybrid proteasome is prepared by an in vitro reconstitution system using purified 26S proteasomes and PA28 (Kopp et al., 2001; Cascio et al., 2001) because it is hardly separated from 26S proteasomes by conventional chromatographic techniques (Tanahashi et al., 2000). In addition, PA200 is found both in complexes of the homologous type PA200-20S proteasome-PA200 and in complexes that also contain PA700, as PA700-20S proteasome-PA200 (Ustrell et al., 2002). To date, the procedures to separate the diverse forms of active proteasomes individually from cells and tissues have not yet been established.
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