The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini - PubMed (original) (raw)
The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini
Dikla Berko et al. Mol Cell. 2012.
Abstract
Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system.
Copyright © 2012 Elsevier Inc. All rights reserved.
Figures
Figure 1. Certain proteins are exclusively degraded from their N or C termini, while others can be degraded from either end
(A) MBP is translocated into the proteasome exclusively from its C terminus. MBP cross-linked to agarose beads through its C (MBP-agarose) or N-terminus (agarose-MBP) was incubated with PAN and 20S proteasomes for the indicated time. The beads were washed three times and boiled with SDS sample buffer. (B) β-casein is degraded exclusively from its N-terminus. Time course of degradation of β-Casein cross-linked to agarose beads through its C (Casein-agarose) or N-terminus (agarose-Casein). Reactions were performed and analyzed as is described in A. (C) Time course of degradation of β-casein linked to biotin or avidin (attached to the biotin) on its N or C terminus by 26S proteasomes. (D) β-casein is degraded from both termini by the SDS-activated mammalian 20S proteasome. β-casein linked to avidin through its C (Casein-avidin) or N terminus (avidin-Casein) was incubated with SDS activated purified rabbit 20S proteasomes. Equal portions were removed and subjected to Western blot analysis using anti-His antibodies. (E) Blocking either terminus of Apo-calmodulin does not prevent its proteasomal degradation. Time course of calmodulin-RFP and RFP-calmodulin degradation by PAN-20S complex. Aliquots of the reaction mixture were removed at the indicated times and analyzed by Western blot using anti-RFP antibody.
Figure 2. p21 and denatured (molten-globule) ovalbumin are degraded by mammalian 20S and 26S proteasomes with either terminus blocked
Time course of p21 N or C terminally fused to RFP degradation by mammalian 20S proteasome (A) or 26S proteasome (B). (C) Large excess of p21-RFP & RFP-21 (3µM) were incubated with 26S proteasome (1nM), a portion (1:5) of the initial material and following one hour of incubation was blotted with anti RFP. (D) Time course of ova-RFP and RFP-ova degradation by mammalian 26S proteasomes. Right: Quantification of the residual amount of protein at each time point, are presented (SD were calculated from four repetitions under similar conditions).
Figure 3. Directionality of proteasomal degradation in living cells
EL4 cells expressing p21 or ovalbumin N or C-terminally fused to RFP were pre-incubated at 37°C in the presence and absence of 2µM bortezomib (Velcade). After 1 hour cycloheximide was added (50 µg/ml) to the medium for the indicated times. Thereafter, equal numbers of cells were lysed, and the supernatants were subjected to Western blot analyses by anti-RFP and anti-actin antibodies. (A) Cycloheximide chase of EL4 cells expressing p21 fused to RFP either through its C or N-terminus. (B) Cycloheximide chase of EL4 cells expressing ovalbumin fused to RFP either through its C or N-terminus. Quantification of the residual amount of protein detected at each time point during the chycloheximide chase are presented (SD were calculated from three repetitions under similar conditions).
Figure 4. Blocking both of p21 termini lead to internal Initiation of its proteasomal degradation and sparing of the up-and downstream neighboring globular domains in vitro and in vivo
(A) Right panel: To capture all of the proteasomal degradation products and intermediates (in vitro) large excess of His-GFP-p21-CFP-Flag (3µM) was incubated with 26S proteasome (1nM). A portion (1:5) of the initial material, and following one hour of incubation were blotted with anti GFP. Left panel: Time course of in vitro degradation of GFP-p21-CFP showing the accumulation of the GFP (anti His) and CFP (anti Flag) domains upon hydrolysis of the full length GFP-p21-CFP. (B) Left panel: Western blot analysis of total cell lysat prepared for EL4 cells expressing His-GFP-p21-CFP-Flag in the presence and absence of Velcade. The large ratio in favor of the intact GFP and CFP domains to the full-length (more than hundred fold) demonstrate the stability and the quantitative release of these domains in the in vivo proteasomal degradation of GFP-p21-CFP. Right panel: Time course of in vivo degradation of GFP-p21-CFP showing the accumulation of the GFP (anti His) and CFP (anti Flag) domains upon hydrolysis of the full length GFP-p21-CFP. (C) Time course of in vivo degradation of p21-RFP & RFP-p21 (as in Figure 2A) demonstrating the processive degradation of the RFP domain upon in vivo proteasomal degradation of p21 blocked on either termini by RFP.
Figure 5
(A) The 26S proteasome generated different spectrum of peptides from p21 upon degradation from its N or C-terminus and much greater diversity in the N to C direction. Following digestion of RFP-p21 and p21-RFP by 26S proteasomes, equal amounts of the degradation products (as estimated by fluorescamine) were analyzed by MS. Prior to the analysis one set of peptides was labeled by reductive dimethylation with deuterium-and 13C, ‘heavy’, formaldehyde and the other set treated with non-isotopic formaldehyde. The samples were mixed, and the isotopic ratios in the individual peptides were analyzed by mass spectrometry. These experiments were repeated three times with standard deviations of less than 10%. The raw data of both analyses is presented in Figure S4. (B) Size distribution of peptides generated from p21-RFP and RFP-p21. Peptides generated during degradation of p21-RFP and RFP-p21 by 20S (without SDS activation) and 26S proteasomes were separated from undigested protein by ultrafiltration through a 10kDa cutoff membrane. Equal amounts of peptide products were reacted with fluorescamine and immediately fractionated on a high-performance size exclusion column. The fluorescence of eluted material was monitored continuously with a fluorescence detector and a blank run (corresponding to time 0) was always subtracted. Molecular weights were calculated from the calibration curve shown in Figure S6. Similar data were obtained in at least four independent experiments.
Figure 6. SIINFEKL is presented on surface H2-Kb molecules of EL4 cells expressing ova-RFP but much less in cells expressing RFP-ova-RFP
(A) Western blot analysis of total cell lysate prepared from EL4 cells expressing HA-RFP-ova-RFP-His in the presence and absence of Velcade. The much greater content (more than a hundred fold) of the intact RFP domains compared to the full-length substrates demonstrates that RFP is quantitatively released and not degraded during the in vivo proteasomal degradation of RFP-ova-RFP. Both the N-terminal and the C-terminal RFP domains were spared, as is evident from the immunoreactivity of the RFP domains with HA and His antibodies. (B) EL4 cells expressing ova-RFP (blue), RFP-ova-RFP (black) or empty vector (red), were stained with 25D1.16 monoclonal antibody, recognizing the H2-Kb/SIINFEKL complex. For detection, Alexa Fluor 647-conjugated anti-mouse was used as a secondary antibody. Live cells were gated based on their light scatter characteristics. To ensure similar levels of expression, a narrow GFP (pMIG vector internal iris) gate was selected.
Figure 7. In silico calculation of the force needed to unravel the N or the C terminus of MBP, apo-calmodulin and ovalbumin
The graphs on the left depict the length of the unstructured tail that was predicated to form (along the simulation) upon pulling the molecule either from its N’ (black) or C’ (red) terminus. The “snapshots” (generated with VMD (Humphrey et al., 1996)) on the right are representative structures of the proteins along the simulations at the time steps specified at the graphs.
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