Protein degradation is the cell’s mechanism of eliminating misfolded or unwanted proteins. The pathway by which proteins are degraded occurs through the ubiquitin-proteasome system. Ubiquitin is a small 9-kilodalton (kDa) protein that is attached to proteins. A minimum of four ubiquitins is required for proteins to be recognized by the degradation machinery, known as the 26S proteasome. Defects in ubiquitination have been identified in a number of diseases, including cancer, neurodegenerative diseases, and metabolic disorders. We sought to exploit the delicate balance between protein synthesis and degradation to treat cancer by designing a chimeric molecule, known as Protac (Proteolysis Targeting Chimeric molecule). Protacs are heterobifunctional nanomolecules that are approximately 10 nanometers (nm) in size and can recruit proteins that cause cancer to the ubiquitin-proteasome machinery for degradation. In this review, we discuss the development of this novel technology for the treatment of cancer.
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
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 cyclin-dependent kinase (CDK) inhibitor p27Kip1 is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCFSKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.
We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.
Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.
We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2VHL substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2VHL or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC50 = 1.7 μm) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.
ATPases; High-throughput Screening (HTS); Proteasome; Protein Degradation; Ubiquitin
Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection.
Most eukaryotic proteins destined for imminent destruction are first tagged with a chain of ubiquitin molecules and are subsequently dismantled by the proteasome. Ubiquitin-independent degradation of substrates by the proteasome, however, also occurs. The number of documented proteasome-dependent, ubiquitin-independent degradation events remains relatively small but continues to grow. Proteins involved in oncogenesis and tumor suppression make up the majority of the known cases for this type of protein destruction. Provocatively, viruses with confirmed or suspected oncogenic properties are also prominent participants in the pantheon of ubiquitin-independent proteasomal degradation events. In this review, we identify and describe examples of proteasome-dependent, ubiquitin-independent protein degradation that occur during tumor virus infections, speculate why this type of protein destruction may be preferred during oncogenesis, and argue that this uncommon type of protein turnover represents a prime target for antiviral and anticancer therapeutics.
Proteasomes, the primary mediators of ubiquitin-protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that Usp14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin-protein conjugates, in vivo and in vitro. A catalytically inactive variant of Usp14 has reduced inhibitory activity, suggesting that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human Usp14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. Usp14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of Usp14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.
Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms α, δ, and ɛ in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC δ. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC α, δ, and ɛ were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC α could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.
The ubiquitin/proteasome system handles the majority of controlled proteolysis in eukaryotes. Defects in the ubiquitin/proteasome system have been implicated in diseases ranging from cancers to neurodegenerative disorders. However, the precise role of ubiquitin/proteasome-mediated degradation in health and disease is far from clear. A major challenge is to link specific substrates directly to a particular degradation pathway. Here, we review genome-wide approaches that have been developed in recent years to comprehensively identify ubiquitylated substrates of a particular pathway. The components of the ubiquitin/proteasome system are attractive drug targets, as illustrated by the efficacy of some proteasome inhibitors in the treatment of multiple myeloma. Information that has emerged from these studies could reveal novel drug targets and strategies for treating human diseases.
Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF.
Ubiquitination is a widely-studied regulatory modification involved in protein degradation, DNA damage repair and the immune response. Conjugation of ubiquitin to a substrate lysine occurs in an enzymatic cascade involving an E1 ubiquitin activating enzyme, and E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, or E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
Ubiquitin Conjugation; Ubiquitin-like proteins; Ubc13-Mms2; ubiquitin-activating enzyme
The Ubiquitin Proteasome System (UPS) is a major regulator of protein abundance in the cell. The UPS influences the functions of multiple biological processes by targeting key regulators for destruction. E3 ubiquitin ligases are a vital component of the UPS machinery, working with E1 and E2 enzymes to bind substrates and facilitate the transfer of ubiquitin molecules onto the target protein. This polyubiquitination, in turn, directs the modified proteins for proteolysis by the 26S proteasome. As the UPS regulates the degradation of multiple oncogenes and tumor suppressors, the dysregulation of this pathway is known to promote various diseases including cancer. While E1 and E2 enzymes have only been minimally linked to cancer development, burgeoning amounts of evidence have implicated loss or gain of E3 function as a key factor in cancer initiation and progression. This review will examine the literature on two SCF-type E3 ligases, SCFFbw7 and SCFbeta-TRCP. In particular, we will highlight novel substrates recently identified for these two E3 ligases, and further discuss how UPS regulation of these targets may promote carcinogenesis.
Ubiquitination; SCF; Fbw7; beta-TRCP; Oncogene; Tumor suppressor; Cancer; E3-ligase; Phosphorylation; Review
The c-Myc oncoprotein is a transcription factor which is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt's lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Here we demonstrate that proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein which are required for rapid proteolysis. We found that a centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. We found that c-Myc is significantly stabilized (two- to sixfold) in many Burkitt's lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitt's lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitt's lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitt's lymphoma cells.
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
The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress-induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the functional roles of p62 and HSP70 were evaluated in conjunction with proteasome inhibitor–induced autophagy in human RPE cells (ARPE-19).
The p62, HSP70, and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay.
Proteasome inhibition evoked the accumulation of perinuclear aggregates that strongly colocalized with p62 and HSP70. The p62 perinuclear accumulation was time- and concentration-dependent after MG-132 proteasome inhibitor loading. The silencing of p62, rather than Hsp70, evoked suppression of autophagy, when related to decreased LC3-II levels after bafilomycin treatment. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently, we showed that hsp70 mRNA depletion increased cell death in ARPE-19 cells. Here, we demonstrate that p62 mRNA silencing has similar effects on cellular viability.
Our findings open new avenues for understanding the mechanisms of proteolytic processes in retinal cells, and could be useful in the development of novel therapies targeting p62 and HSP70.
The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription.
The ubiquitin proteasome system (UPS) orchestrates the turnover of innumerable cellular proteins. In the process of ubiquitination the small protein ubiquitin is attached to a target protein by a peptide bond. The ubiquitinated target protein is subsequently shuttled to a protease complex known as the 26S proteasome and subjected to degradative proteolysis. The UPS facilitates the turnover of proteins in several settings. It targets oxidized, mutant or misfolded proteins for general proteolytic destruction, and allows for the tightly controlled and specific destruction of proteins involved in development and differentiation, cell cycle progression, circadian rhythms, apoptosis, and other biological processes. In neuropathology, alteration of the UPS, or mutations in UPS target proteins may result in signaling abnormalities leading to the initiation or progression of tumors such as astrocytomas, hemangioblastomas, craniopharyngiomas, pituitary adenomas, and medulloblastomas. Dysregulation of the UPS may also contribute to tumor progression by perturbation of DNA replication and mitotic control mechanisms, leading to genomic instability. In neurodegenerative diseases caused by the expression of mutant proteins, the cellular accumulation of these proteins may overload the UPS, indirectly contributing to the disease process, e.g., sporadic Parkinsonism and prion diseases. In other cases, mutation of UPS components may directly cause pathological accumulation of proteins, e.g., autosomal recessive Parkinsonism and spinocerebellar ataxias. Defects or dysfunction of the UPS may also underlie cognitive disorders such as Angelman syndrome, Rett syndrome and autism, and muscle and nerve diseases, e.g., inclusion body myopathy and giant axon neuropathy. This paper describes the basic biochemical mechanisms comprising the UPS and reviews both its theoretical and proven involvement in neuropathological diseases. The potential for the UPS as a target of pharmacological therapy is also discussed.
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
Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.
The cyclin-dependent kinase (CDK) inhibitor p27Kip1 (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E–CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines over-expressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.
p27; Proteolysis; Breast cancer; T187A; Apoptosis; CDK, cyclin-dependent kinase; FITC, fluorescein isothiocyanate; Mur A, muristerone A; PARP, poly(ADP-ribose) polymerase
The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-Luciferase fusion protein. To insure uHTS compatibility, the assay was scaled to 1,536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogenous assay demonstrated robust performance, with a calculated Z′ factor of 0.65±0.05. The assay was screened against a publicly available library of ~218,000 compounds in order to identify Wee1 stabilizers. Nonselective, cytotoxic and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of a N-cyclin B-Luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of four unrelated cell-permeable small molecules that showed selective Wee1-Luciferase stabilization with micromolar potency. One of these compounds, SID4243143, was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Our results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation, and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.
Wee1; degradation; stabilizer; reporter assay; transient transfection; cryopreserved cells; ubiquitin; proteasome
Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.
Failure to eliminate abnormal proteins in the cell is associated with numerous aggregation diseases. Misfolded proteins are normally detected by protein quality control and either refolded or eliminated. The ubiquitin-proteasome system is a major pathway that degrades these unwanted proteins. Ubiquitin ligases are central to these degradation pathways as they recognize aberrant proteins and covalently attach a polyubiquitin chain to target them to the proteasome. We discovered that the Hul5 ubiquitin ligase is a major player in a novel protein quality control pathway that targets cytosolic misfolded proteins. Hul5 is required for the maintenance of cell fitness and the increased ubiquitination of low solubility proteins after heat-shock in yeast cells. We identified several low-solubility substrates of Hul5, including the prion-like protein Pin3. It is now apparent that in the cytoplasm, misfolded proteins can be targeted by multiple degradation pathways. In this review, we discuss how the Hul5 protein quality control pathway may specifically target low solubility cytosolic proteins in the cell.
HECT ligase; Hul5; Pin3; aggregation; misfolding; protein quality control; proteolysis; ubiquitin proteasome system
GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4). GTPCH1 protein degradation has been reported in animal models of several diseases, including diabetes mellitus and hypertension. However, the molecular mechanisms by which GTPCH1 is degraded remain uncharacterized. Here we report a novel non-covalent interaction between polyubiquitin and GTPCH1 in vitro and in vivo. The non-covalent binding of GTPCH1 to polyubiquitin via an ubiquitin-binding domain (UBD) results in ubiquitination and degradation. Ectopic expression of ubiquitin in cultured cells accelerated GTPCH1 degradation. In cultured cells and in vitro assays, Lys48-linked ubiquitin chains, but not Lys63-linked chains, interacted with GTPCH1 and targeted it for degradation. Consistently, proteasome inhibition attenuated GTPCH1 degradation. Finally, direct mutagenesis of an isoleucine (Ile131) in the hydrophobic patch of the GTPCH1 UBD affected its ubiquitin binding and the enzyme stability. Taken together, we conclude that GTPCH1 non-covalently interacts with polyubiquitin via an ubiquitin-binding domain. The polyubiquitin binding directs GTPCH1 ubiquitination and proteasome degradation.