Bortezomib Amplifies Effect on Intracellular Proteasomes by Changing Proteasome Structure.

The proteasome inhibitor Bortezomib is used to treat multiple myeloma (MM). Bortezomib inhibits protein degradation by inactivating proteasomes' active-sites. MM cells are exquisitely sensitive to Bortezomib - exhibiting a low-nanomolar IC(50) - suggesting that minimal inhibition of degradation suffices to kill MM cells. Instead, we report, a low Bortezomib concentration, contrary to expectation, achieves severe inhibition of proteasome activity in MM cells: the degree of inhibition exceeds what one would expect from the small proportion of active-sites that Bortezomib inhibits. Our data indicate that Bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity. Comparing MM cells to other, Bortezomib-resistant, cancer cells shows that the degree of proteasome inhibition is the greatest in MM cells and only there leads to proteasome stress, providing an explanation for why Bortezomib is effective against MM but not other cancers.

Nuclear proteasomes carry a constitutive posttranslational modification which derails SDS-PAGE (but not CTAB-PAGE).

We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttranslational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively-charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components.

Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites.

The 26S proteasome holoenzyme is formed by the association of a 20S core particle (CP) with a 19S regulatory particle (RP). The CP-RP interaction is labile and subject to regulation in vivo, but the factors controlling this association are poorly understood. Here we describe an in vitro proteasome reconstitution assay and a high-resolution, two-dimensional gel electrophoresis system. Using these techniques, we find that a yeast CP-RP complex can contain a substoichiometric amount of tightly bound, essentially non-exchangeable ATP. However, this nucleotide is dispensable for gating of the CP channel, provided that the CP-RP complex is preserved by the Ecm29 protein. Unexpectedly, proteasome inhibitors are potent in stabilizing proteasomes against the dissociation of CP-RP. These data indicate that active sites of the CP communicate with bound RP, despite their spatial separation. We propose that ongoing protein degradation may suppress proteasome disassembly, thereby enhancing the processivity of proteolysis.

The ubiquitin-proteasome system regulates membrane fusion of yeast vacuoles.

Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin-proteasome regulation may be involved in membrane fusion elsewhere.

The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome.

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis (Kleijnen et al., 2000). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2-proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAA receptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a.

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.