Article Figures & Data
Figures
- Fig. 1.
The ubiquitin proteasome pathway. (A) Simplified model of the ubiquitin conjugation system. (B) Primary steps involved in ubiquitinated substrate processing by the 26S proteasome.
- Fig. 2.
Proteasome structure and function. (A) Structures (PDB 4r3o) and cartoon representation of 20S proteasome, highlighting the different β-subunit combinations found in tissue-specific proteasomes discussed in the text. (B) Structure of the 26S proteasome in complex with Ubp6 (PDB 5a5b). A cross-section of 20S proteasome reveals the C terminus of Rpt5 ATPase (dark orange) positioned in the inter-α-subunit pocket (asterisk). Proteolytic sites are marked with yellow stars. Labeled 19S subunits are discussed in the text. (C) 20S proteasomes (blue and gray) complexed with regulatory caps: PA28 homolog PA26 (PDB 1fnt), 19S (PDB 5gjr), and PA200 yeast homolog Blm10 (PDB 4v7o). 19S ATPases are dark orange, and non-ATPase subunits are light orange. PDB, Protein Data Bank.
- Fig. 3.
Examples of cellular functions that depend on proteasome function. Important pathways dependent on proteasome function and exemplar substrates. Bax, bcl-2-like protein 4; Bim, bcl-2-like protein 11; Cdk, cyclin-dependent kinase; Drp1, dynamin-1-like protein; ERAD, endoplasmic-reticulum-associated protein degradation; E2F-1, target of retinoblastoma protein; Fis1, mitochondrial fission 1 protein; GABA, gamma-aminobutyric acid; JNK, C-Jun-amino-terminal kinase; Mfn, mitofusin; MHC-I, major histocompatibility complex-I; Miro, mitochondrial Rho GTPase; NF-κB, nuclear factor–κB; PKA, protein kinase A; PSD-95, postsynaptic density protein 95; Topo II, type II topoisomerase; Wnt, wingless-type.
- Fig. 4.
Proteasome inhibitors and the β5 binding site. (A) Chemical structures of proteasome inhibitors that are FDA approved and/or are in clinical trials with pharmacophores shown in red. For ixazomib, the orally bioavailable prodrug (MLN9708) is shown, with the biologically active metabolite (MLN2238) highlighted in black and red for clarity. (B) X-ray crystallography structures of human 20S proteasomes in complex with carfilzomib (PDB 4r67), bortezomib (PDB 5lf3), and ixazomib (PDB 5lf7); yeast 20S in complex with marizomib (PDB 2fak); and the cryo-EM structure of human 26S in complex with oprozomib (PDB 5m32). PDB, Protein Data Bank.
Tables
Experimental variables Pitfall Buffer ATP and magnesium Stability of the 26S complex is dependent on a high ATP/ADP ratio. For monitoring 26S activity, an adequate amount of ATP and MgCl2 in a 1:5 ratio (we typically use 2 and 10 mM, respectively) should be present in all buffers to maintain the stability of the 26S proteasome complex. At the same time, the levels of ADP should be kept at a minimum Glycerol Glycerol in the buffer stabilizes and maintains the closed, latent gate. Typically, purification buffers for proteasomes contain 10% glycerol, and assay buffers contain 5% SDS Addition of 0.02% SDS to assay buffer mildly stimulates opening of the latent 20S gate Proteasome population Proteasomes are present as individual 20S particles and 26S particles (singly caped 20S-19S and doubly capped 19S-20S-19S). The 26S complex can dissociate into the 19S and 20S constituents over time, after a freeze/thaw, or in response to buffer condition changes When performing experiments with purified 26S proteasomes, it is important to determine the ratio of intact 26S and free 20S proteasomes. This is commonly accomplished by native PAGE electrophoresis as described in the text Microplate The type of microplate (e.g., untreated vs. nonbinding surface treatments, polypropylene vs. polystyrene) can affect the observed activity. Some substrates (e.g., GFP) interact with and “stick” to untreated plate surfaces. Some compounds or small substrates may bind to the plate surface and reduce the effective concentration in the assay. It is worthwhile to confirm results with new compounds or substrates by using two or more types of plates. Bovine serum albumin can be included in the buffer to prevent nonspecific binding to the plate Proteasome gating Experiments probing the role/regulation of the proteasome gate might be facilitated by using the “open-gate” 20S mutant, α3ΔN Suc-LLVY-amc has been shown to mildly stimulate gate opening, so another substrate (e.g., Ac-nLPnLD-amc) should be used in conjunction Substrate Ub/Pathway Localization t1/2 Reference GFPu Yes/CL1 degron 30 min Bence et al. (2001) NLS-GFPu Yes/CL1 degron Nuclear 60 min Bennett et al. (2005) NES-GFPu Yes/CL1 degron Cytosolic 60 min Bennett et al. (2005) PSD95-GFPu Yes/CL1 degron Postsynaptic ND Wang et al. (2008) SNAP25-GFPu Yes/CL1 degron Presynaptic ND Wang et al. (2008) TCR-α–GFP Yes/ERAD ND DeLaBarre et al. (2006) Ub-M-GFP Yes/“normal” “Stable” Dantuma et al. (2000) Ub-R-GFP Yes/N-end rule “Short” Dantuma et al. (2000) UbG76V-GFP Yes/UFD “Short” Dantuma et al. (2000) UbG76V-dendra2 Yes/UFD ND Hamer et al. (2010) GFP-ODC No 2 h Li et al. (1998) M, methionine; ND, not determined; NES, nuclear export signal; NLS, nuclear localization signal; PSD95, postsynaptic density 95; R, arginine; SNAP25, synaptosomal-associated protein 25; t1/2, in vivo half-life; TCR-α, T-cell receptor α chain.
Inhibitor Class IC50 In Vitro Peptidase Activity Half-Life Route Binding Reference CT-L C-L T-L nM min Bortezomib Boronate 7.9 53 590 110 i.v./s.c. Slowly reversible Cauhan et al. (2005) Carfilzomib Epoxyketone 6 2400 3600 <30 i.v. Irreversible Kuhn et al. (2007) Ixazomib Boronate 3.4 31 3500 18 p.o. Reversible Cauhan et al. (2005), Kupperman et al. (2010) Marizomib β-lactone 3.5 430 28 10–15 i.v. Irreversible Feling et al. (2003) Oprozomib Epoxyketone 36 (β5) ND ND 30–90 p.o. Irreversible Zhou et al. (2009), Roccaro et al. (2010) 82 (β5i) C-L, caspase-like activity (β1); CT-L, chymotrypsin-like activity (β5); ND, not determined; T-L, trypsin-like activity (β2).
In this issue
A Practical Review of Proteasome Pharmacology
Jump to section
- Article
- Abstract
- I. Introduction
- II. Proteasome Structure and Function
- III. Development of Proteasome Inhibitors
- IV. Methods for Pharmacological Proteasome Research
- V. Proteasome Inhibitors to Treat Human Disease
- VI. Novel Proteasome Drug Targets
- VII. Conclusions
- Acknowledgments
- Authorship Contributions
- Footnotes
- Abbreviations
- References
- Figures & Data
- Info & Metrics
- eLetters