E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome. Therefore, E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation, proliferation and apoptosis. E3 ubiquitin ligases are often found overexpressed in human cancers, including lung cancer, and their deregulation has been shown to contribute to cancer development. However, the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting. In this review, we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer. Furthermore, we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets. By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis, we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.
E3 ubiquitin ligases; Lung cancer; Ubiquitin-proteasome system; Proteasome inhibitors; Bortezomib; Apoptosis; Gene regulation; DNA repair
Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.
Carcinogenesis; F-box proteins; RING proteins; SCF E3 ligases; Skin; Ubiquitin ligases
The ubiquitination–proteasome and degradation system is an essential process that regulates protein homeostasis. This system is involved in the regulation of cell proliferation, differentiation and survival, and dysregulations in this system lead to pathologies including cancers. The ubiquitination system is an enzymatic cascade that mediates the marking of target proteins by an ubiquitin label and thereby directs their degradation through the proteasome pathway. The ubiquitination of proteins occurs through a three-step process involving ubiquitin activation by the E1 enzyme, allowing for the transfer to a ubiquitin-conjugated enzyme E2 and to the targeted protein via ubiquitin-protein ligases (E3), the most abundant group of enzymes involved in ubiquitination. Significant advances have been made in our understanding of the role of E3 ubiquitin ligases in the control of bone turnover and tumorigenesis. These ligases are implicated in the regulation of bone cells through the degradation of receptor tyrosine kinases, signaling molecules and transcription factors. Initial studies showed that the E3 ubiquitin ligase c-Cbl, a multi-domain scaffold protein, regulates bone resorption by interacting with several molecules in osteoclasts. Further studies showed that c-Cbl controls the ubiquitination of signaling molecules in osteoblasts and in turn regulates osteoblast proliferation, differentiation and survival. Recent data indicate that c-Cbl expression is decreased in primary bone tumors, resulting in excessive receptor tyrosine kinase signaling. Consistently, c-Cbl ectopic expression reduces bone tumorigenesis by promoting tyrosine kinase receptor degradation. Here, we review the mechanisms of action of E3 ubiquitin ligases in the regulation of normal and pathologic bone formation, and we discuss how targeting the interactions of c-Cbl with some substrates may be a potential therapeutic strategy to promote osteogenesis and to reduce tumorigenesis.
ubiquitin ligases; proteasome; receptor tyrosine kinases; bone tumors; Cbl proteins; ubiquitination
Although radiotherapy represents one of the most effective treatment modalities for patients with cancer, inherent and/or acquired resistance of cancer cells to radiotherapy is often an impediment to effective treatment. Diverse strategies have been developed to improve the efficacy of radiotherapy. The ubiquitin-proteasome system (UPS) operates in numerous vital biologic processes by controlling the protein turnover in cells. Ubiquitination is central to the UPS pathway, and it relies on the E3 ubiquitin ligases to catalyze the covalent attachment of ubiquitin to its protein substrates. Cullin-based RING ligases (CRLs) are the largest family of E3 ligases that are responsible for the ubiquitination and destruction of numerous cancer-relevant proteins. Its deregulation has been linked to many human cancers, making it an attractive target for therapeutic intervention. This review discusses how targeting the ubiquitin-proteasome system, particularly CRLs, is an exciting new strategy for radiosensitization in cancer and, specifically, focuses on MLN4924, a recently discovered small-molecule inhibitor of the NEDD8-activating enzyme, which is being characterized as a novel radiosensitizing agent against cancer cells by inactivating CRL E3 ubiquitin ligases.
The ubiquitin–proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.
Isopeptidases; Protein degradation; N terminus; 3D polymerase
The ubiquitin-proteasome system plays a critical role in controlling the level, activity, and location of various cellular proteins. Significant progress has been made in investigating the molecular mechanisms of ubiquitination, particularly in understanding the structure of the ubiquitination machinery and identifying ubiquitin protein ligases, the primary specificity-determining enzymes. Therefore, it is now possible to target specific molecules involved in the ubiquitination and proteasomal degradation to regulate many cellular processes such as signal transduction, proliferation and apoptosis. In particular, alterations in ubiquitination are observed in most, if not all, cancer cells. This is manifested by destabilization of tumor suppressors, such as p53, and overexpression of oncogenes such as c-Myc and c-Jun. In addition to the development and clinical validation of proteasome inhibitor Bortezomib in myeloma therapy, recent studies have demonstrated that it is possible to develop inhibitors for specific ubiquitination and deubiquitination enzymes. With the help of structural studies, rational design, and chemical synthesis, it is conceivable that we will be able to use “druggable” inhibitors of the ubiquitin system to evaluate their effects in animal tumor models in the not-so-distant future.
Molecule targeting; ubiquitin; proteasome; cancer therapeutics
Nrf2 is a transcription factor that has emerged as the cell's main defense mechanism against many harmful environmental toxicants and carcinogens. Nrf2 is negatively regulated by Keap1, a substrate adaptor protein for the Cullin3 (Cul3)-containing E3-ligase complex, which targets Nrf2 for ubiquitination and degradation by the ubiquitin proteasome system (UPS). Recent evidence suggests that constitutive activation of Nrf2, due to mutations in Keap1 or Nrf2, is prominent in many cancer types and contributes to chemoresistance. Regulation of Nrf2 by the Cul3–Keap1-E3 ligase provides strong evidence that tight regulation of Cullin-ring ligases (CRLs) is imperative to maintain cellular homeostasis. There are seven known Cullin proteins that form various CRL complexes. They are regulated by neddylation/deneddylation, ubiquitination/deubiquitination, CAND1-assisted complex assembly/disassembly, and subunit dimerization. In this review, we will discuss the regulation of each CRL using the Cul3–Keap1-E3 ligase complex as the primary focus. The substrates of CRLs are involved in many signaling pathways. Therefore, deregulation of CRLs affects several cellular processes, including cell cycle arrest, DNA repair, cell proliferation, senescence, and death, which may lead to many human diseases, including cancer. This makes CRLs a promising target for novel cancer drug therapies. Antioxid. Redox Signal. 13, 1699–1712.
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.
Uba1; E1; Ubiquitin-activating enzyme; Apoptosis; Proliferation; Drosophila; Autonomous control; Non autonomous control
Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.
Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.
bortezomib; MG132; MLN4924; neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE); proteasome; ubiquitinactivating enzyme; BCA3, breast cancer-associated gene 3; CHO, Chinese-hamster ovary; CRL, cullin-RING ubiquitin ligase; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HA, haemagglutinin; HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate; NAE, NEDD8-activating enzyme; NEDD8, neural-precursor-cell-expressed developmentally down-regulated 8; siRNA, small interfering RNA; SUMO, small ubiquitin-like modifier; TCA, trichloroacetic acid; UBL, ubiquitin-like; WT, wild-type
Posttranslational modification of proteins by ubiquitin has emerged as a critical regulator of synapse development and function. Ubiquitination is a reversible modification mediated by the concerted action of a large number of specific ubiquitin ligases and ubiquitin proteases, called deubiquitinating enzymes (DUBs). The balance of activity of these enzymes determines the localization, function, and stability of target proteins. While some DUBs counter the action of specific ubiquitin ligases by removing ubiquitin and editing ubiquitin chains, other DUBs function more generally to maintain the cellular pool of free ubiquitin monomers. The importance of DUB function at the synapse is underscored by the association of specific mutations in DUB genes with several neurological disorders. Over the last decade, although much research has led to the identification and characterization of many ubiquitin ligases at the synapse, our knowledge of the relevant DUBs that act at the synapse has lagged. This review is focused on highlighting our current understanding of DUBs that regulate synaptic function and the diseases that result from dysfunction of these DUBs.
Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells.1 Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have developed.
ubiquitin; endosome; ligase; lysosome; degradation
The SCF (Skp1, Cullins, F-box proteins) multisubunit E3 ubiquitin ligase, also known as CRL (Cullin-RING ubiquitin Ligase) is the largest E3 ubiquitin ligase family that promotes the ubiquitination of various regulatory proteins for targeted degradation, thus regulating many biological processes, including cell cycle progression, signal transduction, and DNA replication. The efforts to discover small molecule inhibitors of a SCF-type ligase or its components were expedited by the FDA approval of Bortezomib (also known as Velcade or PS-341), the first (and only) class of general proteasome inhibitor, for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma. Although Bortezomib has demonstrated a certain degree of cancer cell selectivity with measurable therapeutic index, the drug is, in general, cytotoxic due to its inhibition of overall protein degradation. An alternative and ideal approach is to target a specific E3 ligase, known to be activated in human cancer, for a high level of specificity and selectivity with less associated toxicity, since such inhibitors would selectively stabilize a specific set of cellular proteins regulated by this E3. Here, we review recent advances in validation of SCF E3 ubiquitin ligase as an attractive anti-cancer target and discuss how MLN4924, a small molecule inhibitor of NEDD8-activating enzyme, can be developed as a novel class of anticancer agents by inhibiting SCF E3 ligase via removal of cullin neddylation. Finally, we discuss under future perspective how basic research on SCF biology will direct the drug discovery efforts surrounding this target.
Ubiquitin-proteasome system; SCF E3 ubiquitin ligase; anticancer target; drug discovery; neddylation; cullins; F-box proteins; RING ligases
Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of ∼1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed.
ubiquitin; E3 ligase; self-ubiquitination; proteasomal degradation
Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin-proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell-proliferation. Ubiquitin ligases called SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8 conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.
Ubiquitination; SCF complex; NEDD8; MLN4924; Ubc12; NUB1
Covalent conjugation of proteins with ubiquitin is one the most important post translational modifications because it controls intracellular protein trafficking typically resulting in protein degradation. Frequently ubiquitinated proteins are targeted to the proteasome for degradation in the cytosol. However, ubiquitinated membrane bound proteins can also be targeted for endocytosis and degradation in the lysosome. Ubiquitin-dependent degradation pathways have clear cancer relevance due to their integral involvement in protein quality control, regulation of immune responses, signal transduction, and cell cycle regulation. In spite of its fundamental importance, little is known regarding how proteins are specifically identified for ubiquitin-dependent degradation. In this article we review a newly discovered family of viral and cellular ubiquitin ligases called MARCH proteins. Recent studies of MARCH proteins define new paradigms showing how ubiquitin E3 ligases determine the intracellular location and fate of proteins.
Degradation pathways; ubiquitination; intracellular trafficking; immune evasion; γherpesvirus-68; Kaposi’s sarcoma-associated herpes virus
The ubiquitin-proteasome system is one of the major protein turnover mechanisms that plays important roles in the regulation of a variety of cellular functions. It is composed of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 ubiquitin ligases that transfer ubiquitin to the substrates that are subjected to degradation in the 26S proteasome. The Skp1, Cullin, F-box protein (SCF) E3 ligases are the largest E3 gene family, in which the F-box protein is the key component to determine substrate specificity. Although the SCF E3 ligase and its F-box proteins have been extensively studied in the model yeast Saccharomyces cerevisiae, only limited studies have been reported on the role of F-box proteins in other fungi. Recently, a number of studies revealed that F-box proteins are required for fungal pathogenicity. In this communication, we review the current understanding of F-box proteins in pathogenic fungi.
Cryptococcus neoformans; E3 ligase; F-box; Fungi; Virulence
Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorβ and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.
RING ligase; breast cancer; BCA2; RNF11; ubiquitin
Ubiquitin conjugation to lysine residues regulates a variety of protein functions, including endosomal trafficking and degradation. While ubiquitin plays an important role in the release of many viruses, the requirement for direct ubiquitin conjugation to viral structural proteins is less well understood. Some viral structural proteins require ubiquitin ligase activity, but not ubiquitin conjugation, for efficient release. Recent evidence has shown that, like other viruses, hepatitis B virus (HBV) requires a ubiquitin ligase for release from the infected cell. The HBV core protein contains two lysine residues (K7 and K96), and K96 has been suggested to function as a potential ubiquitin acceptor site based on the fact that previous studies have shown that mutation of this amino acid to alanine blocks HBV release. We therefore reexamined the potential connection between core lysine ubiquitination and HBV replication, protein trafficking, and virion release. In contrast to alanine substitution, we found that mutation of K96 to arginine, which compared to alanine is more conserved but also cannot mediate ubiquitin conjugation, does not affect either virus replication or virion release. We also found that the core lysine mutants display wild-type sensitivity to the antiviral activity of interferon, which demonstrates that ubiquitination of core lysines does not mediate the interferon-induced disruption of HBV capsids. However, mutation of K96 to arginine alters the nuclear-cytoplasmic distribution of core, leading to an accumulation in the nucleolus. In summary, these studies demonstrate that although ubiquitin may regulate the HBV replication cycle, these mechanisms function independently of direct lysine ubiquitination of core protein.
Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.
Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.
By virtue of its ability to regulate both protein turnover and nonproteolytic signalling functions, ubiquitin protein conjugation has been implicated in the control of multiple cellular processes, including protein localization, cell cycle control, transcription regulation, DNA damage repair and endocytosis. Ubiquitin metabolism enzymes have been identified as either oncogenes or tumor suppressors in a variety of cancers. Given that ubiquitin metabolism is governed by enzymes—E1, E2, E3, E4, deubiquitinases (DUBs) and the proteasome-the system as a whole is ripe for target and drug discovery in cancer. Of the ubiquitin/proteasome system components, the E3s and DUBs can recognize substrates with the most specificity, and are thus of key interest as drug targets in cancer. This review examines the molecular role in cancer, relevant substrates and potential for pharmacologic development, of E3s and DUBs that have been associated thus far with human malignancies as oncogenes or tumor suppressors.
ubiquitin; E3; cancer; DUB; ligase; proteasome
Notch signaling is highly conserved in all metazoan animals and plays critical roles in cell fate specification, cell proliferation, apoptosis, and stem cell maintenance. Although core components of the Notch signaling cascade have been identified, many gaps in the understanding of the Notch signaling pathway remain to be filled. One form of posttranslational regulation, which is controlled by the ubiquitin-proteasome system, is known to modulate Notch signaling. The ubiquitination pathway is a highly coordinated process in which the ubiquitin moiety is either conjugated to or removed from target proteins by opposing E3 ubiquitin ligases and deubiquitinases (DUBs). Several E3 ubiquitin ligases have been implicated in ubiquitin conjugation to the receptors and the ligands of the Notch signaling cascade. In contrast, little is known about a direct role of DUBs in Notch signaling in vivo. Here, we report an in vivo RNA interference screen in Drosophila melanogaster targeting all 45 DUBs that we annotated in the fly genome. We show that at least four DUBs function specifically in the formation of the fly wing margin and/or the specification of the scutellar sensory organ precursors, two processes that are strictly dependent on the balanced Notch signaling activity. Furthermore, we provide genetic evidence suggesting that these DUBs are necessary to positively modulate Notch signaling activity. Our study reveals a conserved molecular mechanism by which protein deubiquitination process contributes to the complex posttranslational regulation of Notch signaling in vivo.
deubiquitinase; Drosophila melanogaster; Notch signaling; ubiquitination
An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.
Arabidopsis thaliana; E3 ubiquitin ligases; RING fingers; gene families; pathogen response; stress response
The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.