The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents.

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline.

How the 26S Proteasome Degrades Ubiquitinated Proteins in the Cell.

The 26S proteasome is the central element of proteostasis regulation in eukaryotic cells, it is required for the degradation of protein factors in multiple cellular pathways and it plays a fundamental role in cell stability. The main aspects of proteasome mediated protein degradation have been highly (but not totally) described during three decades of intense cellular, molecular, structural and chemical biology research and tool development. Contributions accumulated within this time lapse allow researchers today to go beyond classical partial views of the pathway, and start generating almost complete views of how the proteasome acts inside the cell. These views have been recently reinforced by cryo-electron microscopy and mechanistic works that provide from landscapes of proteasomal populations distributed in distinct intracellular contexts, to detailed shots of each step of the process of degradation of a given substrate, of the factors that regulate it, and precise measurements of the speed of degradation. Here, we present an updated digest of the most recent developments that significantly contribute in our understanding of how the 26S proteasome degrades hundreds of ubiquitinated substrates in multiple intracellular environments.

A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125.

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.

Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast.

Despite much evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway.

Rpn10 monoubiquitination orchestrates the association of the ubiquilin-type DSK2 receptor with the proteasome.

Despite the progress made in understanding the roles of proteasome polyubiquitin receptors, such as the subunits Rpn10 (regulatory particle non-ATPase 10) and Rpn13, and the transient interactors Rad23 (radiation sensitivity abnormal 23) and Dsk2 (dual-specificity protein kinase 2), the mechanisms involved in their regulation are virtually unknown. Rpn10, which is found in the cell in proteasome-bound and -unbound pools, interacts with Dsk2, and this interaction has been proposed to regulate the amount of Dsk2 that gains access to the proteasome. Rpn10 monoubiquitination has emerged as a conserved mechanism with a strong effect on Rpn10 function. In the present study, we show that functional yeast proteasomes have the capacity to associate and dissociate with Rpn10 and that Rpn10 monoubiquitination decreases the Rpn10-proteasome and Rpn10-Dsk2 associations. Remarkably, this process facilitates the formation of Dsk2-proteasomes in vivo. Therefore, Rpn10 monoubiquitination acts as mechanism that serves to switch the proteasome from an 'Rpn10 high/Dsk2 low' state to an 'Rpn10 low/Dsk2 high' state. Interestingly, Rpn10-ubiquitin, with an inactivated ubiquitin-interacting motif (UIM), and Dsk2(I45S), with an inactive ubiquitin-like domain (UBL), show temperature-dependent phenotypes with multiple functional interactions.

An intrinsically disordered region of RPN10 plays a key role in restricting ubiquitin chain elongation in RPN10 monoubiquitination.

Despite being a common mechanism in eukaryotes, the process by which protein monoubiquitination is produced and regulated in vivo is not completely understood. We present here the analysis of the process of monoubiquitination of the proteasomal subunit Rpn10 (regulatory particle non-ATPase 10), involved in the recruitment of polyubiquitinated substrates. Rpn10 is monoubiquitinated in vivo by the Nedd4 (neural precursor cell expressed developmentally down-regulated 4) enzyme Rsp5 (reverses SPT-phenotype protein 5) and this modification impairs the interaction of Rpn10 with substrates, having a regulatory effect on proteasome function. Remarkably, a disordered region near the ubiquitin-interacting motif of Rpn10 plays a role in the restriction of the polyubiquitin extension activity of Rsp5. Mutations in this disordered region promote ubiquitin chain extension of Rpn10. Thus, our work sheds light on the molecular basis and the functional relevance of a type of monoubiquitination that is driven by the substrate. Moreover, we uncover a putative role for disordered regions in modulating ubiquitin-protein ligation.

Deubiquitinating enzyme inhibitors and their potential in cancer therapy.

Deubiquitinating enzymes (or DUBs) attack the ubiquitin-based isopeptide bond, thus counteracting ubiquitinprotein ligase activity in vivo. By disassembling ubiquitin-substrate and ubiquitin-ubiquitin covalent links, deubiquitinating enzymes exert a very powerful control of many signaling processes within the ubiquitin-proteasome system (UPS). Very active research in this field in the last decade shows that deubiquitinating enzymes play important regulatory roles in aspects relevant to cancer, such as proteasome activity, p53 stability, the regulation of fanconi anemia related proteins, tumor cell apoptosis induction, to mention a few. Thus, deubiquitinating enzymes have emerged as interesting drug targets in cancer research. Here, the pharmacological inhibition of DUBs and its potential effect in cancer treatment are reviewed.

Ubiquitin signaling: extreme conservation as a source of diversity.

Around 2 × 103-2.5 × 103 million years ago, a unicellular organism with radically novel features, ancestor of all eukaryotes, dwelt the earth. This organism, commonly referred as the last eukaryotic common ancestor, contained in its proteome the same functionally capable ubiquitin molecule that all eukaryotic species contain today. The fact that ubiquitin protein has virtually not changed during all eukaryotic evolution contrasts with the high expansion of the ubiquitin system, constituted by hundreds of enzymes, ubiquitin-interacting proteins, protein complexes, and cofactors. Interestingly, the simplest genetic arrangement encoding a fully-equipped ubiquitin signaling system is constituted by five genes organized in an operon-like cluster, and is found in archaea. How did ubiquitin achieve the status of central element in eukaryotic physiology? We analyze here the features of the ubiquitin molecule and the network that it conforms, and propose notions to explain the complexity of the ubiquitin signaling system in eukaryotic cells.

Defective in mitotic arrest 1 (Dma1) ubiquitin ligase controls G1 cyclin degradation.

Progression through the G(1) phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G(1) cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G(1) cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G(1) cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G(1).

Integration of multiple ubiquitin signals in proteasome regulation.

The ubiquitin-proteasome system has emerged in the last decades as a new paradigm in cell physiology. Ubiquitin is found in fundamental levels of cell regulation, as a target for degradation to the proteasome or as a signal that controls protein function in a complex manner. Even though many aspects of the ubiquitin system remain unexplored, the contributions on the field uncover that ubiquitin represents one of the most sophisticated codes in cellular biology. The proteasome is an ATP-dependent protease that degrades a large number of protein substrates in the cell. The proteasome recruits substrates by a number of receptors that interact with polyubiquitin. Recently, it has been shown that one of these receptors, Rpn10, is regulated by monoubiquitination. In this chapter, we show an overview of the central aspects of the pathway and describe the methodology to characterize in vitro the monoubiquitination of proteasome subunits.

Protein-protein interaction antagonists as novel inhibitors of non-canonical polyubiquitylation.

BACKGROUND: Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-kappaB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. METHODOLOGY/PRINCIPAL FINDINGS: By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-kappaB by TNF-alpha and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.

Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome.

The proteasome recognizes its substrates via a diverse set of ubiquitin receptors, including subunits Rpn10/S5a and Rpn13. In addition, shuttling factors, such as Rad23, recruit substrates to the proteasome by delivering ubiquitinated proteins. Despite the increasing understanding of the factors involved in this process, the regulation of substrate delivery remains largely unexplored. Here we report that Rpn10 is monoubiquitinated in vivo and that this modification has profound effects on proteasome function. Monoubiquitination regulates the capacity of Rpn10 to interact with substrates by inhibiting Rpn10's ubiquitin-interacting motif (UIM). We show that Rsp5, a member of NEDD4 ubiquitin-protein ligase family, and Ubp2, a deubiquitinating enzyme, control the levels of Rpn10 monoubiquitination in vivo. Notably, monoubiquitination of Rpn10 is decreased under stress conditions, suggesting a mechanism of control of receptor availability mediated by the Rsp5-Ubp2 system. Our results reveal an unanticipated link between monoubiquitination signal and regulation of proteasome function.

Searching for the boundaries: unlimited expansion of ubiquitin and ubiquitin-like signals in multiple cellular functions.

The ubiquitin-proteasome field has matured, as is evident from the wide diversity of systems and mechanisms in which it participates and that are the subject of investigation, presented in the Ubiquitin-Proteasome System, Dynamics and Targeting meeting held in Barcelona, co-sponsored by the Biochemical Society, the Spanish Ministry of Science, the Spanish Research Council and the Catalan Academy of Sciences. Several of the aspects dealt with in the meeting are discussed in detail in the collection of review papers included in this issue of Biochemical Society Transactions. These papers reflect the importance of ubiquitin and ubiquitin-like modifiers as enormously versatile signalling entities that modulate and direct pathways in specific directions through modification-induced interactions. One conclusion from the meeting is that the field has become so rich and dense that, in order to be useful and informative, future meetings may need to focus on particular aspects of the ubiquitin-proteasome system.

A new map to understand deubiquitination.

Deubiquitination is a crucial mechanism in ubiquitin-mediated signalling networks. The importance of Dubs (deubiquitinating enzymes) as regulators of diverse cellular processes is becoming ever clearer as new roles are elucidated and new pathways are shown to be affected by this mechanism. Recent work, reviewed in the present paper, provides new perspective on the widening influence of Dubs and a new tool to focus studies of not only Dub interactions, but also potentially many more cellular systems.

Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities.

The ubiquitin ligase Hul5 was recently identified as a component of the proteasome, a multisubunit protease that degrades ubiquitin-protein conjugates. We report here a proteasome-dependent conjugating activity of Hul5 that endows proteasomes with the capacity to extend ubiquitin chains. hul5 mutants show reduced degradation of multiple proteasome substrates in vivo, suggesting that the polyubiquitin signal that targets substrates to the proteasome can be productively amplified at the proteasome. However, the products of Hul5 conjugation are subject to disassembly by a proteasome-bound deubiquitinating enzyme, Ubp6. A hul5 null mutation suppresses a ubp6 null mutation, suggesting that a balance of chain-extending and chain-trimming activities is required for proper proteasome function. As the association of Hul5 with proteasomes was found to be strongly stabilized by Ubp6, these enzymes may be situated in proximity to one another. We propose that through dynamic remodeling of ubiquitin chains, proteasomes actively regulate substrate commitment to degradation.

Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation.

Ubiquitin chains serve as a recognition motif for the proteasome, a multisubunit protease, which degrades its substrates into polypeptides while releasing ubiquitin for reuse. Yeast proteasomes contain two deubiquitinating enzymes, Ubp6 and Rpn11. Rpn11 promotes protein breakdown through its degradation-coupled activity. In contrast, we show here that Ubp6 has the capacity to delay the degradation of ubiquitinated proteins by the proteasome. However, delay of degradation by Ubp6 does not require its catalytic activity, indicating that Ubp6 has both deubiquitinating activity and proteasome-inhibitory activity. Delay of degradation by Ubp6 appears to provide a time window allowing gradual deubiquitination of the substrate by Ubp6. Rpn11 catalyzes en bloc chain removal, and Ubp6 interferes with degradation at or upstream of this step, so that degradation delay by Ubp6 is accompanied by a switch in the mode of ubiquitin chain processing. We propose that Ubp6 regulates both the nature and magnitude of proteasome activity.

Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation.

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.

Proteasome activity is fine-tuned by associating the proteolytic core particle (CP) with stimulatory and inhibitory complexes. Although several mammalian regulatory complexes are known, knowledge of yeast proteasome regulators is limited to the 19-subunit regulatory particle (RP), which confers ubiquitin-dependence on proteasomes. Here we describe an alternative proteasome activator from Saccharomyces cerevisiae, Blm10. Synthetic interactions between blm10Delta and other mutations that impair proteasome function show that Blm10 functions together with proteasomes in vivo. This large, internally repetitive protein is found predominantly within hybrid Blm10-CP-RP complexes, representing a distinct pool of mature proteasomes. EM studies show that Blm10 has a highly elongated, curved structure. The near-circular profile of Blm10 adapts it to the end of the CP cylinder, where it is properly positioned to activate the CP by opening the axial channel into its proteolytic chamber.

Localization to the proteasome is sufficient for degradation.

The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.

Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism.

Aldo-keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all- trans -, 9- cis - and 13- cis -retinal ( k (cat)/ K (m)=1100-10300 mM(-1).min(-1)), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor ( K (i)=80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All- trans -retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from beta-carotene and the control of retinal bioavailability.