Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes - PubMed (original) (raw)
Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes
Paolo Cascio et al. EMBO J. 2002.
Abstract
PA28 is a gamma-interferon-induced complex that associates with the 20S proteasome and stimulates breakdown of small peptides. Recent immunoprecipitation studies indicate that, in vivo, PA28 also exists in larger complexes that also contain the 19S particle, which is required for ATP-ubiquitin-dependent degradation of proteins. However, because of its lability, the structure and properties of this larger complex remain unclear. Here, we demonstrate that, in vitro, PA28 can associate with 'singly capped' 26S (i.e. 19S-20S) proteasomes. Electron microscopy of the resulting structures revealed one PA28 ring at one end of the 20S particle and a 19S complex at the other. These hybrid complexes show enhanced hydrolysis of small peptides, but no significant increase in rates of protein breakdown. Nevertheless, during breakdown of proteins, the complexes containing PA28alphabeta or PA28alpha generated a pattern of peptides different from those generated by 26S proteasomes, without altering mean product length. Presumably, this change in peptides produced accounts for the capacity of PA28 to enhance antigen presentation.
Figures
Fig. 1. PA28 interacts with 26S proteasomes and stimulates peptide hydrolysis. (A) 26S proteasomes (10 pM) were pre-incubated in 20 mM Tris–HCl pH 7.5, 2 mM ATP, 5 mM MgCl2, 1 mM EDTA at 37°C with and without PA28 (20 nM). Reactions were started by adding Suc-LLVY-amc at a final concentration of 100 µM, and the fluorescence of released amc was measured. (B) Native PAGE of 26S and 20S proteasomes. Note the two bands corresponding to singly and doubly capped 26S. (C) A 5 µg aliquot of 26S proteasomes was pre-incubated in the absence and presence of 3 µg of PA28 and separated by native PAGE. The gel was developed by fluorogenic peptide overlay, and the same gel was then stained with Coomassie Blue. (D) After association with PA28, 26S and 20S migrate in completely different positions. A 5 µg aliquot of 26S and 1 µg of 20S were pre-incubated with 3 µg of PA28 and then subjected to native PAGE.
Fig. 2. (A) Electron micrographs of a negatively stained proteasome mixture showing the six different proteasome species: a 20S proteasome, proteasomes that are singly capped (19S–20S) and doubly capped (19S–20S–19S) with 19S complex, proteasomes that are singly capped (PA28–20S) and doubly capped (PA28–20S–PA28) with PA28 rings as well as a hybrid proteasome with a 19S complex and a PA28 ring bound to either end (19S–20S–PA28). For visualization of the different complexes, an excess of PA28 was used due to the weak interaction of PA28α rings with the 20S proteasome. Accordingly, our images showed many top views of unbound PA28 rings. As a result of the weak PA28α–proteasome interaction, PA28 rings often dissociated from the 20S proteasome upon adsorption to the carbon film and could be seen as a top view adjacent to the proteasome (see, for example, the particle denoted PA28–20S–PA28). Such particles were excluded from the multireference alignment procedure and not used for the calculation of averages. (B) Known forms of the proteasome: gallery of negatively stained particles and the corresponding average images. (1) 20S proteasomes with two 19S caps (19S–20S–19S); (2) proteasomes with one 19S cap (19S–20S); (3) 20S proteasomes (20S); (4) proteasomes with one PA28 cap (20S–PA28); and (5) proteasomes with two PA28 caps (20S–PA28). The corresponding averages contain 106, 748, 164, 78 and 66 particles. The length of the side of the individual frames corresponds to 80 nm. (C) Hybrid proteasome complex (19S–20S–PA28): negatively stained particles and average image based upon 266 particles.
Fig. 3. (A) Association of PA28 with the 26S proteasomes does not increase the rate of protein breakdown. Degradation of reductively methylated IGF-1 (1.5 mM), casein (1 mM), ovalbumin (10 µM) and FITC–casein (10 µM) was performed at 37°C in 20 mM Bis-Tris-propane pH 7.5, 2 mM ATP, 5 mM MgCl2, 1 mM EDTA with a concentration of the enzyme variable between 10 and 30 nM depending on the substrate. At the indicated times, aliquots were removed and analyzed for the appearance of amino groups with fluorescamine (for IGF-1, casein and ovalbumin) or for the generation of soluble fluorescence after precipitation with perchloric acid (for FITC–casein). (B) Even after a 6 h incubation at 37°C, only 26S and hybrid complexes could be detected in the digestion assays. 26S and 26S–PA28 were incubated for 6 h in the presence of casein. At time 0 and after 6 h, an aliquot was removed and loaded onto a native polyacrylamide gel. The gel was overlaid with fluorogenic substrate, and then Coomassie Blue stained. Even after 6 h, 26S particles (5 µg) in the presence of PA28 were several fold more active than control 26S alone, and no 20S particles could be detected.
Fig. 4. The association of PA28 with the 26S proteasomes modifies the patterns of peptides generated from proteins. (A) IGF-1 was digested by 26S or hybrid complexes, and peptides generated were separated using a C8 Vydac column as described previously (Cascio et al., 2001), except for the use of an HP 1100 chromatographer (Hewlett-Packard) in the present studies. (B) FITC–casein (10 µM) was degraded as already described (see Figure 3A), and an aliquot was injected onto a C18 Vydac column equilibrated with 10 mM sodium phosphate (pH 6.8) and analyzed by detection of fluorescence. Under these conditions, protein substrate is degraded and peptide products generated at linear rates (Cascio et al., 2001). Peptides were eluted by a gradient of acetonitrile from 0 to 50% in 100 min. Data very similar to those shown in (A) and (B) were obtained in several different analyses.
Fig. 5. Native PA28αβ is able to modify the patterns of peptides generated from IGF-1 in a way similar to recombinant PA28α. Digestion of IGF-1 and separation of peptides were performed as in Figure 4, except that PA28αβ was used at a 3-fold lower concentration than PA28α (0.5 µM instead of 1.5 µM).
Fig. 6. Different peptides generated from IGF-1 by 26S proteasomes and hybrid complexes.
Fig. 7. Size distribution of peptides generated from IGF-1 by 26S and hybrid complexes. Peptides generated during degradation of IGF-1 were separated from undigested protein on a C18 Vydac column as described (Kisselev et al., 1998), and lyophilized. After lyophilization, the resuspended material was used for size-exclusion chromatography with a polyhydroxy-ethyl aspartamide column (0.46 × 20 cm, Poly LC, Columbia, MD), equilibrated with 0.2 M sodium sulfate, 25% acetonitrile pH 3.0 using an HP1090 chromatographer (Hewlett-Packard) and a fluorometer detector. Peptide products were resuspended in 0.1 M HEPES buffer pH 6.8. For each analysis, 3 nmol of peptides in a total volume of 20 µl were added to 10 µl of fluorescamine (0.3 mg/ml acetone). The reaction was terminated after 1 min with 30 µl of H2O and the sample was injected immediately into the HPLC column. To determine the apparent molecular mass of the peptides eluted, the column was calibrated each time before use with 11 standard amino acids and peptides in the 200–3500 Da range that had been derivatized with fluorescamine in the same way as proteasome products. Prior control studies showed that retention times of these fluorescamine-derivatized peptides were highly reproducible, and linearly dependent on the logarithm of their molecular weights (Kohler et al., 2001).
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