No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Pickart CM.. Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-533
Abstract
The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains--a HECT domain or a RING finger--and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.
... Drug resistance is also an important issue, and for these reasons, identifying novel proteins for targeted therapy is continuously necessary. Ubiquitin pathway factors have been steadily investigated, as they are one of the major post-translational modifications (PTMs) that regulate a plethora of proteins in the cell [20][21][22][23][24]. Ubiquitination is essential for tagging proteins for proteolytic degradation, and it is also involved in cell signaling transduction as well as protein-protein interaction (PPI). ...
... There are seven lysine residues on ubiquitin, and poly-ubiquitination at K48 tags its substrate for degradation, whereas K63 functions as a signal for the recruitment of interacting partners. There are three steps to ubiquitinating a substrate protein [20,22,24,27]. Ubiquitination is a three-step event with three enzymes that are involved: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligases. ...
... E3 ligases are more numerous than E1 and E2 enzymes, and have specificity for its substrate. Many of these E3 ligases and their substrates have been defined [20,23,[41][42][43], offering a positive outlook on the development of anticancer therapy. This review will highlight the function of three E3 ubiquitin ligases, RNF168, RNF126 and CUL1, that have multiple roles in DDR, DNA double-strand break (DSB) repair, cell cycle regulation, and cell death. ...
Ubiquitination is a post-translational modification (PTM) that is involved in proteolysis, protein–protein interaction, and signal transduction. Accumulation of mutations and genomic instability are characteristic of cancer cells, and dysfunction of the ubiquitin pathway can contribute to abnormal cell physiology. Because mutations can be critical for cells, DNA damage repair, cell cycle regulation, and apoptosis are pathways that are in close communication to maintain genomic integrity. Uncontrolled cell proliferation due to abnormal processes is a hallmark of cancer, and mutations, changes in expression levels, and other alterations of ubiquitination factors are often involved. Here, three E3 ubiquitin ligases will be reviewed in detail. RNF126, RNF168 and CUL1 are involved in DNA damage response (DDR), DNA double-strand break (DSB) repair, cell cycle regulation, and ultimately, cancer cell proliferation control. Their involvement in multiple cellular pathways makes them an attractive candidate for cancer-targeting therapy. Functional studies of these E3 ligases have increased over the years, and their significance in cancer is well reported. There are continuous efforts to develop drugs targeting the ubiquitin pathway for anticancer therapy, which opens up the possibility for these E3 ligases to be evaluated for their potential as a target protein for anticancer therapy.
... According to many studies, it is also involved in the shedding of membrane and as well as associated proteins as extracellular vesicles that affect not only the amount of certain membrane proteins on the cell surface, but also their potential transport to neighboring cells. Additionally, several diseases such as muscular dystrophy, metabolic syndrome, nervous system disorders and cancer are several disease which can occur due to any kind of abnormality in ubiquitination process [13]. The multistep process of ubiquitination comprises the ubiquitin activation by E1 enzymes, and to conjugate it to E2 enzymes, and the ligation of ubiquitin to the substrate protein by E3 enzymes. ...
... E3 ligases then recognize and bind target proteins, marking them with ubiquitin molecules through isopeptide bond formation. These ubiquitinated proteins are then subjected to degradation in the proteasome [13]. The attachment of the ubiquitin molecule to the cytisine site and production of the intermediate ubiquitin-adenylate can be done with the help of adenosine triphosphate (ATP). ...
... Ubiquitination is a post-translational mechanism of protein modification involved in multiple signaling pathways (1,2). The 76-amino acid ubiquitin is covalently conjugated to lysine (K) residues of target proteins catalyzed by ubiquitin-activating (E1), ubiquitinconjugating (E2), and ubiquitin-ligating (E3) enzymes (3,4) (Figure 1). Ubiquitin molecules are conjugated to target proteins either singly (monoubiquitination) or in the form of polyubiquitin chains. ...
... The most studied E3 ligase subfamilies are really interesting new gene-(RING-) and homologous to E6AP C terminus-(HECT-) domain containing ligases (9). E3s with RING domain catalyze the transfer of ubiquitin from E2 to substrate (10), while E3s with HECT domain transfer ubiquitin to substrate through covalent thioester intermediate between ubiquitin and the cysteine residue of HECT domain (4,11). ...
T cells play critical role in multiple immune processes including antigen response, tumor immunity, inflammation, self-tolerance maintenance and autoimmune diseases et. Fetal liver or bone marrow-derived thymus-seeding progenitors (TSPs) settle in thymus and undergo T cell-lineage commitment, proliferation, T cell receptor (TCR) rearrangement, and thymic selections driven by microenvironment composed of thymic epithelial cells (TEC), dendritic cells (DC), macrophage and B cells, thus generating T cells with diverse TCR repertoire immunocompetent but not self-reactive. Additionally, some self-reactive thymocytes give rise to Treg with the help of TEC and DC, serving for immune tolerance. The sequential proliferation, cell fate decision, and selection during T cell development and self-tolerance establishment are tightly regulated to ensure the proper immune response without autoimmune reaction. There are remarkable progresses in understanding of the regulatory mechanisms regarding ubiquitination in T cell development and the establishment of self-tolerance in the past few years, which holds great potential for further therapeutic interventions in immune-related diseases.
... Ubiquitination, a well-known post-translational modification (PTM), plays a pivotal role in the regulation of a wide range of biological processes, including protein degradation, signal transduction, and DNA repair [14][15][16][17][18]. PTM involves tightly controlled activation, conjugation, ligation, and deubiquitination, which are each carried out and regulated by specific enzymes [15,19]. The ubiquitin system includes (1) all ubiquitinated proteins, (2) enzymes that catalyse the addition/removal of ubiquitin, and (3) proteins that bind to ubiquitinated proteins. ...
... The deubiquitinating activity of the resulting product was further mapped to aa 149-285, as predicted ( Figure 5C). Seven lysine (Lys) residues and the N-terminal methionine (Met) of ubiquitin can be ubiquitinated to generate eight linkage types of ubiquitin chains (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) [16,19]. Most mammalian OTUs strongly prefer to cleave polyubiquitin chains with specific linkage types [54,55]. ...
Toxoplasma gondii is among the most important parasites worldwide. The apicoplast is a unique organelle shared by all Apicomplexan protozoa. Increasing lines of evidence suggest that the apicoplast possesses its own ubiquitination system. Deubiquitination is a crucial step executed by deubiquitinase (DUB) during protein ubiquitination. While multiple components of ubiquitination have been identified in T. gondii, the deubiquitinases involved remain unknown. The aim of the current study was to delineate the localization of TgOTU7 and elucidate its functions. TgOTU7 was specifically localized at the apicoplast, and its expression was largely regulated during the cell cycle. Additionally, TgOTU7 efficiently breaks down ubiquitin chains, exhibits linkage-nonspecific deubiquitinating activity and is critical for the lytic cycle and apicoplast biogenesis, similar to the transcription of the apicoplast genome and the nuclear genes encoding apicoplast-targeted proteins. Taken together, the results indicate that the newly described deubiquitinase TgOTU7 specifically localizes to the apicoplast and affects the cell growth and apicoplast homeostasis of T. gondii.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13567-023-01261-y.
... This process is integral to cell cycle advancement, signal transduction, and membrane protein transportation, serving as a significant form of post-translational protein modification [1]. The enzymatic reaction of ubiquitination is carried out through the sequential action of the ubiquitin-activating enzyme E1, the ubiquitin-binding enzyme E2, and the ubiquitin-protein ligase E3 [2,3]. E1 initiates the activation of ubiquitin, followed by the transfer of the activated ubiquitin to E2 to create the E2-Ub complex. ...
TRAF6 is an E3 ubiquitin ligase that plays a crucial role in cell signaling. It is known that MMP is involved in tumor metastasis, and TRAF6 induces MMP-9 expression by binding to BSG. However, inhibiting TRAF6’s ubiquitinase activity without disrupting the RING domain is a challenge that requires further research. To address this, we conducted computer-based drug screening to identify potential TRAF6 inhibitors. Using a ligand–receptor complex pharmacophore based on the inhibitor EGCG, known for its anti-tumor properties, we screened 52,765 marine compounds. After the molecular docking of 405 molecules with TRAF6, six compounds were selected for further analysis. By replacing fragments of non-binding compounds and conducting second docking, we identified two promising molecules, CMNPD9212-16 and CMNPD12791-8, with strong binding activity and favorable pharmacological properties. ADME and toxicity predictions confirmed their potential as TRAF6 inhibitors. Molecular dynamics simulations showed that CMNPD12791-8 maintained a stable structure with the target protein, comparable to EGCG. Therefore, CMNPD12791-8 holds promise as a potential inhibitor of TRAF6 for inhibiting tumor growth and metastasis.
... Oncogene was not solely dependent on types but also on the ubiquitylated sites of substrates [25], we conducted in vitro ubiquitination assays in the lysine-mutated FABP5 protein and identified the TRIM45adding ubiquitin chain conjugated to 24th lysine residues of FABP5 (Fig. 6E). Consistently, immunoprecipitation results indicated that the nuclear location signal (NLS) domain was necessary for TRIM45-FABP5 interaction (Fig. 6F), and the substitution of 24th lysine to arginine also impaired the interaction (Fig. S6B). ...
Non-alcoholic steatohepatitis (NASH) is rapidly surpassing viral hepatitis as the primary cause of hepatocellular carcinoma (HCC). However, understanding of NASH-progressed HCC remains poor, which might impede HCC diagnosis and therapy. In this study, we aim to identify shared transcriptional changes between NASH and HCC, of which we focused on E3 ligase TRIM45. We found TRIM45 exacerbates HCC cells proliferation and metastasis in vitro and in vivo. Further transcriptome analysis revealed TRIM45 predominantly affects fatty acid metabolism and oleic acid restored impaired proliferation and metastasis of TRIM45-deficient HCC cells. IP-tandem mass spectrum and FABP5 depriving experiment indicated that TRIM45 enhance fatty acid synthesis depending on FABP5 presence. Interestingly, we found TRIM45 directly added K33-type and K63-type poly-ubiquitin chains to FABP5 NLS domain, which ultimately promoted FABP5 nuclear translocation. Nuclear FABP5 interacted with PPARγ to facilitate downstream lipid synthesis gene expression. We observed TRIM45 accelerated NASH-to-HCC transition and exacerbated both NASH and NASH-HCC with the enhanced fatty acid production in vivo. Moreover, high concentration of fatty acid increased TRIM45 expression. The established mechanism was substantiated by gene expression correlation in TCGA-LIHC. Collectively, our research revealed a common lipid reprograming process in NASH and HCC and identified the cyclical amplification of the TRIM45-FABP5-PPARγ-fatty acid axis. This signaling pathway offers potential therapeutic targets for therapeutic intervention in NASH and NASH-progressed HCC.
... Ubiquitination is an important type of posttranslational modification of proteins that requires ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligase enzymes (E3), and through three-step sequential actions, it transfers the activated ubiquitin from the E2 to the substrate [17,18]. The ubiquitin-conjugating enzyme E2C is a ubiquitin-binding enzyme that accepts ubiquitin from E1 and transfers it to a substrate associated with E3. ...
Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of UBE2C inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of UBE2C promotes C2C12 cell differentiation. Additionally, knockdown of UBE2C, specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration in vivo. Mechanistically, we show that UBE2C knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.
... Post-translational protein modifications play an important role in living organisms [1]. Ubiquitination, one of the most common posttranslational modifications of proteins, plays a crucial role in the process of selective protein degradation in plants [2][3][4]. Protein ubiquitination is involved in various processes, such as photomorphogenesis, vascular differentiation, flower development, phytohormone and light signaling, and biotic and abiotic stress responses [5][6][7][8]. The process of protein ubiquitination is mediated by the action of three enzymes, termed ubiquitin-activating enzyme (E1s), ubiquitin-conjugating enzyme (E2s), and ubiquitin-ligating enzyme (E3s) [9]. ...
Background
Ubiquitination is an important regulatory step of selective protein degradation in the plant UPS (ubiquitin–proteasome system), which is involved in various biological processes in eukaryotes. Ubiquitin-conjugating enzymes play an intermediate role in the process of protein ubiquitination reactions and thus play an essential role in regulating plant growth and response to adverse environmental conditions. However, a genome-wide analysis of the UBC gene family in wheat (Triticum aestivum L.) has not yet been performed.
Results
In this study, the number, physiochemical properties, gene structure, collinearity, and phylogenetic relationships of TaUBC family members in wheat were analyzed using bioinformatics methods. The expression pattern of TaUBC genes in different tissues/organs and developmental periods, as well as the transcript levels under abiotic stress treatment, were analyzed using RNA-Seq data and qRT-PCR. Meanwhile, favorable haplotypes of TaUBC25 were investigated based on wheat resequencing data of 681 wheat cultivars from the Wheat Union Database. The analyses identified a total of 93 TaUBC family members containing a UBC domain in wheat genome. These genes were unevenly distributed across 21 chromosomes, and numerous duplication events were observed between gene members. Based on phylogenetic analysis, the TaUBC family was divided into 13 E2 groups and a separate UEV group. We investigated the expression of TaUBC family genes under different tissue/organ and stress conditions by quantitative real-time PCR (qRT-PCR) analysis. The results showed that some TaUBC genes were specifically expressed in certain tissues/organs and that most TaUBC genes responded to NaCl, PEG6000, and ABA treatment with different levels of expression. In addition, we performed association analysis for the two haplotypes based on key agronomic traits such as thousand-kernel weight (TKW), kernel length (KL), kernel weight (KW), and kernel thickness (KT), examining 122 wheat accessions at three environmental sites. The results showed that TaUBC25-Hap II had significantly higher TKW, KL, KW, and KT than TaUBC25-Hap I. The distribution analysis of haplotypes showed that TaUBC25-Hap II was preferred in the natural population of wheat.
Conclusion
Our results identified 93 members of the TaUBC family in wheat, and several genes involved in grain development and abiotic stress response. Based on the SNPs detected in the TaUBC sequence, two haplotypes, TaUBC25-Hap I and TaUBC25-Hap II, were identified among wheat cultivars, and their potential value for wheat breeding was validated by association analysis. The above results provide a theoretical basis for elucidating the evolutionary relationships of the TaUBC gene family and lay the foundation for studying the functions of family members in the future.
... By targeting specific proteins for degradation via ubiquitination and subsequent proteasomal processing, the UPS ensures the timely removal of damaged or surplus proteins, thereby influencing various cellular processes and signaling pathways (Ciechanover 1994). Ubiquitination, a complex post-translational modification process, is characterized by the covalent linkage of ubiquitin molecules to designated lysine sites on target proteins, thereby regulating a vast array of cellular signaling networks crucial for maintaining protein balance and cellular homeostasis (Pickart 2001). The process of attaching ubiquitin to substrates is a sequential, multi-phase event that includes the participation of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). ...
Elongin B (ELOB), a pivotal element in the ELOB/c-Cullin2/5-SOCS-box E3 ubiquitin-protein ligase complex, plays a significant role in catalyzing the ubiquitination and subsequent degradation of a broad spectrum of target proteins. Notably, it is documented to facilitate these processes. However, the regulatory role of ELOB in breast cancer remains ambiguous. In this study, through bio-informatic analysis of The Cancer Genome Atlas and Fudan University Shanghai Cancer Center database, we demonstrated that ELOB was over-expressed in breast cancer tissues and was related to unfavorable prognosis. Additionally, pathway enrichment analysis illustrated that high expression of ELOB was associated with multiple cancer promoting pathways, like cell cycle, DNA replication, proteasome and PI3K − Akt signaling pathway, indicating ELOB as a potential anticancer target. Then, we confirmed that both in vivo and in vitro, the proliferation of breast cancer cells could be significantly suppressed by the down-regulation of ELOB. Mechanically, immunoprecipitation and in vivo ubiquitination assays prompted that, as the core element of Cullin2-RBX1-ELOB E3 ligase (CRL2) complex, ELOB regulated the ubiquitination and the subsequent degradation of oncoprotein p14/ARF. Moreover, the anticancer efficacy of erasing ELOB could be rescued by simultaneous knockdown of p14/ARF. Finally, through analyzing breast cancer tissue microarrays and western blot of patient samples, we demonstrated that the expression of ELOB in tumor tissues was elevated in compared to adjacent normal tissues. In conclusion, ELOB is identified to be a promising innovative target for the drug development of breast cancer by promoting the ubiquitination and degradation of oncoprotein p14/ARF.
Graphical headlights
ELOB is highly expressed in breast cancer.
High ELOB levels were positively associated with poor prognosis.
ELOB promotes p14/ARF degradation as part of the Cullin2-RBX1-ELOB E3 ligase complex.
ELOB is a promising biomarker for breast cancer.
Graphical Abstract
... protein-protein interactions and protein localisation). Ubiquitin (Ub) signalling revolves around the addition of the small (8.5 kDa), highly conserved 76 amino acid long protein Ub to target substrates via an isopeptide bond between its C-terminal glycine to, predominantly, substrate lysine residues [5,6]. The covalent attachment occurs via an enzymatic cascade, with an initial ATP-dependent E1-activation step, followed by E2-conjugation, and E3-ligase mediated substrate modification. ...
Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.
... Non-canonical protein ubiquitination can induce non-proteolytic modifications impacting target protein activity or receptor recognitionfor example during endosomal sorting of plasma membrane target proteins ubiquitinated at lysine 63 (K63) residues. Intracellular signalling is predominantly associated with ubiquitin chains ( poly-Ub) linked through lysine 48 (K48), which usually initiates proteolysis of the target proteins via the 26S proteasome [85,86]. Ubiquitin tagging of substrates is preceded by an ATP dependent Ub conjugation cascade involving a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase (E3). ...
The plant macronutrient phosphorus is a scarce resource and plant-available phosphate is limiting in most soil types. Generally, a gene regulatory module called the phosphate starvation response (PSR) enables efficient phosphate acquisition by roots and translocation to other organs. Plants growing on moderate to nutrient-rich soils need to co-ordinate availability of different nutrients and repress the highly efficient PSR to adjust phosphate acquisition to the availability of other macro- and micronutrients, and in particular nitrogen. PSR repression is mediated by a small family of single SYG1/Pho81/XPR1 (SPX) domain proteins. The SPX domain binds higher order inositol pyrophosphates that signal cellular phosphorus status and modulate SPX protein interaction with PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcriptional regulator of PSR. Sequestration by SPX repressors restricts PHR1 access to PSR gene promoters. Here we focus on SPX4 that primarily acts in shoots and sequesters many transcription factors other than PHR1 in the cytosol to control processes beyond the classical PSR, such as nitrate, auxin, and jasmonic acid signalling. Unlike SPX1 and SPX2, SPX4 is subject to proteasomal degradation not only by singular E3 ligases, but also by SCF–CRL complexes. Emerging models for these different layers of control and their consequences for plant acclimation to the environment will be discussed.
... There are two subfamilies of E3 ubiquitin ligases, which are distinguished by the presence of either a RING (Really Interesting New Gene)/U box or HECT (Homologous to the E6-AP Carboxyl Terminus) domain, each with distinct domains and mechanisms of action [11]. The RING domain contains essential Cys and His conserved key residues (Cys-X 2 -Cys-X 9-39 -Cys-X 1-3 -His-X 2-3 -Cys/His-X 2 -Cys-X 4-48 -Cys-X 2 -Cys) that form an active site for the E2-ubiquitin intermediate, functioning as a zinc-binding domain. ...
These authors contributed equally to this work. Abstract: The ubiquitin/26S proteasome system is a crucial regulatory mechanism that governs various cellular processes in plants, including signal transduction, transcriptional regulation, and responses to biotic and abiotic stressors. Our study shows that the RING-H2-type E3 ubiquitin ligase, Arabidopsis Tóxicos en Levadura 2 (ATL2), is involved in response to fungal pathogen infection. Under normal growth conditions, the expression of the ATL2 gene is low, but it is rapidly and significantly induced by exogenous chitin. Additionally, ATL2 protein stability is markedly increased via chitin treatment, and its degradation is prolonged when 26S proteasomal function is inhibited. We found that an atl2 null mutant exhibited higher susceptibility to Alternaria brassicicola, while plants over-expressing ATL2 displayed increased resistance. We also observed that the hyphae of A. brassicicola were strongly stained with trypan blue staining, and the expression of A. brassicicola Cutinase A (AbCutA) was dramatically increased in atl2. In contrast, the hyphae were weakly stained, and AbCutA expression was significantly reduced in ATL2-overexpressing plants. Using bioinformatics, live-cell confocal imaging, and cell fractionation analysis, we revealed that ATL2 is localized to the plasma membrane. Further, it is demonstrated that the ATL2 protein possesses E3 ubiquitin ligase activity and found that cysteine 138 residue is critical for its function. Moreover, ATL2 is necessary to successfully defend against the A. brassicicola fungal pathogen. Altogether, our data suggest that ATL2 is a plasma membrane-integrated protein with RING-H2-type E3 ubiquitin ligase activity and is essential for the defense response against fungal pathogens in Arabidopsis.
... The E3 enzyme typically conjugates ubiquitin's C-terminal glycine to the ε-amino group of lysine residues in a substrate by the formation of an isopeptide bond ( Figures 1A, B) (Hershko and Ciechanover, 1992;Hershko et al., 1983). Strict control of ubiquitination is achieved by the coordinated action of two E1 enzymes, approximately 40 E2 enzymes and more than 600 E3 enzymes, ensuring spatiotemporal specificity (Pickart, 2001;Clague et al., 2015). ...
Ubiquitination is a dynamic post-translational modification that regulates virtually all cellular processes by modulating function, localization, interactions and turnover of thousands of substrates. Canonical ubiquitination involves the enzymatic cascade of E1, E2 and E3 enzymes that conjugate ubiquitin to lysine residues giving rise to monomeric ubiquitination and polymeric ubiquitination. Emerging research has established expansion of the ubiquitin code by non-canonical ubiquitination of N-termini and cysteine, serine and threonine residues. Generic methods for identifying ubiquitin substrates using mass spectrometry based proteomics often overlook non-canonical ubiquitinated substrates, suggesting that numerous undiscovered substrates of this modification exist. Moreover, there is a knowledge gap between in vitro studies and comprehensive understanding of the functional consequence of non-canonical ubiquitination in vivo . Here, we discuss the current knowledge about non-lysine ubiquitination, strategies to map the ubiquitinome and their applicability for studying non-canonical ubiquitination substrates and sites. Furthermore, we elucidate the available chemical biology toolbox and elaborate on missing links required to further unravel this less explored subsection of the ubiquitin system.
... The effect of USP10 on TDP-43 cytoplasmic aggregation is independent of its deubiquitinase activity, but dependent on its binding to G3BP1 USP10's deubiquitinase activity is known to be critical in its involvement in regulating different cellular processes (Pickart, 2001) (Clague et al., 2013) (Yuan et al., 2010) (Liu et al., 2011). However, USP10 is also known to have critical functions that are independent of its catalytic activity, including in the regulation of SG formation (Kedersha et al., 2016) (Yuan et al., 2010) (Liu et al., 2011). ...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by the progressive degeneration of motor neurons in the cerebral cortex and spinal cord, with a rapid progression from diagnosis to death. The great majority of ALS cases and around 50% of FTD cases present with TDP-43 pathology, leading to mislocalization and cytoplasmic aggregation of TDP-43, which can result in both its loss of nuclear functions and a gain of toxicity in the cytoplasm. TDP-43 and other RNA-binding proteins accumulate in stress granules (SGs) under stress conditions. The ubiquitin-specific protease 10 (USP10) is an inhibitor of SGs assembly that has been recently linked to neurodegeneration. Here, we identified a new functional interaction between TDP-43 and USP10, in which USP10 can control multiple aspects of TDP-43 biology that are thought to play important roles in its involvement in disease pathogenesis, such as its cytoplasmic and nuclear aggregation, expression and splicing functionality. In turn, TDP-43 is also able to control diverse aspects of USP10 biology, such as its expression levels, aggregation and function. Critically, we found USP10 dysregulation in ALS and FTD patients, overall suggesting a possible role for USP10 in ALS/FTD pathogenesis.
... Ubiquitin is a conserved, 76-amino-acid protein often found covalently conjugated to proteins to regulate their activity [114]. While AR ubiquitination can result in advancement to this classical pathway, recent investigations have focused on the role of AR non-classical ubiquitination in promoting CRPC. ...
Androgen receptor (AR) transcriptional activity significantly influences prostate cancer (PCa) progression. In addition to ligand stimulation, AR transcriptional activity is also influenced by a variety of post-translational modifications (PTMs). A number of oncogenes and tumor suppressors have been observed leveraging PTMs to influence AR activity. Subjectively targeting these post-translational modifiers based on their impact on PCa cell proliferation is a rapidly developing area of research. This review elucidates the modifiers, contextualizes the effects of these PTMs on AR activity, and connects these cellular interactions to the progression of PCa.
... Ubiquitylation is an essential cellular mechanism that is frequently disrupted in ND/ ID (Ebstein et al, 2021). The mechanism involves the covalent transfer of the 76-aa protein ubiquitin to primarily lysine residues in protein substrates in a three-step enzymatic cascade (Hershko et al, 2000;Petroski, 2008), which requires ATP consumption and involves the activity of E1 activating, E2 conjugating, and E3 ligase enzymes (Pickart, 2001). Among the ubiquitylation genes associated with ND/ID, E3 ligases are the largest subgroup of which the 1 prototype UBE3A is associated with Angelman disease (105830; MIM) (Kishino et al, 1997;Matsuura et al, 1997). ...
Neurodevelopmental disorders with intellectual disability (ND/ID) are a heterogeneous group of diseases driving lifelong deficits in cognition and behavior with no definitive cure. X-linked intellectual disability disorder 105 (XLID105, #300984; OMIM) is a ND/ID driven by hemizygous variants in the USP27X gene encoding a protein deubiquitylase with a role in cell proliferation and neural development. Currently, only four genetically diagnosed individuals from two unrelated families have been described with limited clinical data. Furthermore, the mechanisms underlying the disorder are unknown. Here, we report 10 new XLID105 individuals from nine families and determine the impact of gene variants on USP27X protein function. Using a combination of clinical genetics, bioinformatics, biochemical, and cell biology approaches, we determined that XLID105 variants alter USP27X protein biology via distinct mechanisms including changes in developmentally relevant protein–protein interactions and deubiquitylating activity. Our data better define the phenotypic spectrum of XLID105 and suggest that XLID105 is driven by USP27X functional disruption. Understanding the pathogenic mechanisms of XLID105 variants will provide molecular insight into USP27X biology and may create the potential for therapy development.
... The UPS-dependent pathway serves as the core cellular system for degrading over 80% of intracellular misfolded or damaged proteins via a two-stage process [6]. In the first stage [7], a high-energy thiol-ester bond forms between a ubiquitin-activating enzyme (E1) and ubiquitin (Ub) to activate Ub. Activated Ub is then transferred to the ubiquitinconjugating enzyme (E2), which, under the guidance of ubiquitin ligase (E3), transfers Ub to the target protein. ...
Proteolysis-targeting chimera (PROTAC) technology is a groundbreaking therapeutic approach with significant clinical potential for degrading disease-inducing proteins within targeted cells. However, challenges related to insufficient target selectivity raise concerns about PROTAC toxicity toward normal cells. To address this issue, researchers are modifying PROTACs using various approaches to enhance their target specificity. This review highlights innovative optically controlled PROTACs as anti-cancer therapies currently used in clinical practice and explores the challenges associated with their efficacy and safety. The development of optically controlled PROTACs holds the potential to significantly expand the clinical applicability of PROTAC-based technology within the realm of drug discovery.
... Ubiquitination is a posttranslational modification which tags ubiquitin via its C terminus to a target protein directing it either for degradation or becoming scaffolds, and consequently regulates signalling pathways. Ubiquitin chains is formed by constitutive attachment of ubiquitin to either of seven different lysine (K) residues (K6, K11, K27, K29, K33, K48, K63) or the N-terminal methionine (M1, Met1, linear chains) 6 . Remodelling of ubiquitin (Ub) loads of proteins is performed by specific ubiquitin ligases or deubiquitinases (DUBs). ...
TNF is a potent cytokine known for its involvement in physiology and pathology. In Rheumatoid Arthritis (RA), persistent TNF signals cause aberrant activation of synovial fibroblasts (SFs), the resident cells crucially involved in the inflammatory and destructive responses of the affected synovial membrane. However, the molecular switches that control the pathogenic activation of SFs remain poorly defined. Cyld is a major component of deubiquitination (DUB) machinery regulating the signaling responses towards survival/inflammation and programmed necrosis that induced by cytokines, growth factors and microbial products. Here we follow functional genetic approaches to understand how Cyld affects arthritogenic TNF signaling in SFs. We demonstrate that in spontaneous and induced RA models, SF-Cyld DUB deficiency deteriorates arthritic phenotypes due to increased levels of chemokines, adhesion receptors and bone-degrading enzymes generated by mutant SFs. Mechanistically, Cyld serves to restrict the TNF-induced hyperactivation of SFs by limiting Tak1-mediated signaling, and, therefore, leading to supervised NFkB and JNK activity. However, Cyld is not critically involved in the regulation of TNF-induced death of SFs. Our results identify SF-Cyld as a regulator of TNF-mediated arthritis and inform the signaling landscape underpinning the SF responses.
... Ubiquitination is an important post-translational modification process and is catalysed by several enzymes including ubiquitin activating enzymes (UBE1), ubiquitin binding enzymes (UBE2) and ubiquitin ligating enzymes (UBE3) (Ma et al. 2023;Pickart 2001). Among these enzymes, UBE2 proteins are the central part of the reaction process, interacting with UBE1 and UBE3 enzymes to result in transferring activated ubiquitin to the substrates. ...
Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications. Despite the challenge, effort has been made to develop UBE2T inhibitors. We obtained UBE2T constructs with and without the C-terminal region which is flexible in solution. Herein, we report the backbone resonance assignments for human UBE2T without the C-terminal region. The backbone dynamics of UBE2T was also explored. The available assignments will be helpful for hit identification, determining ligand binding site and understanding the mechanism of action of UBE2T inhibitors.
... The ubiquitin-proteasome system (UPS) plays an essential role in post-translational protein modification and serves many cellular functions through the selective degradation of regulatory proteins. The UPS also plays critical roles in protein trafficking, cell cycle progression, DNA damage repair, and chromatin remodeling [3]. Ubiquitination involves adding ubiquitin molecules to target proteins through ubiquitin ligase enzymes. ...
Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.
... The transthiolation reaction then takes place, allowing E2 to take up the activated Ub. The E2 cooperates with E3 to position the target protein and attaches the ubiquitin moiety to its εamino group in a lysine residue (Pickart, 2001). Deubiquitinating enzymes (DUBs), a vital group of enzymes, are then responsible for determining the fate of ubiquitinated target proteins because, in the absence of DUB to remove the (poly)ubiquitin chain, the 26S proteasome will recognise and degrade the target protein (Komander & Rape, 2012) (Figure 1). ...
Hepatocellular carcinoma (HCC) presents a significant global health challenge due to its high incidence, poor prognosis, and limited treatment options. As a pivotal regulator of protein stability, E3 ubiquitin ligase plays a crucial role in tumorigenesis and development. This review provides an overview of the latest research on the involvement of E3 ubiquitin ligase in hepatocellular carcinoma and elucidates its significance in hepatocellular carcinoma cell proliferation, invasion, and evasion from immune surveillance. Special attention is given to the functions of RING, HECT, and RBR E3 ubiquitin ligases and their association with hepatocellular carcinoma progression. By dissecting the molecular mechanisms and regulatory networks governed by E3 ubiquitin ligase, several potential therapeutic strategies are proposed: including the development of specific inhibitors targeting E3 ligases; augmentation of their tumor suppressor activity through drug or gene therapy; utilization of E3 ubiquitin ligase to modulate immune checkpoint proteins for improved efficacy of immunotherapy; combination strategies integrating traditional therapies with E3 ubiquitin ligase inhibitors; as well as biomarker development based on E3 ubiquitin ligase activity. Furthermore, this review discusses the prospect of overcoming drug resistance in hepatocellular carcinoma treatment through these novel approaches. Overall, this review establishes a theoretical foundation and offers fresh insights into harnessing the potential of E3 ubiquitin ligase for treating hepatocellular carcinoma while highlighting future research directions that pave the way for clinical translation studies and new drug discoveries.
Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by “wet lab” experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.
Preeclampsia (PE) is considered as a disease of placental origin. However, the specific mechanism of placental abnormalities remains elusive. This study identified thrombospondin‐1 (THBS1) is downregulated in preeclamptic placentae and negatively correlated with blood pressure. Functional studies show that THBS1 knockdown inhibits proliferation, migration, and invasion and increases the cycle arrest and apoptosis rate of HTR8/SVneo cells. Importantly, THBS1 silencing induces necroptosis in HTR8/SVneo cells, accompanied by the release of damage‐associated molecular patterns (DAMPs). Necroptosis inhibitors necrostatin‐1 and GSK′872 restore the trophoblast survival while pan‐caspase inhibitor Z‐VAD‐FMK has no effect. Mechanistically, the results show that THBS1 interacts with transforming growth factor B‐activated kinase 1 (TAK1), which is a central modulator of necroptosis quiescence and affects its stability. Moreover, THBS1 silencing up‐regulates the expression of neuronal precursor cell‐expressed developmentally down‐regulated 4 (NEDD4), which acts as an E3 ligase of TAK1 and catalyzes K48‐linked ubiquitination of TAK1 in HTR8/SVneo cells. Besides, THBS1 attenuates PE phenotypes and improves the placental necroptosis in vivo. Taken together, the down‐regulation of THBS1 destabilizes TAK1 by activating NEDD4‐mediated, K48‐linked TAK1 ubiquitination and promotes necroptosis and DAMPs release in trophoblast cells, thus participating in the pathogenesis of PE.
Background
Desensitization of G protein–coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of β-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D 2 -like receptors (D 2 R, D 3 R, D 4 R), have suggested the existence of other desensitization mechanisms. The present study employed D 2 R/D 3 R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT.
Results
AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin β1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of β-Arr2. The deubiquitinated β-Arr2 then forms a complex with Gβγ, which serves as a biomarker for GPCRs desensitization. Like in D 3 R, ubiquitination of AKT is also involved in the desensitization of β 2 adrenoceptors.
Conclusion
Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.
Pancreatic development is orchestrated by timely synthesis and degradation of stage‐specific transcription factors (TFs). The transition from one stage to another stage is dependent on the precise expression of the developmentally relevant TFs. Persistent expression of particular TF would impede the exit from the progenitor stage to the matured cell type. Intracellular protein degradation‐mediated protein turnover contributes to a major extent to the turnover of these TFs and thereby dictates the development of different tissues. Since even subtle changes in the crucial cellular pathways would dramatically impact pancreatic β‐cell performance, it is generally acknowledged that the biological activity of these pathways is tightly regulated by protein synthesis and degradation process. Intracellular protein degradation is executed majorly by the ubiquitin proteasome system (UPS) and Lysosomal degradation pathway. As more than 90% of the TFs are targeted to proteasomal degradation, this review aims to examine the crucial role of UPS in normal pancreatic β‐cell development and how dysfunction of these pathways manifests in metabolic syndromes such as diabetes. Such understanding would facilitate designing a faithful approach to obtain a therapeutic quality of β‐cells from stem cells.
Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin‐specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus‐mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27‐linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63‐linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1‐mediated degradation of USP19 reversed USP19‐mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.
MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.
Ubiquitination is a cascade reaction involving E1, E2, and E3 enzymes. The orthogonal ubiquitin transfer (OUT) method has been previously established to identify potential substrates of E3 ligases. In this study, we verified the ubiquitination of five substrates mediated by the E3 ligases CHIP and E4B. To further explore the activity of U‐box domains of E3 ligases, two mutants with the U‐box domains interchanged between CHIP and E4B were generated. They exhibited a significantly reduced ubiquitination ability. Additionally, different E3s recruited similar E2 ubiquitin‐conjugating enzymes when ubiquitinating the same substrates, highlighting that U‐box domains determined the E2 recruitment, while the substrate determined the E2 selectivity. This study reveals the influence of substrates and U‐box domains on E2 recruitment, providing a novel perspective on the function of U‐box domains of E3 ligases.
Bacterial wilt caused by Ralstonia solanacearum is a destructive plant disease, particularly in potato ( Solanum tuberosum ). R . solanacearum deploys a diverse and potent arsenal of type III effectors to inhibit the plant immune system. However, the understanding of individual effectors promoting susceptibility in host plants and interfering with plant immunity responses is still limited. Here, we demonstrated that the type III effector RipV1 functioned as a novel E3 ubiquitin ligase (NEL) effector and exhibited E3 ubiquitin ligase activity in vitro. Transient expression of RipV1 suppressed plant pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) responses in Nicotiana benthamiana , such as the expression of PTI‐related genes and the reactive oxygen species (ROS) burst. Prolonged expression of RipV1 induced cell death in N . benthamiana leaves. Notably, mutating the conserved cysteine residue of RipV1 abolished its E3 ligase activity and its ability to suppress plant PTI responses. This study also revealed the indispensability of RipV1 for R . solanacearum 's full virulence in potato. Transgenic potato plants overexpressing ripV1 but not the catalytic mutant ripV1‐C444A displayed enhanced susceptibility to R . solanacearum . RipV1 was observed to localize specifically to the plant plasma membrane, with its N‐terminus being pivotal in determining this localization. These findings showcase that RipV1 acts as a NEL effector and contributes to R . solanacearum virulence by suppressing plant PTI responses through its E3 activity.
Cancer is a leading cause of death worldwide. Targeted therapies aimed at key oncogenic driver mutations in combination with chemotherapy and radiotherapy as well as immunotherapy have benefited cancer patients considerably. Tumor protein p53 (TP53), a crucial tumor suppressor gene encoding p53, regulates numerous downstream genes and cellular phenotypes in response to various stressors. The affected genes are involved in diverse processes, including cell cycle arrest, DNA repair, cellular senescence, metabolic homeostasis, apoptosis, and autophagy. However, accumulating recent studies have continued to reveal novel and unexpected functions of p53 in governing the fate of tumors, for example, functions in ferroptosis, immunity, the tumor microenvironment and microbiome metabolism. Among the possibilities, the evolutionary plasticity of p53 is the most controversial, partially due to the dizzying array of biological functions that have been attributed to different regulatory mechanisms of p53 signaling. Nearly 40 years after its discovery, this key tumor suppressor remains somewhat enigmatic. The intricate and diverse functions of p53 in regulating cell fate during cancer treatment are only the tip of the iceberg with respect to its equally complicated structural biology, which has been painstakingly revealed. Additionally, TP53 mutation is one of the most significant genetic alterations in cancer, contributing to rapid cancer cell growth and tumor progression. Here, we summarized recent advances that implicate altered p53 in modulating the response to various cancer therapies, including chemotherapy, radiotherapy, and immunotherapy. Furthermore, we also discussed potential strategies for targeting p53 as a therapeutic option for cancer.
Background
The U-box gene family encodes E3 ubiquitin ligases involved in plant hormone signaling pathways and abiotic stress responses. However, there has yet to be a comprehensive analysis of the U-box gene family in maize (Zea mays L.) and its responses to abiotic stress.
Results
In this study, 85 U-box family proteins were identified in maize and were classified into four subfamilies based on phylogenetic analysis. In addition to the conserved U-box domain, we identified additional functional domains, including Pkinase, ARM, KAP and Tyr domains, by analyzing the conserved motifs and gene structures. Chromosomal localization and collinearity analysis revealed that gene duplications may have contributed to the expansion and evolution of the U-box gene family. GO annotation and KEGG pathway enrichment analysis identified a total of 105 GO terms and 21 KEGG pathways that were notably enriched, including ubiquitin-protein transferase activity, ubiquitin conjugating enzyme activity and ubiquitin-mediated proteolysis pathway. Tissue expression analysis showed that some ZmPUB genes were specifically expressed in certain tissues and that this could be due to their functions. In addition, RNA-seq data for maize seedlings under salt stress revealed 16 stress-inducible plant U-box genes, of which 10 genes were upregulated and 6 genes were downregulated. The qRT-PCR results for genes responding to abiotic stress were consistent with the transcriptome analysis. Among them, ZmPUB13, ZmPUB18, ZmPUB19 and ZmPUB68 were upregulated under all three abiotic stress conditions. Subcellular localization analysis showed that ZmPUB19 and ZmPUB59 were located in the nucleus.
Conclusions
Overall, our study provides a comprehensive analysis of the U-box gene family in maize and its responses to abiotic stress, suggesting that U-box genes play an important role in the stress response and providing insights into the regulatory mechanisms underlying the response to abiotic stress in maize.
Human trophoblast cells are crucial for healthy pregnancy. However, whether the defective homologous recombination (HR) repair of dsDNA break (DSB) in trophoblast cells may induce miscarriage is completely unknown. Moreover, the abundance of BRCA1 (a crucial protein for HR repair), its recruitment to DSB foci, and its epigenetic regulatory mechanisms, are also fully unexplored. In this work, it is identified that a novel lnc‐HZ10, which is highly experssed in villous tissues of recurrent miscarriage (RM) vs their healthy control group, suppresses HR repair of DSB in trophoblast cell. Lnc‐HZ10 and AhR (aryl hydrocarbon receptor) form a positive feedback loop. AhR acts as a transcription factor to promote lnc‐HZ10 transcription. Meanwhile, lnc‐HZ10 also increases AhR levels by suppressing its CUL4B‐mediated ubiquitination degradation. Subsequently, AhR suppresses BRCA1 transcription; and lnc‐HZ10 (mainly 1‐447 nt) interacts with γ‐H2AX; and thus, impairs its interactions with BRCA1. BPDE exposure may trigger this loop to suppress HR repair in trophoblast cells, possibly inducing miscarriage. Knockdown of murine Ahr efficiently recovers HR repair in placental tissues and alleviates miscarriage in a mouse miscarriage model. Therefore, it is suggested that AhR/lnc‐HZ10/BRCA1 axis may be a promising target for alleviation of unexplained miscarriage.
Ubiquitination is a reversible post-translational modification based on the chemical addition of ubiquitin to proteins with regulatory effects on various signaling pathways. Ubiquitination can alter the molecular functions of tagged substrates with respect to protein turnover, biological activity, subcellular localization or protein–protein interaction. As a result, a wide variety of cellular processes are under ubiquitination-mediated control, contributing to the maintenance of cellular homeostasis. It follows that the dysregulation of ubiquitination reactions plays a relevant role in the pathogenic states of human diseases such as neurodegenerative diseases, immune-related pathologies and cancer. In recent decades, the enzymes of the ubiquitin–proteasome system (UPS), including E3 ubiquitin ligases and deubiquitinases (DUBs), have attracted attention as novel druggable targets for the development of new anticancer therapeutic approaches. This perspective article summarizes the peculiarities shared by the enzymes involved in the ubiquitination reaction which, when deregulated, can lead to tumorigenesis. Accordingly, an overview of the main pharmacological interventions based on targeting the UPS that are in clinical use or still in clinical trials is provided, also highlighting the limitations of the therapeutic efficacy of these approaches. Therefore, various attempts to circumvent drug resistance and side effects as well as UPS-related emerging technologies in anticancer therapeutics are discussed.
Misfolded and aggregated proteins build up in neurodegenerative illnesses, which causes neuronal dysfunction and ultimately neuronal death. In the last few years, there has been a significant upsurge in the level of interest towards the function of molecular chaperones in the control of misfolding and aggregation. The crucial molecular chaperones implicated in neurodegenerative illnesses are covered in this review article, along with a variety of their different methods of action. By aiding in protein folding, avoiding misfolding, and enabling protein breakdown, molecular chaperones serve critical roles in preserving protein homeostasis. By aiding in protein folding, avoiding misfolding, and enabling protein breakdown, molecular chaperones have integral roles in preserving regulation of protein balance. It has been demonstrated that aging, a significant risk factor for neurological disorders, affects how molecular chaperones function. The aggregation of misfolded proteins and the development of neurodegeneration may be facilitated by the aging-related reduction in chaperone activity. Molecular chaperones have also been linked to the pathophysiology of several instances of neuron withering illnesses, enumerating as Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease. Molecular chaperones have become potential therapy targets concerning with the prevention and therapeutic approach for brain disorders due to their crucial function in protein homeostasis and their connection to neurodegenerative illnesses. Protein homeostasis can be restored, and illness progression can be slowed down by methods that increase chaperone function or modify their expression. This review emphasizes the importance of molecular chaperones in the context of neuron withering disorders and their potential as therapeutic targets for brain disorders.
Graphical abstract
Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organizing protein Dbn1 as a major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCFβ-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized β-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA damage-dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX’s ability to identify targets of stimulus-regulated ubiquitylation and reveal an SCFβ-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.
The term epigenetics was coined by Waddington in 1942 and was used to describe the “interactions of genes with their environment that brings the phenotype into being” (Waddington 2012). In general, genetic factors responsible for change in the appearance of an organism or biological functions without changing the actual DNA sequence is referred as epigenetics (Gavery and Roberts 2017). Therefore, only the expression of genes changes but not the genes. At molecular level, epigenetics refers to the addition or deletion of a methyl group to a DNA base, turning the gene on or off, or to packaging of the chromatin structure by silencing or opening regions of the genome by winding or unwinding the DNA around histones. Environmental factors are known to cause the epigenetic changes, and these changes are more common than mutations. It is also known that epigenetic changes caused by environmental exposures can be transmitted down several generations. The widely studied molecular epigenetic mechanisms are as follows:
G protein–coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to βarrestins. In addition, βarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an in vitro reconstituted system and show that βarrestin1 (βarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that βarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the βarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.
Stimulator of interferon (IFN) genes (STING, also named MITA, ERIS, MPYS, or TMEM173) plays an essential role in DNA virus‐ or cytosolic DNA‐triggered innate immune responses. Here, we demonstrate that the RING‐in‐between RING (RBR) E3 ubiquitin ligase family member RING‐finger protein (RNF) 144A interacts with STING and promotes its K6‐linked ubiquitination at K236, thereby enhancing STING translocation from the ER to the Golgi and downstream signaling pathways. The K236R mutant of STING displays reduced activity in promoting innate immune signal transduction. Overexpression of RNF144A upregulates HSV‐1‐ or cytosolic DNA‐induced immune responses, while knockdown of RNF144A expression has the opposite effect. In addition, Rnf144a ‐deficient cells exhibit impaired DNA virus‐ or cytosolic DNA‐triggered signaling, and RNF144A protects mice from DNA virus infection. In contrast, RNF144A does not affect RNA virus‐ or cytosolic RNA‐triggered innate immune responses. Taken together, our findings identify a new positive regulator of DNA virus‐ or cytosolic DNA‐triggered signaling pathways and a critical ubiquitination site important for fully functional STING during antiviral responses.
Atherosclerosis (AS) is a serious cardiovascular disease. One of its hallmarks is hyperlipidemia. Inhibiting the formation of macrophage foam cells is critical for alleviating AS. Transcription factor EB (TFEB) can limit the formation of macrophage foam cells by upregulating lysosomal activity. We examined whether TFEB SUMOylation is involved in this progress during AS. In this study, we investigated the role of TFEB SUMOylation in macrophages in AS using TFEB SUMOylation deficiency Ldlr−/− (TFEB-KR: Ldlr−/−) transgenic mice and TFEB-KR bone marrow-derived macrophages. We observed that TFEB-KR: Ldlr−/− atherosclerotic mice had thinner plaques and macrophages with higher lysosomal activity when compared to WT: Ldlr−/− mice. TFEB SUMOylation in macrophages decreased after oxidized low-density lipoprotein (OxLDL) treatment in vitro. Compared with wild type macrophages, TFEB-KR macrophages exhibited less lipid deposition after OxLDL treatment. Our study demonstrated that in AS, deSUMOylation of TFEB could inhibit the formation of macrophage foam cells through enhancing lysosomal biogenesis and autophagy, further reducing the accumulation of lipids in macrophages, and ultimately alleviating the development of AS. Thus, TFEB SUMOylation can be a switch to modulate macrophage foam cells formation and used as a potential target for AS therapy.
Tripartite motif‐containing protein 21 (TRIM21), identified as both a cytosolic E3 ubiquitin ligase and FcR (Fragment crystallizable receptor), primarily interacts with proteins via its PRY/SPRY domains and promotes their proteasomal degradation to regulate intracellular immunity. But how TRIM21 involves in intracellular immunity still lacks systematical understanding. Herein, it is probed into the TRIM21‐related literature and raises an interacting model about how TRIM21 orchestrates proteins in cytosol. In this novel model, TRIM21 generally interacts with miscellaneous protein in intracellular immunity in two ways: For one, TRIM21 solely plays as an E3, ubiquitylating a glut of proteins that contain specific interferon‐regulatory factor, nuclear transcription factor kappaB, virus sensors and others, and involving inflammatory responses. For another, TRIM21 serves as both E3 and specific FcR that detects antibody‐complexes and facilitates antibody destroying target proteins. Correspondingly delineated as Fc‐independent signaling and Fc‐dependent signaling in this review, how TRIM21's interactions contribute to intracellular immunity, expecting to provide a systematical understanding of this important protein and invest enlightenment for further research on the pathogenesis of related diseases and its prospective application is elaborated.
Melanogenesis is a critical biochemical process in which melanocytes produce melanin, a crucial element involved in the formation of coat colour in mammals. According to several earlier studies, melanocytes' post‐translational modifications of proteins primarily control melanogenesis. Among the many post‐translational changes that can affect melanin production, ubiquitination and deubiquitination can keep melanin production going by changing how proteins that are related to melanin are broken down or kept stable. Ubiquitination and deubiquitination maintain ubiquitin homeostasis, which is a highly dynamic process in balance under the action of E3 ubiquitin ligase and deubiquitinating enzymes. However, the regulatory mechanisms underlying ubiquitination and deubiquitination in melanogenesis are yet to be thoroughly investigated. As a result, there has been a growing focus on exploring the potential correlation between melanogenesis, ubiquitination and deubiquitination. This study discusses the mechanisms of ubiquitination and deubiquitination in the context of melanogenesis, a crucial process for enhancing mammalian coat coloration and addressing pigment‐related diseases.
Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.
SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD ⁺ ‐dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl 2 ‐induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4‐eGFP decrease upon CoCl 2 treatment of HEK293 cells. Co‐treatment with BafA1, an inhibitor of autophagosome–lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl 2 ‐induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl 2 treatment and were more resistant to pseudohypoxia‐induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild‐type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress‐induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.
Ubiquitin, a conserved protein in eukaryotic cells, exists as a monomer or polyubiquitin chains known as isopeptide-linked polymers. These chains are attached to a substrate or other ubiquitin molecules through a covalent bond between the α-amino group of lysine in ubiquitin and glycine in the C-terminal of the subsequent ubiquitin unit. The choice of the specific lysine residue in ubiquitin for forming ubiquitin–ubiquitin chains determines its biochemical and biological function. A detailed chemical structure–function evaluation of the respective polyubiquitin chain is required. Interestingly, specific lysine linkage polyubiquitin chains become covalently bonded to many pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so the interaction between polyubiquitin chains and ubiquitin antibodies is very useful. For example, the neurofibrillary tangles of Alzheimer's disease and the Lewy bodies seen in Parkinson's disease are heavily ubiquitinated and can be readily visualized using specific ubiquitin antibodies. This study utilized synthetic ubiquitin building block peptides that contained various lysine residues (K6, K11, K33, K48, and K63) linked to a Gly–Gly dipeptide, with the aim of exploring the recognition specificity of the Lys63-polyubiquitin antibody. The interaction studies between different ubiquitin building blocks and the specific Lys63-ubiquitin (K63-Ub) antibody were performed by affinity-mass spectrometry (Affinity-MS) and immunoblotting which enables direct protein identification from biological material with unprecedented selectivity. Affinity-MS and dot blot data proved the specific binding of the K63-Ub antibody to the ubiquitin peptides containing Lys6 or Lys63 residues. In epitope excision for mass spectrometric epitope identification, the ubiquitin building block with Lys63 residue bound to the immobilized K63-Ub antibody was proteolytically cleaved using pronase. The resulting epitope and non-epitope fractions were subjected to matrix-assisted laser desorption/ionization-time of flight analysis, revealing that the epitope is located within the sequence ubiquitin(60–66). Epitope extraction-MS consistently confirmed these findings.
E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases play important roles in the growth and development of plants and animals. To date, the systematic analysis of E2 and E3 genes in Rhodophyta is limited. In this study, 14 E2 genes and 51 E3 genes were identified in Gracilariopsis lemaneiformis, an economically important red alga. E2 genes were classified into four classes according to the structure of the conserved domain, UBC. E3 genes were classified into 12 subfamilies according to individual conserved domains. A phylogenetic tree of seven algae species showed that functional differentiation of RING-type E3s was the highest, and the similarity between orthologous genes was high except in Chlamydomonas reinhardtii and Chara braunii. RNA-seq data analysis showed significant differential expression levels of E2 and E3 genes under the life stages of tetraspore formation and release, especially GlUBCN and GlAPC3. According to GO and KEGG analysis of two transcriptomes, GlUBCN and GlAPC3 were involved in ubiquitin-mediated proteolysis, and other subunits of the anaphase promoting complex or cyclosome (APC/C) and its activators GlCDC20 and GlCDH1 were also enriched into this process. The CDH1 and CDC20 in 981 were down-regulated during tetraspores formation and release, with the down-regulation of CDH1 being particularly significant; CDH1 and CDC20 in WLP-1, ZC, and WT were up-regulated during tetraspores formation and release, with CDC20 being more significantly up-regulated. Therefore, GlCDH1, rather than GlCDC20, in ‘981’ might play the leading role in the activation of the APC/C, and GlCDC20 might play the leading role rather than GlCDH1 in strains WLP-1, ZC and wild type. The low fertility of cultivar 981 might be highly correlated with the inactivity of activators CDH1 and CDC20. This study provided a basic and comprehensive understanding of characteristic of E2 and E3 genes in Gp. lemaneiformis and set a foundation for further understanding of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligase in regulating tetrasporophytes development of Gp. lemaneiformis.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12864-023-09639-0.
Simple Summary
As we age or develop certain brain diseases, our body’s ability to maintain a balanced protein system, termed “proteostasis”, is disturbed. Proteostasis is essential for keeping proteins at the correct levels and functioning properly, as well as avoiding a buildup of proteins that form aggregates involved in neurodegenerative diseases, like Alzheimer’s disease and Parkinson’s disease. One of the major pathways used by our cells to eliminate such proteins is the “ubiquitin proteasome system” (UPS). In this review, we discuss how problems with the UPS are linked to aging and neurodegenerative diseases. We also explore the idea that increasing the activity of NFE2L1, a protein that increases expression of UPS components, could be a strategy to enhance proteasome function and reduce the impact of neurodegenerative diseases.
Abstract
A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis (“proteostasis”) that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced “bounce-back effect” regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.
Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ∼35–40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+, and the interaction is direct and optimal at 1 μM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.
The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases, Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-alpha (PDGFR alpha) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRa signaling machinery strongly suggested that Cbl negatively regulates PDGFR alpha signaling. Here, we show that, similar to PDGFR alpha, selective stimulation of PDGFR beta induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFR beta, as observed with PDGFR alpha. We show that Cbl-dependent negative regulation of PDGFR alpha and beta results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.
Regulation of NF-κB occurs through phosphorylation-dependent ubiquitination of IκBα, which is degraded by the 26S proteasome.
Recent studies have shown that ubiquitination of IκBα is carried out by a ubiquitin-ligase enzyme complex called SCFβTrCP. Here we show that Nedd8 modification of the Cul-1 component of SCFβTrCP is important for function of SCFβTrCP in ubiquitination of IκBα. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCFβTrCP, phosphorylated IκBα and β-catenin, indicating that Nedd8–Cul-1 conjugates are part of SCFβTrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically
expressed βTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IκBα required Nedd8
and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCFβTrCPcontaining a K720R mutant of Cul-1 only weakly supported IκBα ubiquitination compared to SCFβTrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCFβTrCP. These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification
and show how they operate together to selectively target the signal-dependent degradation of IκBα.
Ubiquitin Ligases are generally assumed to play a major role in substrate recognition and thus provide specificity to a particular ubiquitin modification system. The multicopy maintenance protein (Mcm) 7 subunit of the replication licensing factor-M was identified as a substrate of the ES-ubiquitin ligase/E6-AP by its interaction with human papillomavirus-18E6. Mcm7 is ubiquitinated in vivo in both an E6-AP-dependent and -independent manner. EG-AP functions in these reactions independently of the viral oncogene E6, We show that recognition of Mcm7 by EG-AP is mediated by a homotypic interaction motif present in both proteins, called the L2G box. These findings served as the basis for the definition of substrate specificity for EG-AP. A small cluster of proteins whose function is intimately associated with the control of cell growth and/or proliferation contains the L2G box and is thereby implicated in an EG-AP and, by default, HPV-EG-dependent ubiquitination pathway.
The inhibitor protein IkappaBalpha controls the nuclear import of the transcription factor NF-kappaB. The inhibitory activity of IkappaBalpha is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets IkappaBalpha to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of IkappaBalpha. Conservative Lys longrightarrow Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of IkappaBalpha in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-kappaB in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys longrightarrow Arg mutations prevent signal-dependent degradation of IkappaBalpha in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated IkappaBalpha at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-kappaB.
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the
ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal
residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the
cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that
contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the
UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions
of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain.
Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos,
the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.
The cerebral cortex of Alzheimer's and Down syndrome patients is characterized by the presence of protein deposits in neurofibrillary
tangles, neuritic plaques, and neuropil threads. These structures were shown to contain forms of β amyloid precursor protein
and ubiquitin-B that are aberrant (+1 proteins) in the carboxyl terminus. The +1 proteins were not found in young control
patients, whereas the presence of ubiquitin-B+1 in elderly control patients may indicate early stages of neurodegeneration. The two species of +1 proteins displayed cellular
colocalization, suggesting a common origin, operating at the transcriptional level or by posttranscriptional editing of RNA.
This type of transcript mutation is likely an important factor in the widely occurring nonfamilial early- and late-onset forms
of Alzheimer's disease.
Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted
in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53–F-box protein)
complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates
Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel–Lindau
tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.
Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s)
and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s.
To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were
identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the
presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between
the two proteins. In vitrobinding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in
the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that
both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it
is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3
activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome
pathway.
Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein-glycine-conserved (CAP-GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).
The recently characterized ubiquitin activating enzyme catalyzes the first step in ubiquitin-protein isopeptide bond formation and as uch may be central to a
The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.
The MDM2 oncoprotein has transforming potential that can be activated by overexpression, and it represents a critical regulator of the p53 tumor suppressor protein. To identify other factors with a potential role in influencing the expression and/or function of MDM2, we utilized a yeast two-hybrid screening protocol. Here we report that MDM2 physically interacts with a structurally related protein termed MDMX. The results obtained in these studies provide evidence that C-terminal RING finger domains, contained within both of these proteins, play an important role in mediating the association between MDM2 and MDMX. The interaction of these proteins interferes with MDM2 degradation, leading to an increase in the steady-state levels of MDM2. MDMX also inhibits MDM2-mediated p53 degradation, with subsequent accumulation of p53. Taken together, these data indicate that MDMX has the potential to regulate the expression and function of the MDM2 oncoprotein.
The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of
E6-AP, an E3 ubiquitin-protein ligase. Since the C terminus of E6-AP mediates thioester complex formation with ubiquitin,
a necessary intermediate step in E6-AP-dependent ubiquitination, it was proposed that members of the hect domain family in
general have E3 activity. The hect domain is approximately 350 amino acids in length, and we show here that the hect domain
of E6-AP is necessary and sufficient for ubiquitin thioester adduct formation. Furthermore, the human genome encodes at least
20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of
E3s, several of these are shown to form thioester complexes with ubiquitin. In addition, some hect domain proteins interact
preferentially with UbcH5, whereas others interact with UbcH7, indicating that human hect domain proteins can be grouped into
at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the
presence of a large family of E3s should contribute to ensure the specificity and selectivity of ubiquitin-dependent proteolytic
pathways.
SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.
All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability.
The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta
gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of
branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48
of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same
Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta
gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein
is essential for the degradation of the protein.
To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.
The covalent ligation of the 8.6-kDa protein ubiquitin to histones within transcriptionally poised regions is believed to participate in the localized regulation of chromatin structure. This unique post-translational modification is thought to be distinct from similar cytosolic reactions in requiring ubiquitin-activating enzyme (E1) and one or more putative ubiquitin carrier proteins (E2) but not isopeptide ligase (E3). Apparently homogeneous preparations of the E2 isozymes were tested for their ability to catalyze the E3-independent conjugation of ubiquitin to linker histone H1 and core histones H2A, H2B, H3, and H4 in the presence of catalytic amounts of E1. Significant rates of nonprocessive core histone monoubiquitination were catalyzed by the E2(14kDa), E2(20kDa), and E2(32kDa) isozymes but not by either E2(17kDa) or E2(24kDa). The former three E2 isozymes also supported slow rates of direct multiple ubiquitination to secondary ligation sites on the histones. Rate studies for the monoubiquitination of H2A and H2B revealed that: 1) E2(14kDa) catalyzed a second order reaction with respect to histone concentration; 2) E2(32kDa)-mediated ligation proceeded by hyperbolic kinetics, yielding Km values of 2.8 and 12 microM for H2A and H2B, respectively; and 3) E2(20kDa) exhibited complex kinetics composed of both second order and hyperbolic pathways, the latter having Km values of 0.83 and 1.5 microM for H2A and H2B, respectively. Pulse-chase kinetics suggested that both ubiquitin thiol esters formed to E2(20kDa) were catalytically competent in H2A ligation. The active E2 isozymes also catalyzed the processive multiple ubiquitination of calf thymus H1. Other rate studies determined that Kd values for binding of the active E2 species to E1 ternary complex were 0.1 nM for E2(14kDa), 0.4 nM for E2(32kDa), and 3.6 nM for E2(20kDa). The data indicate that E2(20kDa) and E2(32kDa) are specific but mechanistically distinct ligation enzymes responsible for the conjugation of ubiquitin to nucleosomal proteins.
In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.
By affinity chromatography of a crude reticulocyte extract on ubiquitin-Sepharose, three enzymes required for the conjugation of ubiquitin with proteins have been isolated. One is the ubiquitin-activating enzyme (E1), which is covalently linked to the affinity column in the presence of ATP and can be specifically eluted with AMP and pyrophosphate (Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. Chem. 257, 2537-2542). A second enzyme, designated E2, is bound to the ubiquitin column when E1 and ATP are present, and is eluted with a thiol compound at high concentration. The third enzyme, designated E3, is adsorbed to the affinity column by noncovalent interactions and can be eluted with high salt or increased pH. The presence of all three enzymes is absolutely required for the conjugation of 125I-ubiquitin with proteins. All three affinity-purified enzymes are also required for the breakdown of 125I-albumin to acid-soluble material in the presence of ubiquitin, ATP, and the unadsorbed fraction of the affinity column. The following observations indicate that the function of E2 is the transfer of activated ubiquitin to the site of conjugation in the form of an E2-ubiquitin thiol ester intermediate. (a) E2 is rapidly inactivated by iodoacetamide, but can be protected against inactivation by a prior incubation with E1, ATP, and ubiquitin. This suggests an E1-mediated transfer of activated ubiquitin to an iodoacetamide-sensitive thiol site of E2. (b) The requirements for the binding of E2 to the ubiquitin column and the mode of its elution, cited above, are consistent with the notion that a covalent linkage is formed between E2 and Sepharose-bound ubiquitin. (c) Upon the incubation of 125I-ubiquitin with E1 and ATP, followed by the addition of purified E2, activated ubiquitin is transferred from E1 to several low molecular weight forms of E2, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linkage of ubiquitin to all these forms has the characteristics of a thiol ester bond. In a further incubation with E3 and a protein substrate for conjugation, activated ubiquitin was transferred from the different forms of E2-ubiquitin to stable ubiquitin-protein conjugates. Thus, E3 is involved in the last step of the ligase system.
The inhibitor protein I kappa B alpha controls the nuclear import of the transcription factor NF-kappa B. The inhibitory activity of I kappa B alpha is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets I kappa B alpha to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of I kappa B alpha. Conservative Lys-->Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of I kappa B alpha in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-kappa B in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys-->Arg mutations prevent signal-dependent degradation of I kappa B alpha in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated I kappa B alpha at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-kappa B.
Previous work has shown that a fusion protein bearing a “nonremovable” N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing
Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma
viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain
N-terminal residues). We also show that a Lys Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation
of Ub fusions to β-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase
are inhibited by the Ub but not by the Ub moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys, whereas ufd2 mutants are impaired in the ubiquitination at Lys. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the
previously identified Lys but also Lys of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
A global cellular reorganization occurs during the reticulocyte stage of erythroid differentiation. This reorganization is accomplished partly through programmed protein degradation. The selection of proteins for degradation can be mediated by covalent attachment of ubiquitin. We have cloned cDNAs encoding two ubiquitin-conjugating (E2) enzymes, E2-20K and E2-230K, and found their genes to be strongly induced during the differentiation of erythroblasts into reticulocytes. Induction of the E2-20K and E2-230K genes is specific, as transcript levels for at least two other ubiquitinating enzymes fall during erythroblast differentiation. In contrast to most proteins induced in reticulocytes, E2-20K and E2-230K enzymes are present at strongly reduced levels in erythrocytes and thus decline in abundance as reticulocyte maturation is completed. This result suggests that both enzymes function during the reticulocyte stage, when enhanced protein degradation has been observed. These data implicate regulated components of the ubiquitin conjugation machinery in erythroid differentiation.
The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.
Ubiquitin-mediated proteolysis proceeds via the formation and degradation of ubiquitin-protein conjugates. Ubiquitin (Ub)-activating enzyme (E1) catalyzes the first, MgATP-dependent step in the conjugative reaction sequence. With wild type ubiquitin, the product of the E1 reaction is a ternary complex (E1-Ub-AMP-Ub) containing one thiol-linked ubiquitin (via the Ub COOH terminus, Gly-76) and one tightly bound ubiquitin adenylate. The thiol-linked ubiquitin is subsequently transferred to the thiol of a ubiquitin-conjugating enzyme (E2 protein); the latter adduct is the proximal donor of ubiquitin to the target protein. A mutant ubiquitin, bearing a Gly to Ala substitution at the COOH terminus (G76A-ubiquitin), was characterized as a substrate for E1. G76A-ubiquitin 1) supported PPi-ATP exchange poorly (500-fold decrease in kcat/K(m); 2) did not produce detectable AMP-Ub with native E1; 3) produced stoichiometric AMP-Ub with thiol-blocked E1; 4) gave a stoichiometric burst of ATP consumption (1 mol/mol E1) with either native or thiol-blocked E1; 5) supported E1-ubiquitin thiol ester formation with native E1; 6) supported several downstream reactions of the proteolytic pathway at approximately 20% of the rate of wild type ubiquitin. These results indicate that G76A-ubiquitin gives a binary E1 thiol ester complex with native E1, due to the failure of the E1-ubiquitin thiol ester to undergo another round of adenylate synthesis; thus AMP-Ub is detected only if adenylate to thiol transfer is prevented by alkylating E1. The inability of G76A-ubiquitin to support ternary complex formation has implications for E1 active site structure. In other experiments, occupancy of the nucleotide/adenylate site of E1, by either MgATP or AMP-Ub, was found to stimulate ubiquitin transthiolation between E1 and E2 proteins. The intermediacy of ubiquitin adenylate thus provides a previously unrecognized catalytic advantage in the E1 mechanism.
Ubiquitin-conjugating enzymes catalyze the covalent attachment of ubiquitin to cellular substrates. Here we describe the isolation of a novel ubiquitin-conjugating enzyme from human placenta and the cloning of the corresponding cDNA. DNA sequencing revealed that this gene, UbcH2, encodes a protein with significant sequence similarity to yeast UBC8. In contrast to a previous report (Qin, S., Nakajima, B., Nomura, M., and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554), we discovered that UBC8 is interrupted by a single intron bearing an unusual branch point sequence. The revised amino acid sequence of yeast UBC8 exhibits 54% amino acid sequence identity to human UbcH2. Moreover, full-length UbcH2 and UBC8 enzymes expressed from their cDNAs show similar enzymatic activities in vitro by catalyzing the ubiquitination of histones, suggesting that the two enzymes may fulfill similar functions in vivo. Interestingly, comparison of the enzymatic activities of a truncated UBC8 (Qin, S., Nakajima, B., Nomura, M. and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554) and of the full-length enzyme (this report) suggests, that the first 12 amino-terminal residues of UBC8 are required for ubiquitination of histones in vitro but not for thiolester formation with ubiquitin. This suggests that the NH2 terminus of UBC8 may be necessary either for substrate recognition or for the transfer of ubiquitin onto substrates. The UbcH2 gene is located on chromosome 7 and shows a complex expression pattern with at least five different mRNAs.
E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).
Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized. Ubiquitinated H2B (uH2B) has been identified in the yeast Saccharomyces cerevisiae, and mutation of the conserved ubiquitination site is shown to confer defects in mitotic cell growth and meiosis. uH2B was not detected in rad6mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast.
Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.
Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer.
Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2K446R is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.
Activation of NF-κB is achieved by ubiquitination and proteasome-mediated degradation of IκBα. We have detected modified IκBα, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugated SUMO-1 to IκBα primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IκBα cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-κB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IκBα is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation.
Activation of the transcription factor NF-κB in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-κB inhibitor, IκBα. Here, we show the in vitro reconstitution of the phosphorylation-dependent ubiquitination of IκBα with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quaternary SCFHOS–ROC1 holoenzyme (with Skp1 and the β-TRCP homolog HOS). SCFHOS–ROC1 binds IKKβ-phosphorylated IκBα and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization.
During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermatozoa. During this process there occurs remodeling of cell structure and loss of most of the cytoplasm and a large fraction of cellular proteins. To evaluate the role of the ubiquitin proteolytic system in this protein loss, we measured levels of ubiquitinated proteins and rates of ubiquitin conjugation in extracts of testes from rats of different ages. Endogenous ubiquitin–protein conjugates increased till day 30 and then reached a plateau. In parallel, there was a progressive increase in the rate of conjugation of ubiquitin to proteins in testis extracts from these animals. To test the importance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extracts was carried out and the remaining conjugation activity in supernatants was assayed. Depletion of either enzyme family resulted in decreased conjugation. However, most of the conjugation activity and, more importantly, the increased conjugation during development were UBC4-dependent. Immunocytochemistry demonstrated a marked increase in expression of UBC4 in spermatids, consistent with the UBC4-dependent activation of conjugation seen in vitro. In situ hybridization studies evaluated the contribution of various UBC4 isoforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 mRNA was restricted to germ cells with high levels of expression in round and elongated spermatids. UBC4-testis had previously been shown to be expressed only in spermatids. Our data suggest that induction of various UBC4 isoforms activates overall conjugation and plays an important role in the cellular remodeling and protein loss occurring during spermatogenesis.
Protein degradation is deployed to modulate the steady-state abundance of proteins and to switch cellular regulatory circuits from one state to another by abrupt elimination of control proteins. In eukaryotes, the bulk of the protein degradation that occurs in the cytoplasm and nucleus is carried out by the 26S proteasome. In turn, most proteins are thought to be targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. Ubiquitination of proteins requires a multienzyme system. A key component of ubiquitination pathways, the ubiquitin ligase, controls both the specificity and timing of substrate ubiquitination. This review is focused on a conserved ubiquitin ligase complex known as SCF that plays a key role in marking a variety of regulatory proteins for destruction by the 26S proteasome.
Protein degradation by the ubiquitin system controls the intracellular concentrations of many regulatory proteins. A protein substrate of the ubiquitin system is conjugated to ubiquitin through the action of three enzymes, E1, E2 and E3, with the degradation signal (degron) of the substrate recognized by E3 (refs 1-3). The resulting multi-ubiquitylated substrate is degraded by the 26S proteasome. Here we describe the physiological regulation of a ubiquitin-dependent pathway through allosteric modulation of its E3 activity by small compounds. Ubr1, the E3 enzyme of the N-end rule pathway (a ubiquitin-dependent proteolytic system) in Saccharomyces cerevisiae mediates the degradation of Cup9, a transcriptional repressor of the peptide transporter Ptr2 (ref. 5). Ubr1 also targets proteins that have destabilizing amino-terminal residues. We show that the degradation of Cup9 is allosterically activated by dipeptides with destabilizing N-terminal residues. In the resulting positive feedback circuit, imported dipeptides bind to Ubr1 and accelerate the Ubr1-dependent degradation of Cup9, thereby de-repressing the expression of Ptr2 and increasing the cell's capacity to import peptides. These findings identify the physiological rationale for the targeting of Cup9 by Ubr1, and indicate that small compounds may regulate other ubiquitin-dependent pathways.
Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.
A nonhydrolyzable analogue of ubiquitin adenylate has been synthesized for use as a specific inhibitor of the ubiquitination of proteins. Ubiquitin adenylate is a tightly bound intermediate formed by the ubiquitin activating enzyme. The inhibitor adenosyl-phospho-ubiquitinol (APU) is the phosphodiester of adenosine and the C-terminal alcohol derived from ubiquitin. APU is isosteric with the normal reaction intermediate, the mixed anhydride of ubiquitin and AMP, but results from the replacement of the carbonyl oxygen of Gly76 with a methylene group. This stable analogue would be expected to bind to both ubiquitin and adenosine subsites and result in a tightly bound competitive inhibitor of ubiquitin activation. APU inhibits the ATP-PPi exchange reaction catalyzed by the purified ubiquitin activating enzyme in a manner competitive with ATP (Ki = 50 nM) and noncompetitive with ubiquitin (Ki = 35 nM). AMP has no effect on the inhibition, confirming that the inhibitor binds to the free form of the enzyme and not the thiol ester form. This inhibition constant is 10-fold lower than the dissociation constants for each substrate and 30-1000-fold lower than the respective Km values for ubiquitin and ATP. APU also effectively inhibits conjugation of ubiquitin to endogenous proteins catalyzed by reticulocyte fraction II with an apparent Ki of 0.75 microM. This weaker inhibition is consistent with the fact that activation of ubiquitin is not rate limiting in the conjugation reactions catalyzed by fraction II. APU is similarly effective as an inhibitor of the ubiquitin-dependent proteolysis of beta-lactoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)
The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.
The temperature-sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human-ts A1S9 somatic cell hybrids. We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus. The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons. Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast. Northern blot analysis of serum-starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle. Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.
We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85 , in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, short-lived proteins is also specifically temperature sensitive in ts85 . We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.
Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex.
E6-AP is a 100-kDa cellular protein that interacts with the E6 protein
of the cancer-associated human papillomavirus types 16 and 18. The
E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein
for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein
ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme
in the form of a thioester and then directly transfers the ubiquitin to
targeted substrates. The amino acid sequence of E6-AP shows similarity
to a number of protein sequences over an ≈350-aa region corresponding
to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of
particular note is a conserved cysteine residue within the last 32-34
aa, which in E6-AP is likely to be the site of ubiquitin thioester
formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and
a yeast 95-kDa protein (RSP5), both of previously unknown function, are
shown here to form thioesters with ubiquitin. Mutation of the conserved
cysteine residue of these proteins destroys their ability to accept
ubiquitin. These data strongly suggest that the rat 100-kDa protein and
RSP5, as well as the other E6-AP-related proteins, belong to a class of
functionally related E3 ubiquitin-protein ligases, defined by a domain
homologous to the E6-AP carboxyl terminus (hect domain).
Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.
The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.
The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for postreplication repair of UV-damaged DNA, DNA damage induced mutagenesis, sporulation, and amino-end rule protein degradation. RAD6 interacts physically with the UBR1 gene product in carrying out the multiubiquitination of amino-end rule proteolytic substrates. In mediating postreplication repair, it has remained unclear whether RAD6 acts in a pleiotropic manner distal from the site of DNA damage or is targeted to the damage site via interaction with another repair component. Here, we show that RAD6 forms a specific complex with the product of the DNA repair gene RAD18. The biological significance of this interaction is attested by the observation that overproduction of the rad6 Ala-88 mutant protein, which lacks ubiquitin-conjugating activity but retains the ability to interact with RAD18 protein, confers a high level of UV sensitivity on wild-type RAD+ cells that can be corrected by the concomitant overexpression of RAD18. We demonstrate that whereas RAD6 has no affinity for DNA, RAD18 binds single-stranded DNA. Thus, association of RAD6 with RAD18 could provide a means for targeting RAD6 to damage-containing DNA regions, where the RAD6 ubiquitin-conjugating function could modulate the activity of a stalled DNA replication machinery. We also show that RAD6 forms separate complexes with RAD18 and with UBR1, and the extremely conserved amino terminus of RAD6 that is required for complex formation with UBR1 is dispensable for complex formation with RAD18.
The strict evolutionary conservation of ubiquitin suggests an essential role for each residue in the folding, stability, or function of the protein but precludes identification of such contributions through interspecies comparison of ubiquitin sequences. However, site-directed mutagenesis potentially allows assignment of specific function(s) for each residue. The four arginines present on ubiquitin at positions 42, 54, 72, and 74 were independently mutated to leucine and their effects on the interaction of the resulting polypeptides with ubiquitin-activating enzyme (E1) were characterized. All of the mutants except UbR54L exhibited altered kinetics for E1-catalyzed ATP:PPi exchange compared to wild-type ubiquitin. In addition, the UbR72L mutant altered the mechanism of E1 from strictly order addition of substrates to random addition with respect to ATP and ubiquitin. Values for the intrinsic Kd of ubiquitin binding were determined by coupling the net forward reaction of E1 to the E232K-catalyzed conjugation of histone H2B. Only R54 and R72 residues participate in the initial binding of free ubiquitin, resulting in a 6- or 58-fold increase in Kd for UbR54L or UbR72L, respectively, compared to wild type. More significant effects of the UbR42L and UbR72L mutants were observed for binding of their respective ubiquitin adenylate intermediates within the E1 active site. Wild-type ubiquitin adenylate binds to E1 with an estimated Kd < or = 8 x 10(-12) M while intermediates formed with UbR42L or UbR72L each bind with ca. 10(3)-fold lower affinity, representing a destabilization of > or = 7 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
The role of the ubiquitin-dependent proteolysis system in c-Jun breakdown was investigated. Using in vitro experiments and a novel in vivo assay that utilizes molecularly-tagged ubiquitin and c-Jun proteins, it was shown that c-Jun, but not its transforming counterpart, retroviral v-Jun, can be efficiently multiubiquitinated. Consistently, v-Jun has a longer half-life than c-Jun. Mutagenesis experiments indicate that the reason for the escape of v-Jun from multiubiquitination and its resulting stabilization is the deletion of the delta domain, a stretch of 27 amino acids that is present in c-Jun but not in v-Jun. c-Jun sequences containing the delta domain, when transferred to the bacterial beta-galactosidase protein, function as a cis-acting ubiquitination and degradation signal. The correlation between transforming ability and the escape from ubiquitin-dependent degradation described here suggests a novel route to oncogenesis.