The 26S proteasome is an approximately 2.5 MDa multi-catalytic protease complex that is responsible for most non-lysosomal protein degradation in eukaryotic cells. It is composed of a barrel-like catalytic 20S core particle (CP) that consists of four stacked heteroheptameric rings capped on each side by a 19S regulatory particle (RP) (Figure 1), 1 which contains a putative hetero-hexameric ring of ATPases (Rpt1-6) as well as several other proteins. The RP binds polyubiquitylated proteins, unfolds them, and feeds the polypeptide chain into the interior of the barrel where the proteins are degraded into small peptides. 2 Inhibition of the 20S CP proteolytic activity has emerged as an attractive pharmacological target for cancer and inflammatory diseases 3,4 as well as useful as mechanistic probes of proteasome function in a variety of biological processes. 3 Recently, a number of pioneering studies have revealed that stimulating proteasome-mediated proteolysis is but one of several activities of the Rpt proteins. It is now clear that these ATPases play non-proteolytic roles in RNA polymerase II transcription, DNA repair, structural modification of chromatin and other nuclear processes. 5-10 Therefore, pharmacological inhibitors of the proteasomal ATPases (Rpts) would be of great interest. Here we report the isolation of the first compound of this type from a library of nucleoside-capped peptoids. We show that this peptoid derivative inhibits the protein unfolding activity of the Rpt proteins in vitro and inhibits the proteasome activity in living cells.A "one bead one compound" peptoid library was constructed by split and pool synthesis (see Supporting Information Figure S1). 11 Each peptoid molecule was capped with a purine analogue ( Figure 2A) in hope of biasing the library toward targeting one of the ATPases. The theoretical diversity of the library was 8 5 (32 768 compounds). Approximately 100 000 beads, representing about 3-fold coverage of the library, were used in the screen for compounds that bind to the proteasome. The screen employed a whole cell extract 12 prepared from a yeast strain that expressed a FLAG-tagged 4 proteasome subunit (one of the 20S proteins). The beads were exposed to the extract, washed thoroughly (see Supporting Information), probed with anti-FLAG monoclonal antibody, and then washed again. Finally, putative proteasomebinding peptoids were visualized by addition of a secondary antiIgG antibody labeled with red-emitting quantum dots. 13 Three beads evinced an obvious red halo, consistent with retention of the quantum-dot-conjugated secondary antibody ( Figure 2B). Note that the library had been prescreened to eliminate peptoids that bound directly to the antibodies or the quantum dot before exposing it to the yeast extract. Edman degradation revealed that all three hits were identical, indicating the presence of a single proteasome-binding peptoid in the library with sufficient affinity and specificity to register under these relatively demanding conditions. We called this compound ...

Destabilization of activator-DNA complexes by the proteasomal ATPases can inhibit transcription by limiting activator interaction with DNA. Modification of the activator by monoubiquitylation protects the activator from this destabilization activity. In this study, we probe the mechanism of this protective effect of monoubiquitylation. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and Rpt1. This interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. A model is proposed in which activator monoubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.

A new label transfer method is presented that overcomes most of the limitations of current systems. A protein of interest is tagged with tetra-cysteine sequence (FlAsH Receptor Peptide (FRP)) that binds tightly and specifically to a chimeric molecule 3,4-dihydroxyphenylalanine-biotin-4′,5′-bis (1,3,2-dithioarsolan-2-yl)fluorescein (DOPA-biotin-FlAsH). Upon brief periodate oxidation, the DOPA moiety is cross-linked to nearby surface-exposed nucleophiles. Boiling the products in excess dithiol dissolves the FlAsH-FRP interaction, resulting in transfer of the biotin tag to the partner proteins, allowing them to be identified by standard methods. Most methods to characterize protein-protein interactions require the protein of interest to be displayed as an artificial fusion outside of its native environment. To overcome such limitations, a biochemical method called label transfer has been developed, in which a latent cross-linking agent and some easily detectable tag such as a biotin or a radiolabel are connected to the protein of interest via a cleavable linker. Once the modified protein is incorporated into its native complex, the cross-linking moiety is activated and the linker arm is then cleaved, resulting in transfer of the tag to the partner protein 1. While this approach has been employed in the study of some protein complexes, 2,3 the utility of current label transfer technology is severely limited by the requirement to modify the protein of interest covalently and then reconstitute the complex of interest with the modified protein. The inefficient cross-linking chemistry commonly used in such systems (usually photolysis of aryl azides) also complicates the characterization of partner proteins 3. We report here a new type of label transfer system that eliminates the need for covalent modification of the protein of interest (see Scheme 1). It involves tagging the "bait" protein with a tetracysteine-containing peptide (CCPGCC, here called FRP for FlAsH Receptor

The Saccharomyces cerevisiae Gal4 protein is a paradigmatic transcriptional activator containing a C-terminal acidic activation domain (AD) of 34 amino acids. A mutation that results in the truncation of about two-thirds of the Gal4AD (gal4D) results in a crippled protein with only 3% the activity of the wild-type activator. We show here that although the Gal4D protein is not intrinsically deficient in DNA binding, it is nonetheless unable to stably occupy GAL promoters in vivo. This is because of the activity of the proteasomal ATPases, including Sug1/Rpt6, which bind to Gal4D via the remainder of the AD and strip it off of DNA. A mutation that suppressed the Gal4D "no growth on galactose" phenotype repressed the stripping activity of the ATPase complex but not other activities. We further demonstrate that Gal4D is hypersensitive to this stripping activity because of its failure to be monoubiquitylated efficiently in vivo and in vitro. Evidence is presented that the piece of the AD that is deleted in Gal4D protein is likely a recognition element for the E3 ubiquitin-protein ligase that modifies Gal4. These data argue that acidic ADs comprise at least two small peptide subdomains, one of which is responsible for activator monoubiquitylation and another that interacts with the proteasomal ATPases, coactivators and other transcription factors. This study validates the physiological importance of Gal4 monoubiquitylation and clarifies its major role as that of protecting the activator from being destabilized by the proteasomal ATPases.

This study describes the identification of the protein target of the first chemical inhibitor (RIP-1) of the 19S regulatory particle (RP) of the 26S proteasome. Periodate-triggered chemical cross-linking of DOPA-conjugated RIP-1 and the 26S proteasome identified Sug2/Rpt4, one of the six ATPases in the 19S RP as the molecular target of RIP-1. The specificity of RIP-1 for Sug2/Rpt4 was demonstrated by examining cross-linking reactions with each ATPase of the 19S RP. RIP-1 should provide a useful biological tool to probe the various biological roles of Rpt4. We recently reported the first 19S RP inhibitor, called RIP-1 (Regulatory Particle Inhibitor Peptoid-1) (Figure 1). 13 RIP-1 was isolated from a combinatorial library of approximately 33,000 peptoids, all of which were capped with a purine molecule in an attempt to bias the library towards binding one or more of the six ATPases in the 19S RP. It was demonstrated that RIP-1 inhibited the protein unfolding activity of the 19S RP in vitro and blocked proteasome-mediated turnover of the cell cycle protein p27 in HeLa cells. In this report, we describe experiments designed to identify the molecular target of RIP-1. RIP-1 was identified originally in a screen that registered binding of the peptoid to the intact 26S proteasome, which contains at least 34 different polypeptides. 13 To identify the RIP-1 receptor, we used a chemical cross-linking reaction that involves the oxidation of a