Deubiquitinating enzymes: their functions and substrate specificity

Conjugation of one or more molecules of ubiquitin to target proteins can signify one of several fates, including degradation by the 26S proteasome, or trafficking via the secretory or endocytic pathways. Whereas much attention in recent years has focussed on the mechanisms of forming these different ubiquitin conjugates, far less is known about the removal of ubiquitin, which is performed by deubiquitinating enzymes (DUBs). While it has been appreciated for some 10 years that DUBs constitute large gene families in eukaryotes, and known for much longer that ubiquitination is a reversible process, information on the exact role of DUBs has been slow in coming. This review will attempt to summarise results from the last few years that shows that DUBs are an essential regulatory step of both protein degradation by the proteasome, and of other ubiquitin-dependent processes, by virtue of their ability to regulate protein ubiquitination in a target-specific manner.     

Roles of p97-Associated Deubiquitinases in Protein Quality Control at the Endoplasmic Reticulum

To maintain protein homeostasis in the ER, an ER protein quality control system retains unfolded polypeptides and misassembled membrane proteins, allowing only properly folded proteins to exit the ER. Misfolded proteins held in the ER are retrotranslocated into the cytosol, ubiquitinated, and degraded by the proteasome through the ER-associated degradation pathway (ERAD). By timely eliminating misfolded proteins, the ERAD system alleviates cytotoxic stress imposed by protein misfolding. It is well established that ER-associated ubiquitin ligases play pivotal roles in ERAD by assembling ubiquitin conjugates on retrotranslocation substrates, which serve as degradation signals for the proteasome. Surprisingly, recent studies have revealed an equally important function for deubiquitinases (DUBs), enzymes that disassemble ubiquitin chains, in ERAD. Intriguingly, many ERAD specific DUBs are physically associated with the retrotranslocation- driving ATPase p97. Here we discuss the potential functions of p97-associated DUBs including ataxin-3 and YOD1. Our goal is to integrate the emerging evidence into models that may explain how protein quality control could benefit from deubiquitination, a process previously deemed destructive for proteasomal degradation.     

Conjugation and deconjugation of ubiquitin regulating the destiny of proteins

The homeostasis for a number of cellular proteins is regulated by not only phosphorylation and dephosphorylation, but also ubiquitination and deubiquitination. A number of proteins involved in the degradation of polypeptides have been isolated in various eukaryotic organisms from Saccharomyces cerevisiae to human. Recently, several deubiquitinating enzymes, classified into either the Ub C-terminal hydrolase (UCH) or the Ub-specific processing protease (UBP), have been reported. It has been shown that they contain conserved domains including Cys, His, and Asp residues throughout the enzyme. These proteins have been demonstrated that Cys and His domains are critical for deubiquitinating enzymatic activity. Recently, we have shown that the Asp domain localized between Cys and His domains is also essential for cleaving the ubiquitin from protein substrates. Mouse deubiquitinating enzymes including DUB-1, DUB-2, and DUB-2A have been isolated and they showed the expression specificity. Of these, DUB- 1 and DUB-2 are expressed in lymphocytes depending on the presence of cytokines (interleukin-3 in B-lymphocytes and interleukin-2 in T- lymphocytes, respectively), indicating that they are involved in cytokine signaling pathways. Isolation of all putative DUBs will help to identify their substrates and to regulate the homeostasis of cellular proteins, especially in proliferative cells.     

An inhibitor of ubiquitin conjugation and aggresome formation

Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches. Furthermore, proteasome inhibitors lead to the formation of aggresomes that clear misfolded proteins via the autophagy–lysosome degradation pathway. Importantly, aggresome formation depends on the presence of polyubiquitin tags on misfolded proteins. We therefore hypothesized that inhibitors of ubiquitin conjugation should inhibit both degradation of misfolded proteins, and ubiquitin dependent aggresome formation, thus outlining the path forward toward more effective anticancer therapeutics. To explore the therapeutic potential of targeting the UPS to treat solid cancers, we have developed an inhibitor of ubiquitin conjugation (ABP A3) that targets ubiquitin and Nedd8 E1 enzymes, enzymes that are required to maintain the activity of the entire ubiquitin system. We have shown that ABP A3 inhibits conjugation of ubiquitin to intracellular proteins and prevents the formation of cytoprotective aggresomes in A549 lung cancer cells. Furthermore, ABP A3 induces activation of the unfolded protein response and apoptosis. Thus, similar to proteasome inhibitors MG132, bortezomib, and carfilzomib, ABP A3 can serve as a novel probe to explore the therapeutic potential of the UPS in solid and hematological malignancies.     

Nonenzymatic Rubylation and Ubiquitination of Proteins for Structural and Functional Studies

Uncovering the mechanisms that allow conjugates of ubiquitin (Ub) and/or Ub‐like (UBL) proteins such as Rub1 to serve as distinct molecular signals requires the ability to make them with native connectivity and defined length and linkage composition. A novel, effective, and affordable strategy for controlled chemical assembly of fully natural UBL–Ub, Ub–UBL, and UBL–UBL conjugates from recombinant monomers is presented. Rubylation of Ub and Rub1 and ubiquitination of Rub1 was achieved without E2/E3 enzymes. New residue‐specific information was obtained on the interdomain contacts in naturally‐occurring K48‐linked Rub1–Ub and Ub–Rub1, and K29‐linked Rub1–Ub heterodimers, and their recognition by a K48‐linkage‐specific Ub receptor. The disassembly of these heterodimers by major deubiquitinating enzymes was examined and it was discovered that some deubiquitinases also possess derubylase activity. This unexpected result suggests possible crosstalk between Ub and Rub1/Nedd8 signaling pathways.     

Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn²⁺ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn²⁺ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.     

Biochemical properties of K11,48-branched ubiquitin chains

The affinities of chemically synthetic linkage- and length-defined K_11,48-branched ubiquitin chains binding to ubiquitin receptor S5a were quantitatively measured. Proteasome-associated deubiquitinase Rpn11 showed a higher activity towards K_11,48-branched ubiquitin chains.     

泛素-蛋白酶体与药物应用

In eukaryotes, the ubiquitin-proteasome pathway degrades the majority of intracellular proteins tagged with polyubiquitin chains. It participates in regulation of key cellular activities, such as cell proliferation, cell differentiation, apoptosis, DNA repair, etc. through the degradation of malformed or misfolded proteins. Dysfunctions of the ubiquitin-proteasome pathway have been linked to many diseases, including cancer and neurodegeneration, etc. The commercially available proteasome inhibitors have been successfully used to treat multiple myeloma and mantle cell lymphoma. In addition, novel inhibitors against other components of the ubiquitin-proteasome pathway, such as those enzymes that drive ubiquitination and deubiquitination in preclinical testing or clinical trials, exhibit promising therapeutic effects in vivo. This paper briefly introduces the ubiquitin-proteasome pathway related drug discovery progress.     

Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes

Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family

The ubiquitin (Ub)-proteasome system includes a large family of deubiquitinating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but without evidence for biological activity. A panel of novel DUB-specific probes was generated by a chemical ligation method. These probes allowed identification of DUBs and associated components by tandem mass spectrometry, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure. We identified 23 active DUBs in EL4 cells, including the tumor suppressor CYLD1. At least two DUBs tightly interact with the proteasome 19S regulatory complex. An OTU domain-containing protein, with no sequence homology to any known DUBs, was isolated. We show that this polypeptide reacts with the C terminus of Ub, thus demonstrating DUB-like enzymatic activity for this novel superfamily of proteases.     

Proteasomes: a complex story

Protein degradation in eukaryotic cells is important for regulation of metabolism, progression through the division cycle, in cell signalling pathways, and in mammals also for generation of antigen fragments for presentation on the major histocompatibility complex (MHC) class I. Most cell proteins are degraded via the ubiquitin/proteasome pathway where an elaborate enzyme system recognises the protein substrates and marks them for destruction by attachment of a chain of ubiquitin. The substrates are then bound to 26S proteasomes, unfolded, and threaded into the cylindrical central part of the 26S proteasome, where they are cleaved to peptides. Recently many proteins, which associate with proteasomes, have been found. One of them controls the cellular contents of proteasomes by regulating their synthesis. Others ubiquitylate substrates or transfer substrates to proteasomes. Others again seem to unfold the substrates or release ubiquitin and glycans from them during degradation, stabilise proteasomes, regulate their cellular localisation, and modify their activity. It therefore appears that proteasomes are centres in macromolecular clusters, which degrade cell proteins in a tightly regulated manner.     

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be readily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small‐molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activity, can be targeted instead, thus increasing the number of druggable targets. Whereas targeting of the proteasome itself can lead to a global increase in protein levels, the targeting of other components of the UPS (e.g., the E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small‐molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small‐molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets, such as enzymes and receptors.     

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (K_D) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display K_D values in the range 0.3–1.2 nm , which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.     

Electrophoretic separation of ubiquitin and single amino acid residue ubiquitin extensions using a commercial modified acrylamide gel electrophoresis system: an assay to determine catalytic capacities of deubiquitinating enzymes

We have chemically synthesised a number of ubiquitin extension proteins, with carboxyl-terminal single amino acid residue extensions, to use as substrates to assess the catalytic capacities of deubiquitinating enzymes (DUBs). Here we describe a modified acrylamide gel electrophoresis system which allows separation of peptide- or isopeptide-linked ubiquitin-lysine from ubiquitin (77 and 76 residue proteins respectively) in only 2 h. Western blotting, using antibodies against ubiquitin, allows both substrate (i.e. ubiquitin-lysine) and product (i.e. ubiquitin) of DUB-catalyzed cleavage reactions to be detected. Catalytic capacities of DUBs may be indicative of in vivo functions of these proteases.     

Targeting reversible disassembly as a mechanism of controlling V-ATPase activity

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved proton pumps consisting of a peripheral membrane subcomplex called V1, which contains the sites of ATP hydrolysis, attached to an integral membrane subcomplex called Vo, which encompasses the proton pore. V-ATPase regulation by reversible dissociation, characterized by release of assembled V1 sectors into the cytosol and inhibition of both ATPase and proton transport activities, was first identified in tobacco hornworm and yeast. It has since become clear that modulation of V-ATPase assembly level is also a regulatory mechanism in mammalian cells. In this review, the implications of reversible disassembly for V-ATPase structure are discussed, along with insights into underlying subunit-subunit interactions provided by recent structural work. Although initial experiments focused on glucose deprivation as a trigger for disassembly, it is now clear that V-ATPase assembly can be regulated by other extracellular conditions. Consistent with a complex, integrated response to extracellular signals, a number of different regulatory proteins, including RAVE/rabconnectin, aldolase and other glycolytic enzymes, and protein kinase A have been suggested to control V-ATPase assembly and disassembly. It is likely that multiple signaling pathways dictate the ultimate level of assembly and activity. Tissue-specific V-ATPase inhibition is a potential therapy for osteoporosis and cancer; the possibility of exploiting reversible disassembly in design of novel V-ATPase inhibitors is discussed.     

The Lysine48‐Based Polyubiquitin Chain Proteasomal Signal: Not a Single Child Anymore

The conjugation of ubiquitin (Ub) to proteins is involved in the regulation of many processes. The modification serves as a recognition element in trans, in which downstream effectors bind to the modified protein and determine its fate and/or function. A polyUb chain that is linked through internal lysine (Lys)‐48 of Ub and anchored to an internal Lys residue of the substrate has become the accepted “canonical” signal for proteasomal targeting and degradation. However, recent studies show that the signal is far more diverse and that chains based on other internal linkages, as well as linear or heterologous chains made of Ub and Ub‐like proteins and even monoUb, are recognized by the proteasome. In addition, chains linked to residues other than internal Lys were described, all challenging the current paradigm.     

Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif

The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif.     

Effects of covalent modification by 4‐hydroxy‐2‐nonenal on the noncovalent oligomerization of ubiquitin

When lipid membranes containing ω‐6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4‐hydroxy‐2‐nonenal (HNE)—a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non‐covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.