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Targeting of substrates to the 26S proteasome
Abstract
The ubiquitin-proteasome pathway is the principal mechanism for the turnover of short-lived proteins in eukaryotic cells. In this pathway, the covalent ligation of ubiquitin to the substrate is a signal for recognition by the 26S proteasome. Recent studies indicate that targeting of substrates of the ubiquitin pathway to the proteasome is usually accomplished by the ligation of a polyubiquitin chain assembled through K48-G76 isopeptide bonds, rather than by ligation of monoubiquitin. In addition to providing benefits in signal generation, recognition, and persistence, assigning the proteolytic targeting function to a specific specific type of polyubiquitin chain may allow monoubiquitin or polyubiquitin chains of novel structures to serve distinct targeting functions. Besides polyubiquitinated substrates, the proteasome also degrades an unknown number of proteins that are recognized without undergoing ubiquitination. Ornithine decarboxylase is the prototype ubiquitin-independent substrate; it is targeted to the proteasome through noncovalent interaction with a specific protein factor known as antizyme. The existence of ubiquitin-independent substrates of the proteasome raises important questions about the nature of the substrate- and proteasome-based elements that cooperate to bring about the targeting of substrates to this novel proteolytic complex.
... We confirmed that overexpression of COP1, but not the E3 ligase-dead COP1 C136/139A mutant, enhances GATA2 ubiquitination in HEK293T cells (Fig. 2C). We next performed GATA2 ubiquitination assays in HEK293T cells co-transfected with either the wild-type ubiquitin or the ubiquitin K48R mutant that blocks the formation of K48-linked polyubiquitin chains (34). We observed that the K48R mutation totally abolished COP1-induced GATA2 ubiquitination in HEK293T cells (Fig. 2D). ...
... We observed that the K48R mutation totally abolished COP1-induced GATA2 ubiquitination in HEK293T cells (Fig. 2D). This indicates that COP1 promotes the K48-linked polyubiquitination of GATA2, which is consist with the previous reports of K48-linked polyubiquitination as a key pathway for 26S proteasome-mediated degradation (34). Since we observed significant levels of GATA2 ubiquitination in the HEK293T cells without ectopic expression of COP1, we also performed knockdown of COP1 in HEK293T cells using three independent siRNAs and similarly performed ubiquitination assays on the transfected Flag-tagged GATA2. ...
Androgen receptor (AR) signaling is crucial for driving prostate cancer (PCa), the most diagnosed and the second leading cause of death in male patients with cancer in the United States. Androgen deprivation therapy is initially effective in most instances of AR-positive advanced or metastatic PCa. However, patients inevitably develop lethal castration-resistant PCa (CRPC), which is also resistant to the next-generation AR signaling inhibitors. Most CRPCs maintain AR expression, and blocking AR signaling remains a main therapeutic approach. GATA2 is a pioneer transcription factor emerging as a key therapeutic target for PCa because it promotes AR expression and activation. While directly inhibiting GATA2 transcriptional activity remains challenging, enhancing GATA2 degradation is a plausible therapeutic strategy. How GATA2 protein stability is regulated in PCa remains unknown. Here, we show that constitutive photomorphogenesis protein 1 (COP1), an E3 ubiquitin ligase, drives GATA2 ubiquitination at K419/K424 for degradation. GATA2 lacks a conserved [D/E](x)xxVP[D/E] degron but uses alternate BR1/BR2 motifs to bind COP1. By promoting GATA2 degradation, COP1 inhibits AR expression and activation and represses PCa cell and xenograft growth and castration resistance. Accordingly, GATA2 overexpression or COP1 mutations that disrupt COP1-GATA2 binding block COP1 tumor-suppressing activities. We conclude that GATA2 is a major COP1 substrate in PCa and that COP1 promotion of GATA2 degradation is a direct mechanism for regulating AR expression and activation, PCa growth, and castration resistance.
... Homotypic K48-linked ubiquitin chains canonically signal for proteasomal degradation and also represent the most abundant linkage-type (135,137,141). Additional linkage-types that mediate proteasomal targeting include K29, which may also drive lysosomal degradation, and perhaps surprisingly, monoubiquitination and K63 which have been predominantly associated with non-proteasomal functions including endocytosis and autophagy (141)(142)(143)(144). Indeed, K63polyubiquitin chains have been primarily implicated in protein complex assembly which includes TRAF6, Ubc13-Uev1A and TRIKA2 (TAK1, TAB1 and TAB2) that associates with IkB kinase (IKK). ...
... Homotypic K48-linked ubiquitin chains canonically signal for proteasomal degradation and also represent the most abundant linkage-type (135,137,141). Additional linkage-types that mediate proteasomal targeting include K29, which may also drive lysosomal degradation, and perhaps surprisingly, monoubiquitination and K63 which have been predominantly associated with non-proteasomal functions including endocytosis and autophagy (141)(142)(143)(144). Indeed, K63polyubiquitin chains have been primarily implicated in protein complex assembly which includes TRAF6, Ubc13-Uev1A and TRIKA2 (TAK1, TAB1 and TAB2) that associates with IkB kinase (IKK). ...
Glioblastoma is the most common primary brain tumor in adults with poor overall outcome and 5-year survival of less than 5%. Treatment has not changed much in the last decade or so, with surgical resection and radio/chemotherapy being the main options. Glioblastoma is highly heterogeneous and frequently becomes treatment-resistant due to the ability of glioblastoma cells to adopt stem cell states facilitating tumor recurrence. Therefore, there is an urgent need for novel therapeutic strategies. The ubiquitin system, in particular E3 ubiquitin ligases and deubiquitinating enzymes, have emerged as a promising source of novel drug targets. In addition to conventional small molecule drug discovery approaches aimed at modulating enzyme activity, several new and exciting strategies are also being explored. Among these, PROteolysis TArgeting Chimeras (PROTACs) aim to harness the endogenous protein turnover machinery to direct therapeutically relevant targets, including previously considered “undruggable” ones, for proteasomal degradation. PROTAC and other strategies targeting the ubiquitin proteasome system offer new therapeutic avenues which will expand the drug development toolboxes for glioblastoma. This review will provide a comprehensive overview of E3 ubiquitin ligases and deubiquitinating enzymes in the context of glioblastoma and their involvement in core signaling pathways including EGFR, TGF-β, p53 and stemness-related pathways. Finally, we offer new insights into how these ubiquitin-dependent mechanisms could be exploited therapeutically for glioblastoma.
... Several intracellular proteolytic pathways may participate in AP degradation, including the calcium-activated calpains, the lysosomal pathways and the ubiquitin-proteasome system. Of particular interest is the ubiquitin-proteasome system, a proteolytic pathway responsible for degrading abnormal, short-lived or misfolded proteins, and which appears to be disrupted or defective in AD (for reviews see Pickart, 1997;Haas and Siepmann, 1997;Komitzer and Ciechanover, 2000). Degradation of a protein by this pathway involves two distinct and successive steps. ...
... The proteasome is widely recognised as the principal enzyme complex involved in non-lysosomal protein degradation. It is an essential component of the ATP-dependent proteolytic pathway, catalysing the rapid degradation of many ratelimiting enzymes, transcriptional regulators and many key regulatory proteins (for reviews see Pickart, 1997;Haas and Siepmann, 1997;Komitzer and Ciechanover, 2000). It is also essential for the rapid elimination of highly abnormal proteins arising via mutations, post-translational damage or misfolding. ...
Cardiovascular (CV) risk factors may be implicated in the pathogenesis of Alzheimer's disease (AD). The aim of this project was to examine potential pathological mechanisms whereby a common CV risk factor, hypercholesterolaemia, may modulate the onset and development of AD. A pathological hallmark of AD is the deposition of the cytotoxic peptide, beta amyloid (Aβ), in the walls of cerebral blood vessels. Platelets release Aβ upon activation and therefore subjects with conditions associated with platelet hyperactivity may be at increased risk of developing vascular amyloid deposits. Hypercholesterolaemia is associated with increased platelet activity, and affected individuals were investigated to establish if abnormal Aβ status was evident. Platelet Aβ release under resting and stimulated conditions was increased in hypercholesterolaemics in comparison with normocholesterolaemic individuals and correlated positively with plasma total cholesterol and low-density lipoprotein (LDL) cholesterol These data indicate that abnormal platelet Aβ release occurs in hypercholesterolaemia. Amyloid deposition in cerebral vessels is accompanied by smooth muscle cell degeneration, endothelial dysfunction and narrowing of the vessel lumen. Although soluble Aβ may produce pathological changes, it is generally accepted that Aβ toxicity relates to its aggregation state. Factors that promote Aβ aggregation are therefore potentially important with respect to the development of Aβ toxicity. Since plasma cholesterol has been shown to correlate with cerebral Aβ content, the influence of plasma lipoproteins upon Aβ fibrillogenesis was examined. In view of evidence that oxidative stress plays a role in the pathogenesis of both cardiovascular disease and AD, the influence of lipoprotein oxidation upon Aβ fibrillogenesis was also investigated. The chemical interactions between native and oxidised plasma lipoproteins and Aβ were studied extensively and therapeutically relevant strategies which may prevent lipoprotein mediated fibrillogenesis, including antioxidant and beta-sheet breaker peptide treatments, examined. Evidence for functional and pathophysiological roles for Aβ, including actions upon platelet and endothelial function as well as the modulation of vessel tone has been reported. In the present study, to establish if the biological effects of Aβ are potentiated by native and oxidised plasma lipoproteins, the actions of soluble, fibrillar and lipoprotein-treated Aβ preparations upon platelet function (aggregation and serotonin release), human aortic endothelial cell viability and rat aorta vasoactivity were studied. The results of these studies were compared with respect to the Aβ polymerisation state.
... Protein ubiquitination regulates diverse biological functions by virtue of both the modified target protein site and nature of the ubiquitin-to-ubiquitin linkages within the added polymer chains. For instance, the addition of ubiquitin monomers linked together at Lysine residue 48 (K48-linkage) often marks a target protein for degradation by the 26S proteasome (17). Other types of ubiquitination, such as those involving K63-linked ubiquitin molecules, can be important for the activation of signaling molecules and protein trafficking events (18)(19). ...
... Here we report a distinct pathway of K63-type Foxp3 ubiquitination capable of promoting Foxp3 nuclear localization, regulatory activity and Treg stability and function, mediated by the TNF receptor associated factor protein family member, TRAF6. Mice lacking TRAF6 specifically in Foxp3+ Tregs displayed compromised immune tolerance and robust immune activation at baseline compared to wild type controls (17)(18)(19)(20)(21)(22)(23). But the mechanism of the regulation of TRAF6 to Foxp3 was not determined. ...
Regulatory T cells (Treg) are crucial mediators of immune control. The characteristic gene expression and suppressive function of Treg depend considerably on the stable expression and activity of the transcription factor Foxp3. While transcriptional regulation of the Foxp3 gene has been studied in depth, both the expression and function of Foxp3 are also modulated at the protein level. However, the molecular players involved in posttranslational Foxp3 regulation are just beginning to be elucidated. Here we found TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to B16 melanomas and displayed enhanced anti-tumor immunity. We further determined that Foxp3 undergoes lysine-63 chain (K63) ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. When deprived of TRAF6 activity or rendered insensitive to K63 ubiquitination, Foxp3 displayed aberrant, perinuclear accumulation, disrupted function. Thus, Foxp3 ubiquitination by TRAF6 ensures proper localization of Foxp3 and facilitates Foxp3's gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing force that may be targeted in novel tolerance-breaking therapies.
... These include the nuclear receptor family. [20] The ubiquitin-proteosome pathway has functions in many cellular processes. These functions include; regulation of cell cycle, signal transduction, differentiation, antigen processing, tumor suppressor degradation and directing some proteins to cellular localization. ...
... Then ubiquitin is transferred from E1 to one of the enzymes in the E2 class and the high energy of the thioester bond is preserved. [20,21] In some cases, ubiquitin can be directly transferred from E2 to the target protein with the formation of a peptide bond between the target protein's lysine residues -amino and the carboxy terminal of ubiquitin. Another alternative pathway is the transfer of ubiquitin from one of the UBC (E2) to the target protein via E3ubiquitin protein ligase. ...
... The ubiquitin-proteasome system (UPS) is the principal negative regulatory mechanism for short-lived proteins in eukaryotes (Hershko and Ciechanover, 1998). Proteins destined for degradation are post-translationally tagged with ubiquitin (Ub) or a chain of Ub molecules (polyUb) attached to a lysine side chain on a protein through the action of the E1, E2, E3 enzyme cascade (Pickart, 1997). Provided the protein is appropriately modified with Ub, shuttling factors facilitate its transport to the 26S proteasome where dedicated receptors initiate its degradation (Finley et al., 2012;Pickart, 1997). ...
... Proteins destined for degradation are post-translationally tagged with ubiquitin (Ub) or a chain of Ub molecules (polyUb) attached to a lysine side chain on a protein through the action of the E1, E2, E3 enzyme cascade (Pickart, 1997). Provided the protein is appropriately modified with Ub, shuttling factors facilitate its transport to the 26S proteasome where dedicated receptors initiate its degradation (Finley et al., 2012;Pickart, 1997). ...
The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface patch and the basic/polar residues surrounding it. Ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin receptor and shows strong preference for K48 linkages over K11 and K63. Furthermore, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S proteasome. Finally, ubistatin B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.
... The length and the linkage type of the polyubiquitin chains determine the fate of the modified protein. For example, poly-ubiquitin chains linked through lysines 48, 63, or 11 can direct a protein for proteasomal degradation (Chowdhury et al., 2023;Ohtake et al., 2018;Pickart, 1997;Zheng et al., 2023;Zhu et al., 2023). ...
Deltex proteins are a family of E3 ubiquitin ligases that encode C‐terminal RING and DTC domains that mediate interactions with E2 ubiquitin‐conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N‐terminal domain architecture. The N‐terminal D1 and D2 domains of DTX3L mediate homo‐oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP‐ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP‐ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N‐terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Ų and a smaller one of 415 Ų. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1‐D2 deletion mutant showed the domain is dispensable for DTX3L‐PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP‐ribosylated androgen receptor. Our results suggest that homo‐oligomerization of DTX3L is important for the DTX3L‐PARP9 complex to read mono‐ADP‐ribosylation on a ligand‐regulated transcription factor.
... The length and the linkage type of the poly-ubiquitin chains determine the fate of the modified protein. For example, polyubiquitin chains linked through lysines 48, 63, or 11 can direct a protein for proteasomal degradation (Chowdhury et al., 2023;Ohtake et al., 2018;Pickart, 1997;Zheng et al., 2023;Zhu et al., 2023). ...
Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å ² and a smaller one of 415 Å ² . Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.
... Covalent linkages among ubiquitins to form poly-ubiquitin chains can utilize any of the seven lysine residues of ubiquitin, or the initial methionine residue (Komander and Rape 2012). The most frequently used, K48-and K29-linked poly-ubiquitin chains, are canonical signals for 26S proteasomal degradation and endoplasmic reticulum (ER) associated degradation (ERAD) (Leto et al. 2019), whereas K63-linked poly-ubiquitin chains (and mono-ubiquitination) are the code enabling a diverse array of non-proteasomal functions, including protein translation, sorting, complex formation, and phosphorylation, as well as RNA splicing, DNA repair, endocytosis, autophagy, and transcription (Pickart 1997;Saeki et al. 2009;Wang et al. 2001;Deng et al. 2000;Doil et al. 2009;Huang et al. 2013;Lauwers et al. 2009;Song et al. 2010;Spence et al. 2000;Kodadek et al. 2006). Mono-ubiquitination of transcriptional activators, followed by poly-ubiquitination, has been reported to regulate transcription cycling in the 'timer' or so called 'black widow' models (Kodadek et al. 2006). ...
We have mined public genomic datasets to identify genes coding for components of the ubiquitin proteasome system (UPS) that may qualify as potential diagnostic and therapeutic targets in the three major glioma types, astrocytoma (AS), glioblastoma (GBM), and oligodendroglioma (ODG). In the Sun dataset of glioma (GEO ID: GSE4290), expression of the genes UBE2S and UBE2C, which encode ubiquitin conjugases important for cell-cycle progression, distinguished GBM from AS and ODG. KEGG analysis showed that among the ubiquitin E3 ligase genes differentially expressed, the Notch pathway was significantly over-represented, whereas among the E3 ligase adaptor genes the Hippo pathway was over-represented. We provide evidence that the UPS gene contributions to the Notch and Hippo pathway signatures are related to stem cell pathways and can distinguish GBM from AS and ODG. In the Sun dataset, AURKA and TPX2, two cell-cycle genes coding for E3 ligases, and the cell-cycle gene coding for the E3 adaptor CDC20 were upregulated in GBM. E3 ligase adaptor genes differentially expressed were also over-represented for the Hippo pathway and were able to distinguish classic, mesenchymal, and proneural subtypes of GBM. Also over-expressed in GBM were PSMB8 and PSMB9, genes encoding subunits of the immunoproteasome. Our transcriptome analysis provides a strong rationale for UPS members as attractive therapeutic targets for the development of more effective treatment strategies in malignant glioma.
Graphical abstract
Ubiquitin proteasome system and glioblastoma:
E1—ubiquitin-activating enzyme, E2—ubiquitin-conjugating enzyme, E3—ubiquitin ligase. Ubiquitinated substrates of E3 ligases may be degraded by the proteasome. Expression of genes for specific E2 conjugases, E3 ligases, and genes for proteasome subunits may serve as differential markers of subtypes of glioblastoma.
... The nature of the ubiquitin modification determines the fate of a particular substrate: ubiquitin can be attached as a monomer to one or several sites within a substrate (monoubiquitylation or multi-monoubiquitylation), or it can be attached in the form of ubiquitin chains (polyubiquitylation). Polyubiquitin chains can be interconnected by ubiquitin conjugation to one or more of the seven lysine residues or the N-terminal methionine (K6, K11, K27, K29, K33, K48, K63, M1) of any ubiquitin monomer, resulting in a staggering complexity of homotypic and heterotypic architectures, each of which has the biochemical capacity to elicit distinct downstream functional consequences 15 (Box 1). Notable ubiquitin modifications are the homotypic K48-linked ubiquitin chains that mediate degradation by the proteasome 16 , and the homotypic and heterotypic M1-K63-linked ubiquitin chains that assemble multi-protein signalling platforms during immune responses 17,18 . Different types of ubiquitylation are recognized by a variety of proteins with ubiquitin-binding domains (ubiquitin receptors) that translate specific ubiquitin linkages into cellular responses 19,20 . ...
Ubiquitylation is an essential post-translational modification that regulates intracellular signalling networks by triggering proteasomal substrate degradation, changing the activity of substrates or mediating changes in proteins that interact with substrates. Hundreds of enzymes participate in reversible ubiquitylation of proteins, some acting globally and others targeting specific proteins. Ubiquitylation is essential for innate immune responses, as it facilitates rapid regulation of inflammatory pathways, thereby ensuring sufficient but not excessive responses. A growing number of inborn errors of immunity are attributed to dysregulated ubiquitylation. These genetic disorders exhibit broad clinical manifestations, ranging from susceptibility to infection to autoinflammatory and/or autoimmune features, lymphoproliferation and propensity to malignancy. Many autoinflammatory disorders result from disruption of components of the ubiquitylation machinery and lead to overactivation of innate immune cells. An understanding of the disorders of ubiquitylation in autoinflammatory diseases could enable the development of novel management strategies.
... [2,4] Modification by Lys48-polyubiquitin is read by cells as a strong signal for substrate degradation by the proteasome. [5] Ubiquitin-dependent protein degradation is crucial for the health of neurons, where protein remodeling is at the basis of specialized brain processes such as memory and learning, and synaptic plasticity. [6] The age-dependent dysfunction of the mechanisms controlling protein clearance determines the accumulation of damaged and misfolded proteins within neurons. ...
The multi‐site ubiquitination of Tau protein found in Alzheimer's disease filaments hints at the failed attempt of neurons to remove early toxic species. The ubiquitin‐dependent degradation of Tau is regulated in vivo by the E3 ligase CHIP, a quality controller of the cell proteome dedicated to target misfolded proteins for degradation. In our study, by using site‐resolved NMR, biochemical and computational methods, we elucidate the structural determinants underlying the molecular recognition between the ligase and its intrinsically disordered substrate. We reveal a multi‐domain dynamic interaction that explains how CHIP can direct ubiquitination of Tau at multiple sites even in the absence of chaperones, including its typical partner Hsp70/Hsc70. Our findings thus provide mechanistic insight into the chaperone‐independent engagement of a disordered protein by its E3 ligase.
... Central to the ubiquitin-proteasomal proteolysis process is the relative balance between activation and inhibition of ubiquitin following binding of various signaling proteins. Ubiquitin has a major role in targeting cellular proteins for degradation by the 26S proteosome (Pickart, 1997). Degradation of proteins via the proteasome involves an initial polyubiquitination of substrate proteins targeted for elimination through the ubiquitination enzyme cascade involving the ubiquitin activation (E1), conjugation (E2), and protein substrate labeling (E3) enzymes (Ciechanover, 2005). ...
Background: Malaria remains one of the leading global causes of childhood morbidity and mortality. In holoendemic Plasmodium falciparum transmission regions, such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dl] is the primary form of severe disease. Ubiquitination is essential for regulating intracellular processes involved in innate and adaptive immunity. Although dysregulation in ubiquitin molecular processes is central to the pathogenesis of multiple human diseases, the expression patterns of ubiquitination genes in SMA remain unexplored.
Methods: To examine the role of the ubiquitination processes in pathogenesis of SMA, differential gene expression profiles were determined in Kenyan children (n = 44, aged <48 mos) with either mild malarial anemia (MlMA; Hb ≥9.0 g/dl; n = 23) or SMA (Hb <6.0 g/dl; n = 21) using the Qiagen Human Ubiquitination Pathway RT² Profiler PCR Array containing a set of 84 human ubiquitination genes.
Results: In children with SMA, 10 genes were down-regulated (BRCC3, FBXO3, MARCH5, RFWD2, SMURF2, UBA6, UBE2A, UBE2D1, UBE2L3, UBR1), and five genes were up-regulated (MDM2, PARK2, STUB1, UBE2E3, UBE2M). Enrichment analyses revealed Ubiquitin-Proteasomal Proteolysis as the top disrupted process, along with altered sub-networks involved in proteasomal, protein, and ubiquitin-dependent catabolic processes.
Conclusion: Collectively, these novel results show that protein coding genes of the ubiquitination processes are involved in the pathogenesis of SMA.
... 6 While a variety of polyubiquitin chain topologies are possible, K48-linked ubiquitin serves as the canonical recognition signal for the 26S proteasome and generally leads to substrate degradation. 7 The fact that E3s govern substrate specificity and often exhibit remarkable plasticity has made these enzymes the component of choice in the majority of proteome editing technologies described to date. Most notable among these technologies is PROTACs (proteolysis targeting chimeras), 8,9 which are heterobifunctional small molecules that effectively bridge the E3 and the POI, forming a ternary complex that facilitates polyubiquitination and subsequent proteasomal degradation of target proteins in cultured cells and mice. ...
... They include: (1) really interesting new gene (RING) finger, (2) homologous to E6AP carboxyl terminus (HECT), (3) Skp1-Cul1-F box (SCF), and (4) anaphase-promoting complex (APC) families [7,21,22]. The efficiency for degradation of a targeted protein relies on the assembly of multiple ubiquitin molecules ultimately forming polyubiquitin chains [23][24][25]. For every newly added ubiquitin there is an exponential increase in the number of different positions that the subsequent ubiquitin ligation can occur [11]. ...
Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival.
... In contrast to 20S proteasomal degradation, the J o u r n a l P r e -p r o o f 26S proteasomal degradation is an ATP-dependent process. Moreover, 26S proteasome substrates are required to be labeled by a K48-linked polyubiquitin-tag of at least four ubiquitin molecules (8,9). Thus, this degradation system is also referred to as ubiquitin-proteasome system (UPS) (1,10). ...
The proteasomal degradation system is one of the most important protein degradation systems in the cytosol and nucleus. This system is present in two major forms: the ATP-stimulated 26S/30S proteasome or the ATP-independent 20S core proteasome. While the first recognize ubiquitin-tagged target proteins and degrade them, the 20S proteasome works also independent from ATP, but requires partially unfolded substrates. While the role of the proteasome in the selective removal of oxidized proteins is undoubted, the debate about a selective ubiquitination of oxidized proteins is still ongoing.
Here we demonstrate, that under some conditions of oxidative stress an accumulation of oxidized and of K48-ubiquitinated proteins occurs. However, the removal of oxidized proteins seems not to be linked to ubiquitination. In further experiments, we could show that the accumulation of ubiquitinated proteins under certain oxidative stress conditions is rather a result of a different sensitivity of the 26S proteasome and the ubiquitination machinery towards oxidants.
... It has been previously shown that both Hsp40 and Hsp70 expression is elevated due to the expression of UBB^^ in SH-SY5Y cells (Chapter 6). However, in pontine neurons of the PSP brains studied, Hsp70 and Hsp40 immunoreactivity did not co-localise with polyubiquitin chains, as has been described elsewhere (Pickart, 1997). Also, there may be an in vivo failure to deubiquitinate the Ubn-UBB^ ^ substrate by a component of the 19S regulatory complex of the proteasome, i.e. ...
Tau is a microtubule-associated protein, normally binding to tubulin, in an interaction mediated by chaperones. Unwanted tau is degraded by proteasomes. The tauopathies are neurodegenerative disorders characterized by a tau-predominant neuropathology. Tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP-17, which can be caused by mutations in the tau gene). Pathological tau is hyperphosphorylated, and has reduced microtubule binding capacity. Tau mutations in the microtubule-binding region have similar effects. In some tauopathies, tau deposition is isoform-specific, and pathogenic mutations can cause isoform imbalances. Oxidative stress may also be a factor in tauopathies. In addition, tau pathology has been associated with an ubiquitin variant, which causes proteasome inhibition. There is evidence of aberrations in the chaperone system in diseased cells, reducing tau-microtubule binding, and degradation. To investigate the role of UBB+1 in tauopathies, a model system was developed with SH-SY5Y neuroblastoma cells, which showed that UBB+1 inhibits the proteasome. UBB+1 expression did not affect tau, but was coupled with a chaperone expression response. Subsequently, contrary to expectations, cells with induced UBB+1 expression coped better with oxidative insults. These proteins were studied in sections from PSP brains; UBB+1 and abnormal tau were only present in the diseased tissue. Chaperones and UBB+1 did not co-localise, indicating a failure in the chaperone response. In cells, mutant-tau localized with microtubules as effectively as wild-type. Through neuronal differentiation, tau concentrations were greatly increased, and phosphorylation was decreased. Proteasome inhibition did not increase tau concentration or phosphorylation, or affect isoform balance. A failure in the chaperone response in the brains of tauopathies could explain an increase in unbound cytosolic-tau, tau aggregation, decreased proteasome activity, and susceptibility to oxidative stress.
... 24 Moreover, multi mono-ubiquitination and chains of four or more ubiquitin molecules linked through K 48 can lead to proteasomal degradation of target proteins. 17,[25][26][27] Likewise, the anaphase-promoting complex/cyclosome (APC/C) marks its substrates for proteasomal degradation through K 11 -linked ubiquitin chain formation 28 and chains linked through other lysines may also have the same outcome. 29,30 However, in NF-κB signaling pathway, K 63 -linked and linear ubiquitin chains are associated with nonproteasomal outcomes. ...
The ubiquitin‐proteasome system (UPS) is a complex process that regulates protein stability and activity by the sequential actions of E1, E2 and E3 enzymes to influence diverse aspects of eukaryotic cells. However, due to the diversity of proteins in cells, substrate selection is a highly critical part of the process. As a key player in UPS, E3 ubiquitin ligases recruit substrates for ubiquitination specifically. Among them, RING E3 ubiquitin ligases which are the most abundant E3 ubiquitin ligases contribute to diverse cellular processes. The multisubunit cullin‐RING ligases (CRLs) are the largest family of RING E3 ubiquitin ligases with tremendous plasticity in substrate specificity and regulate a vast array of cellular functions. The F‐box protein Skp2 is a component of CRL1 (the prototype of CRLs) which is expressed in many tissues and participates in multiple cellular functions such as cell proliferation, metabolism, and tumorigenesis by contributing to the ubiquitination and subsequent degradation of several specific tumor suppressors. Most importantly, Skp2 plays a pivotal role in a plethora of cancer‐associated signaling pathways. It enhances cell growth, accelerates cell cycle progression, promotes migration and invasion, and inhibits cell apoptosis among others. Hence, targeting Skp2 may represent a novel and attractive strategy for the treatment of different human cancers overexpressing this oncogene. In this review article, we summarized the known roles of Skp2 both in health and disease states in relation to the UPS.
... For example, K48 ubiquitin is the most abundant linkage type and the canonical signal for proteasomal degradation [61]. K48 ubiquitin consists of a ubiquitin chain covalently bonded to the ε-amino group of the lysine in the 48th position of the preceding ubiquitin molecule [62]. In regards to translation, K48 ubiquitin possesses an important function in the degradation of ribosomal units by the proteasome, ensuring proper ribosomal composition, and removal of excess free protein [49]. ...
The eukaryotic proteome has to be precisely regulated at multiple levels of gene expression, from transcription, translation, and degradation of RNA and protein to adjust to several cellular conditions. Particularly at the translational level, regulation is controlled by a variety of RNA binding proteins, translation and associated factors, numerous enzymes, and by post-translational modifications (PTM). Ubiquitination, a prominent PTM discovered as the signal for protein degradation, has newly emerged as a modulator of protein synthesis by controlling several processes in translation. Advances in proteomics and cryo-electron microscopy have identified ubiquitin modifications of several ribosomal proteins and provided numerous insights on how this modification affects ribosome structure and function. The variety of pathways and functions of translation controlled by ubiquitin are determined by the various enzymes involved in ubiquitin conjugation and removal, by the ubiquitin chain type used, by the target sites of ubiquitination, and by the physiologic signals triggering its accumulation. Current research is now elucidating multiple ubiquitin-mediated mechanisms of translational control, including ribosome biogenesis, ribosome degradation, ribosome-associated protein quality control (RQC), and redox control of translation by ubiquitin (RTU). This review discusses the central role of ubiquitin in modulating the dynamism of the cellular proteome and explores the molecular aspects responsible for the expanding puzzle of ubiquitin signals and functions in translation.
... However, formation of the engineered E3 ligase and protein substrate complex requires an accurate spatial geometric structure, which allows ubiquitin-conjugating enzymes (E2s) to catalyze the addition of ubiquitin molecules onto specific lysine residues in the protein substrate (20,21). The precise requirements for the structure indicate that valid ubiquitination of protein substrates may be difficult due to mismatched rigidity between protein substrates and E3 ligases (22)(23)(24). ...
The oncogene KRAS not only promotes the tumorigenesis of pancreatic cancers but also is required for the malignant progression and metastasis of these cancers. Many methods have been explored to influence the malignant biological behavior of these cancers by targeting mutant KRAS. The ornithine decarboxylase/antizyme (ODC/AZ) system is another protein degradation pathway that exists in nature. The formation of an ODC and protein substrate complex through direct combination can promote its degradation by the 26S proteasome without ubiquitination, and this process can be catalyzed by AZ. In this study, we designed and reconstructed a chimeric fusion protein (named RC‐ODC). The engineered fusion protein RC‐ODC was confirmed to interact with the mutant KRAS oncoprotein in a co‐immunoprecipitation assay, and the introduction of both RC‐ODC and AZ resulted in degradation of the exogenous and endogenous mutant KRAS oncoprotein at the post‐translational level independent of ubiquitination in vitro. Along with a decreased KRAS level, suppression of PANC‐1 cell proliferation was detected in vitro and in vivo, and meanwhile downregulation of phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) was also observed. Targeted degradation of the KRAS oncoprotein through the ODC/AZ pathway at the post‐translational level may reflect a more effective future therapeutic strategy for pancreatic cancer patients. © 2018 The Authors. IUBMB Life published by Wiley Periodicals,Inc. on behalf of International Union of Biochemistry and Molecular Biology, 1‐9, 2018
... Ubiquitination is an enzymatically catalized PTM that targets and marks proteins for degradation by the attachment of ubiquitin (Ub) [16]. Ubiquitination consists of three main enzyme classes including, Ub activating enzyme (E1), Ub conjugating enzyme (E2), and Ub ligase (E3) [17]. Proteins tagged for degradation can be monoubiquitinated or poly-ubiqutinated where the latter targets proteins to the 26S proteasome for degradation. ...
The term proteostasis reflects the fine-tuned balance of cellular protein levels, mediated through a vast network of biochemical pathways. This requires the regulated control of protein folding, post-translational modification, and protein degradation. Due to the complex interactions and intersection of proteostasis pathways, exposure to stress conditions may lead to a disruption of the entire network. Incorrect protein folding and/or modifications during protein synthesis results in inactive or toxic proteins, which may overload degradation mechanisms. Further, a disruption of autophagy and the endoplasmic reticulum degradation pathway may result in additional cellular stress which could ultimately lead to cell death. Neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and Amyotrophic Lateral Sclerosis all share common risk factors such as oxidative stress, aging, environmental stress, and protein dysfunction; all of which alter cellular proteostasis. The differing pathologies observed in neurodegenerative diseases are determined by factors such as location-specific neuronal death, source of protein dysfunction, and the cell’s ability to counter proteotoxicity. In this review, we discuss how the disruption in cellular proteostasis contributes to the onset and progression of neurodegenerative diseases.
... Although individual E3 ligases of the UPS have been implicated previously [7,8], using a comprehensive approach employing K48R transgenic mice to inhibit poly-ubiquitin conjugation, we now demonstrate partial dependency of pulmonary inflammation-driven muscle atrophy on UPS-mediated proteolysis. The Ub K48R substitution interferes with assembly of ubiquitin chains with the topology required for proteasomal targeting [32], but not with upstream activation of the UPS proteolytic program. Accordingly, the induction of Atrogin-1 and MuRF1 expression is similar to UBWT mice. ...
Background:
Pulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation.
Methods:
Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice.
Results:
Pulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass.
Conclusion:
Pulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.
... (Peng et al., 2003). La liaison de ces chaînes sur les substrats entraine essentiellement leur dégradation par le protéasome (Pickart, 1997). Les chaînes de type K11 sont moins exprimées dans les cellules mais favorisent également la dégradation des protéines. ...
Themis est une protéine de signalisation des récepteurs des lymphocytes T (TCR) essentielle pour la sélection positive des cellules T. La fonction moléculaire de Themis a été controversée mais de récentes études suggèrent qu'il est un régulateur positif des voies de signalisation des TCR. Nous avons montré dans une étude préliminaire que Themis interagit avec des déubiquitinases et qu'il est ubiquitiné dans les thymocytes. L'objectif de ma thèse était de caractériser les mécanismes moléculaires qui régulent l'ubiquitination de Themis et de déterminer si ces processus affectent la fonction de Themis durant le développement des lymphocytes T. Nous avons montré que si l'expression des ARNm codant pour Themis diminue dans les stades précoces de la sélection positive, son expression protéique est parallèlement augmentée, suggérant une stabilisation de Themis par des modifications post-traductionnelles durant cette étape. Nous avons montré que la déubiquitinase USP9X déubiquitine Themis pour stabiliser son expression durant la stimulation des TCR. L'ensemble de nos résultats proposent qu'USP9X soit activé durant la stimulation des TCR grâce à son recrutement dans les complexes proximaux des TCR par l'intermédiaire de l'adaptateur Grb2 et Themis, entrainant la stabilisation de l'expression de Themis. Nous pensons que ce mécanisme est important pour maintenir l'expression de Themis durant la sélection positive afin de favoriser l'induction d'un signal des TCR soutenu, requis pour l'efficacité de ce processus.
... After the monoubiquitination of the substrate protein, the E2-E3 ligase complex can build a polyubiquitin chain, in which the C-terminus of each ubiquitin unit is linked by an isopeptide bond to a specific Lysine residue (generally Lys48) of the previous ubiquitin in the same way as in ubiquitin-substrate reaction (Pickart, 1997;Pickart and Fushman, 2004). ...
Our genome is continuously exposed to DNA damaging agents. In order to preserve the integrity of their genome, cells have evolved a DNA damage signalling pathway known as checkpoint. Some lesions may persist when cells enter the S-phase and halt the progression of replicative DNA polymerases. This can cause prolonged replication forks stalling which threaten the stability of the genome. To preserve the integrity of genetic information, cells have developed a tolerance pathway which involves specialized DNA polymerases, called translesion DNA polymerases (TLS Pols). These polymerases have the unique ability to accommodate the damaged bases thanks to their catalytic site. In this process, PCNA acts as a scaffold for many proteins involved in DNA metabolism. The mechanisms governing the exchanges between different PCNA partners are not well understood. Among the proteins that interact with PCNA, CDT1, p21 and PR-Set7/set8 are characterized by a high binding affinity. These proteins have a particular interaction domain with PCNA, called "PIP degron", which promotes their proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. After UV-C irradiation, the replication initiation factor CDT1 is rapidly degraded in a PIP degron-dependent manner. During the first part of my work, we wanted to understand the functional role of this degradation. Our results have shown that inhibition of CDT1 degradation by CRL4Cdt2 in mammalian cells, compromises the relocalisation of TLS Pol eta and Pol kappato nuclear foci after UV-C irradiation. We also found that only the proteins which contain a PIP degron interfere with the formation of Pol eta foci. Mutagenesis experiments on CDT1 PIP degron revealed that a threonine residue conserved among PIP degrons is essential for inhibiting foci formation of at least two TLS polymerases. This results suggest that CRL4Cdt2-dependent degradation of proteins containing PIP degrons regulates the recruitment of TLS polymerases at sites of UV-induced DNA damage.During the second part of my thesis, we studied DNA damage checkpoint regulation during embryogenesis. Indeed, in early embryos, the DNA damage checkpoint is silent until the mid-blastula transition (MBT) due to maternal inhibiting factors. In this work, we have shown, both in vitro and in vivo, that the E3 ubiquitin ligase RAD18, a major regulator of translesion DNA synthesis, is a limiting factor for the checkpoint activation in Xenopus embryos. We have also shown that RAD18 depletion leads to the activation of DNA damage checkpoints by inducing replication fork uncoupling in front of the lesions. Furthermore, we showed that the abundance of RAD18 and PCNA monoubiquitination (PCNAmUb) is regulated during embryonic development. Near the MBT, the increased abundance of DNA limits the availability of RAD18, thereby reducing the amount of PCNAmUb and inducing the de-repression of the checkpoint. Moreover, we have shown that this embryonic-like regulation can be reactivated in somatic mammalian cells by ectopic expression of RAD18, conferring resistance to DNA damaging. Finally, we found high RAD18 levels in glioblastoma cancer stem cells highly resistant to DNA damage. All together, these data propose RAD18 as a critical factor that inhibits DNA damage checkpoint in early embryos and suggests that dysregulation of RAD18 expression may have an unexpected oncogenic potential
... We show that Themis is ubiquitylated predominantly by K48linked chains that have a well-established function in the targeting of proteins to proteasomes for degradation (32). Supporting this mechanism, ubiquitylated Themis accumulates when thymocytes are incubated with the proteasome inhibitor MG132. ...
Themis is a new component of the TCR signaling machinery that plays a critical role during T cell development. The positive selection of immature CD4(+)CD8(+) double-positive thymocytes and their commitment to the CD4(+)CD8(-) single-positive stage are impaired in Themis(-/-) mice, suggesting that Themis might be important to sustain TCR signals during these key developmental processes. However, the analysis of Themis mRNA levels revealed that Themis gene expression is rapidly extinguished during positive selection. We show in this article that Themis protein expression is increased in double-positive thymocytes undergoing positive selection and is sustained in immature single-positive thymocytes, despite the strong decrease in Themis mRNA levels in these subsets. We found that Themis stability is controlled by the ubiquitin-specific protease USP9X, which removes ubiquitin K48-linked chains on Themis following TCR engagement. Biochemical analyses indicate that USP9X binds directly to the N-terminal CABIT domain of Themis and indirectly to the adaptor protein Grb2, with the latter interaction enabling recruitment of Themis/USP9X complexes to LAT, thereby sustaining Themis expression following positive selection. Together, these data suggest that TCR-mediated signals enhance Themis stability upon T cell development and identify USP9X as a key regulator of Themis protein turnover.
... It does so by interacting directly with TRAF6 and preventing its K48linked ubiquitination. In the absence of this interaction, ubiquitinated TRAF6 is degraded by the proteasome (Hjerpe and Rodriguez, 2008;Pickart, 1997). Thus, myeloid cellspecific IPMK deletion in mice reduces excessive inflammation thereby protecting mice against polymicrobial sepsis and endotoxemic shock. ...
The inositol polyphosphates are a group of multifunctional signaling metabolites whose synthesis is catalyzed by a family of inositol kinases that are evolutionarily conserved from yeast to humans. Inositol polyphosphate multikinase (IPMK) was first identified as a subunit of the arginine-responsive transcription complex in budding yeast. In addition to its role in the production of inositol tetrakis- and pentakisphosphates (IP4 and IP5), IPMK also exhibits phosphatidylinositol 3-kinase (PI3-kinase) activity. Through its PI3-kinase activity, IPMK activates Akt/PKB and its downstream signaling pathways. IPMK also regulates several protein targets non-catalytically via protein- protein interactions. These non-catalytic targets include cytosolic signaling factors and transcription factors in the nucleus. In this review, we highlight the many known functions of mammalian IPMK in controlling cellular signaling networks and discuss future challenges related to clarifying the unknown roles IPMK plays in physiology and disease.
... Ubiquitin (Ub) chain extension can occur on a number of different lysine (K) residues on the Ub molecule, but only chain polymerization at K48 is important for substrate degradation by the proteasome [20,21]. To examine whether OVA12 promotes K48linked polyubiquitin chain formation of p53, we performed a ubiquitination assay with the ubiquitin mutant UbK48R, which cannot form K48-conjugated polyubiquitin chains. ...
Ovarian cancer-associated antigen 12 (OVA12) was first identified in an ovarian carcinoma complementary DNA (cDNA) expression library and has been shown to play an important role in tumor growth. Here, we found that overexpression of OVA12 accelerated tumor growth in different tumor cells, whereas OVA12 depletion was associated with the opposite effect. Moreover, knocking down OVA12 led to a significant increase in the protein levels of p53, and the overexpression of OVA12 significantly decreased endogenous p53 levels. In addition, OVA12 stimulated p53 polyubiquitination and degradation by the proteasome and promoted tumor growth at least partially through the p53 pathway. Taken together, these results indicate that OVA12 is a negative regulator of p53 and that inhibition of OVA12 expression might serve as a therapeutic target to restore tumor suppression.
... Ubiquitination is the covalent attachment of ubiquitin to proteins by ubiquitin ligases and is a multifunctional protein modification that controls diverse biological phenomena including innate immunity. The covalent linkage of ubiquitin or a polyubiquitin chain to lysine 48 (K48) of a protein is a well-characterized signal that targets the ubiquitinated protein to degradation by the 26S proteasome (14,15). K63linked ubiquitin can act as a scaffold that controls conformational changes and as molecular interactions (16). ...
Toll-like receptor (TLR) signaling is tightly controlled to protect hosts from microorganisms while simultaneously preventing uncontrolled immune responses. Tumor necrosis factor receptor–associated factor 6 (TRAF6) is a critical mediator of TLR signaling, but the precise mechanism of how TRAF6 protein stability is strictly controlled still remains obscure. We show that myeloid-specific deletion of inositol polyphosphate multikinase (IPMK), which has both inositol polyphosphate kinase activities and noncatalytic signaling functions, protects mice against polymicrobial sepsis and lipopolysaccharide-induced systemic inflammation. IPMK depletion in macrophages results in decreased levels of TRAF6 protein, thereby dampening TLR-induced signaling and proinflammatory cytokine production. Mechanistically, the regulatory role of IPMK is independent of its catalytic function, instead reflecting its direct binding to TRAF6. This interaction stabilizes TRAF6 by blocking its K48-linked ubiquitination and subsequent degradation by the proteasome. Thus, these findings identify IPMK as a key determinant of TRAF6 stability and elucidate the physiological function of IPMK in TLR-induced innate immunity.
... These small proteins (8.5 kDa) act usually as a recognition signal for 26S proteasome. The ubiquitin-proteasome pathway is the major route for the selective degradation of short-lived regulatory proteins in eukaryotic cells [72]. ...
Vitamin D receptor (VDR) is a member of the nuclear receptor (NR) superfamily of ligand-activated transcription factors. Activated VDR is responsible for maintaining calcium and phosphate homeostasis, and is required for proper cellular growth, cell differentiation and apoptosis. The expression of both phases I and II drug-metabolizing enzymes is also regulated by VDR, therefore it is clinically important.
Post-translational modifications of NRs have been known as an important mechanism modulating the activity of NRs and their ability to drive the expression of target genes. The aim of this mini review is to summarize the current knowledge about post-transcriptional and post-translational modifications of VDR.
The heat shock response is an evolutionarily conserved mechanism that protects cells or organisms from the harmful effects of various stressors such as heat, chemicals toxins, UV radiation, and oxidizing agents. The heat shock response triggers the expression of a specific set of genes and proteins known as heat shock genes/proteins or molecular chaperones, including HSP100, HSP90, HSP70, HSP60, and small HSPs. Heat shock proteins (HSPs) play a crucial role in thermotolerance and aiding in protecting cells from harmful insults of stressors. HSPs are involved in essential cellular functions such as protein folding, eliminating misfolded proteins, apoptosis, and modulating cell signaling. The stress response to various environmental insults has been extensively studied in organisms from prokaryotes to higher organisms. The responses of organisms to various environmental stressors rely on the intensity and threshold of the stress stimuli, which vary among organisms and cellular contexts. Studies on heat shock proteins have primarily focused on HSP70, HSP90, HSP60, small HSPs, and ubiquitin, along with their applications in human biology. The current review highlighted a comprehensive mechanism of heat shock response and explores the function of heat shock proteins in stress management, as well as their potential as therapeutic agents and diagnostic markers for various diseases.
The ubiquitin-proteasome system (UPS) is a pivotal cellular mechanism responsible for the selective degradation of proteins, playing an essential role in proteostasis, protein quality control, and regulating various cellular processes, with ubiquitin marking proteins for degradation through a complex, multi-stage process. The shuttle proteins family is a very unique group of proteins that plays an important role in the ubiquitin-proteasome system. Ddi1, Dsk2, and Rad23 are shuttle factors that bind ubiquitinated substrates and deliver them to the 26S proteasome. Besides mediating the delivery of ubiquitinated proteins, they are also involved in many other biological processes. Ddi1, the least-studied shuttle protein, exhibits unique physicochemical properties that allow it to play non-canonical functions in the cells. It regulates cell cycle progression and response to proteasome inhibition and defines MAT type of yeast cells. The Ddi1 contains UBL and UBA domains, which are crucial for binding to proteasome receptors and ubiquitin respectively, but also an additional domain called RVP. Additionally, much evidence has been provided to question whether Ddi1 is a classical shuttle protein. For many years, the true nature of this protein remained unclear. Here, we highlight the recent discoveries, which shed new light on the structure and biological functions of the Ddi1 protein.
The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined lengths and connectivity. While in vitro enzymatic assembly using expressed E1-activating and E2-conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48- and K63-biased E2-conjugating enzymes and two new carbamate protecting groups-pyridoxal 5'-phosphate (PLP)-cleavable aminobutanamide carbamate (Abac group) and periodate-cleavable aminobutanol carbamate (Aboc group)-provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers by buffered solutions of PLP or NaIO4. Through the incorporation of a cleavable C-terminal His-tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.
Ubiquitination is a protein post-translational modification that affects protein localisation, stability and interactions. E3 ubiquitin ligases regulate the final step of the ubiquitination reaction by recognising target proteins and mediating the ubiquitin transfer from an E2 enzyme. DTX3L is a multi-domain E3 ubiquitin ligase in which the N-terminus mediates protein oligomerisation, a middle D3 domain mediates the interaction with PARP9, a RING domain responsible for recognising E2 ∼ Ub and a DTC domain has the dual activity of ADP-ribosylating ubiquitin and mediating ubiquitination. The activity of DTX3L is known to be modulated by at least two different factors: the concentration of NAD+, which dictates if the enzyme acts as a ligase or as an ADP-ribosyltransferase, and its binding partners, which affect DTX3L activity through yet unknown mechanisms. In light of recent findings it is possible that DTX3L could ubiquitinate ADP-ribose attached to proteins. Different DTX3L–protein complexes have been found to be part of multiple signalling pathways through which they promote the adhesion, proliferation, migration and chemoresistance of e.g. lymphoma, glioma, melanoma, and prostate cancer. In this review, we have covered the literature available for the molecular functions of DTX3L especially in the context of cancer biology, different pathways it regulates and how these relate to its function as an oncoprotein.
Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.
The multi‐site ubiquitination of Tau protein found in Alzheimer’s disease filaments hints at the failed attempt of neurons to remove early toxic species. The ubiquitin‐dependent degradation of Tau is regulated in vivo by the E3 ligase CHIP, a quality controller of the cell proteome dedicated to target misfolded proteins for degradation. In our study, by using site‐resolved NMR, biochemical and computational methods, we elucidate the structural determinants underlying the molecular recognition between the ligase and its intrinsically disordered substrate. We reveal a multi‐domain dynamic interaction that explains how CHIP can direct ubiquitination of Tau at multiple sites even in the absence of chaperones, including its typical partner Hps70/Hsc70. Our findings thus provide mechanistic insight into the chaperone‐independent engagement of a disordered protein by its E3 ligase.
Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. Here, we present the structure of a chemically trapped complex of the E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3. The structure reveals the basis for acceptor Ub recognition by UBE2K active site residues and the C-terminal Ub-associated (UBA) domain, to impart K48-linked Ub specificity and catalysis. Furthermore, the structure unveils multiple Ub-binding surfaces on the UBA domain that allow distinct binding modes for K48- and K63-linked Ub chains. This multivalent Ub-binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K.
Sphingolipids (SL) are both membrane building blocks and potent signaling molecules regulating a variety of cellular functions in both physiological and pathological conditions. Under normal physiology, sphingolipid levels are tightly regulated, whereas disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiovascular and autoimmune diseases. Yet, mechanisms governing cellular sensing of SL, and according regulation of their biosynthesis remain largely unknown.
In yeast, serine palmitoyltransferase (SPT), catalyzing the first and rate limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orosomucoid 1 and 2 (Orm) proteins. Lowering sphingolipid levels triggers Orms phosphorylation, resulting in the removal of the inhibitory brake on SPT to enhance sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. This study is aimed at filling this knowledge gap.
Here, we identify sphingosine-1-phosphate (S1P) as the key sphingolipid sensed by endothelial cells via S1PRs. The increase of S1P-S1PR signaling stabilizes ORMDLs, which downregulates SPT activity to maintain SL homeostasis. These findings reveal the S1PR/ORMDLs axis as the sensor-effector unit regulating SPT activity accordingly. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, therefore preserving SPT activity at steady state. The disruption of the S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, and impaired signal transduction, all leading to endothelial dysfunction, which is an early event in the onset of cardio- and cerebrovascular diseases.
The disruption of S1P-ORMDL-SPT signaling may be implicated in the pathogenesis of conditions such as diabetes, cancer, cardiometabolic disorders, and neurodegeneration, all characterized by deranged sphingolipid metabolism. Our discovery may provide the molecular basis for a therapeutic intervention to restore sphingolipid homeostasis.
Androgen and estrogen synthesis is necessary for human growth and sexual maturation. Hormone-dependent cancers, such as breast and prostate cancer, however, use these sex steroids to drive cellular proliferation. Cytochrome P450 17A1 (CYP17A1), an essential enzyme for sex steroid synthesis, represents a clinically established drug target. Inhibiting CYP17A1 decreases androgen and estrogen biosynthesis and thereby blocks the growth of hormone-dependent cancers. The first part of this dissertation characterizes the previously unreported interaction between the FDA approved CYP17A1 inhibitor, abiraterone, and the estrogen receptor (ER). We show for the first time that abiraterone is a weak ER agonist in preclinical models of ER-positive breast cancer cells. Abiraterone induces cellular growth and expression of the ER response gene, GREB1, by binding to ER, and these effects are inhibited with the ER antagonist fulvestrant (ICI 182,780). To further investigate the impact of CYP17A1 expression in breast cancer, we engineered ER-positive MCF-7 cells to express CYP17A1 (MCF-7/CYP17A1). Progesterone treatment induces cell growth and GREB1 expression in these cells but not in the parental MCF-7 cells, which do not express CYP17A1. Tandem mass spectrometry (LC-MS/MS) analysis confirmed that following progesterone treatment, MCF-7/CYP17A1 cells synthesize downstream steroid products that require CYP17A1 activity including 17OH-progesterone, androstenedione, and testosterone. Treatment of these cells with either abiraterone or a novel CYP17A1 inhibitor decreases progesterone metabolite-induced GREB1 expression in a dose-dependent manner. In addition to studies of CYP17A1 in breast cancer, we further hypothesized that characterization of CYP17A1 genetic variants may lead to insights on enzyme structure and function. We therefore utilized a HEK-293T cell-based expression system to characterize the enzymatic properties of two CYP17A1 gene variants, D216H (rs200063521) and G162R (rs141821705). Our results show that the D216H variant selectively alters 16OH-progesterone production, while no effect on 17OH-progesterone synthesis was observed. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to wild-type. Proteasome inhibition with MG132 indicated that this variant is preferentially ubiquitinated and degraded prematurely. Overall, these studies have broadened our understanding of CYP17A1 enzymatic activity in breast cancer, as well as led to new insights into how CYP17A1 structure relates to enzyme function and stability.
Excessive bone resorption induced by increased osteoclast activity in postmenopausal women often causes osteoporosis. Although the pharmacological treatment of osteoporosis has been extensively developed, a safer and more effective treatment is still needed. Here, we found that curcumenol (CUL), an anti-oxidant sesquiterpene isolated from Curcuma zedoaria, impaired receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro, whereas the osteoblastogenesis of MC3T3-E1 cells was not affected. We further demonstrated that CUL treatment during RANKL-induced osteoclastogenesis promotes proteasomal degradation of TRAF6 by increasing its K48-linked polyubiquitination, leading to suppression of mitogen-activated protein kinases (MAPKs) and NF-κB pathways, and the production of reactive oxygen species (ROS). We also showed that inositol polyphosphate multikinase (IPMK) binds with TRAF6 to reduce its K48-linked polyubiquitination under RANKL stimulation. Concurrently, IPMK deficiency inhibits osteoclast differentiation. The binding between IPMK and TRAF6 blocked by CUL treatment was found in our study. Finally, we confirmed that CUL treatment prevented ovariectomy (OVX)-induced bone loss in mice. In summary, our study demonstrates that CUL could impair the stability of TRAF6 enhanced by IPMK and suppress excessive osteoclast activity in estrogen-deficient mice to treat osteoporosis. This article is protected by copyright. All rights reserved.
Ubiquibodies (uAbs) are a customizable proteome editing technology that utilizes E3 ubiquitin ligases genetically fused to synthetic binding proteins to steer otherwise stable proteins of interest (POIs) to the proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a posttranslational manner offers a simple yet robust tool for dissecting diverse functional properties of cellular proteins as well as for expanding the druggable proteome to include tumorigenic protein families that have yet-to-be successfully drugged by conventional inhibitors. Here, we describe the engineering of uAbs comprised of a highly modular human E3 ubiquitin ligase, human carboxyl terminus of Hsc70-interacting protein (CHIP), tethered to different designed ankyrin repeat proteins (DARPins) that bind to nonphosphorylated (inactive) and/or doubly phosphorylated (active) forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Two of the resulting uAbs were found to be global ERK degraders, pan-specifically capturing all endogenous ERK1/2 protein forms and redirecting them to the proteasome for degradation in different cell lines, including MCF7 breast cancer cells. Taken together, these results demonstrate how the substrate specificity of an E3 ubiquitin ligase can be reprogrammed to generate designer uAbs against difficult-to-drug targets, enabling a modular platform for remodeling the mammalian proteome.
All prokaryotic and eukaryotic cells in response to stressful insults, such as chemical exposure or hyperthermic treatment upregulate a well conserved set of genes. These genes encode a family of proteins known as stress proteins whose induction is an adaptive response which serves to protect the cells by interacting with denatured or damaged proteins. The aim of the current thesis was to determine the effect of induction of stress proteins on the subsequent toxicity of chemicals in isolated hepatocytes. A further aim was to determine whether the induction of stress proteins following chemical exposure could be used as a sensitive marker of chemical exposure. These studies were performed in two hepatocyte models, hepatocyte monolayers and liver spheroids, and the hepatic effect was also determined in vivo. Hyperthermic treatment of hepatocytes had a bi-phasic effect on the toxicity of subsequent chemical exposure. Exposure of cells to chemicals immediately following hyperthermic treatment resulted in a sensitisation of the cells to the toxin. If the cells were allowed a recovery period prior to chemical exposure the hepatocytes became tolerant to the toxic actions of the chemicals. The reasons for this are thought to be alterations in stress protein and GSH levels. The use of stress proteins as markers was problematic as hepatocyte monolayers had elevated basal stress protein levels resulting in poor induction following chemical exposure. However, liver spheroids had low basal stress protein levels which were significantly induced following chemical exposure to a greater extent than the response observed in vivo. However, in all situations only certain stress proteins were induced following exposure to specific chemicals. Differences in the activation of the transcription factor responsible for stress protein induction (HSF-1) were thought to be responsible for these differences. Therefore, measurement of these components of stress response may not be an all encompassing marker of toxicity but may be of use in determining the toxicity of certain chemicals. However, it does not rule out the possibility that other components of the stress response may be of use as such a marker.
Hematopoiesis is responsible for numerous functions, ranging from oxygen transportation to host defense, to injury repair. This process of hematopoiesis is maintained throughout life by hematopoietic stem cells and requires a controlled balance between self-renewal, differentiation, and quiescence. Disrupting this balance can result in hematopoietic malignancies, including anemia, immune deficiency, leukemia, and lymphoma. Recent work has shown that FBOX E3 ligases, a substrate recognition component of the ubiquitin proteasome system (UPS), have an integral role in maintaining this balance. In this review, we detail how FBOX proteins target specific proteins for degradation to regulate hematopoiesis through cell processes, such as cell cycle, development, and apoptosis.
Ubiquitination is an enzymatic process that involves the covalent attachment of the conserved protein ubiquitin to a substrate. Protein ubiquitination is carried out by the sequential and coordinate actions of three enzymes and can be removed by deubiquitinases. Modification of proteins by addition of ubiquitin exerts both proteolytic‐dependent and proteolytic‐independent functions. Polyubiquitin chains act as a signal for 26S proteasome‐mediated degradation. Ubiquitination plays a central regulatory role in a number of cellular processes such as receptor endocytosis, cell cycle control, transcription, DNA repair, gene silencing and stress response. The ubiquitin proteasome system (UPS) is critical for the proper turnover of both oncoproteins and tumour suppressor molecules. As a result, it is not surprising that aberrations within the UPS often result in a neoplastic phenotype.
Key Concepts
Ubiquitination is an ATP‐dependent and reversible process through which ubiquitin is covalently attached to a polypeptide.
The topology of the polyubiquitin chains dictates the fate of the modified protein.
Deubiquitinating enzymes (DUBs) are proteases that reversely modify proteins by removing ubiquitin.
The RING finger type E3 ligases crucially control cell cycle entry and progression.
The proteasome is a proteolytic ‘machinery’ that digests proteins into short polypeptides.
The ubiquitin proteasome system (UPS) degrades 80–90% of all intracellular proteins.
The UPS regulates the timing and extent of protein turnover.
Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by a mutation in the huntingtin gene (HTT). While mutant HTT is present ubiquitously throughout life, HD onset typically occurs in mid-life, suggesting that aging may play an active role in pathogenesis. Cellular aging is defined as the slow decline in stress resistance and accumulation of damage over time. While different cells and tissues can age at different rates, 9 hallmarks of aging have emerged to better define the cellular aging process. Strikingly, many of the hallmarks of aging are also hallmarks of HD pathology. Models of HD and HD patients possess markers of accelerated aging, and processes that decline during aging also decline at a more rapid rate in HD, further implicating the role of aging in HD pathogenesis. Furthermore, accelerating aging in HD mouse and patient-derived neurons unmasks HD-specific phenotypes, suggesting an active role for the aging process in the onset and progression of HD. Here, we review the overlap between the hallmarks of aging and HD and discuss how aging may contribute to pathogenesis in HD.
Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.
Evolution of macromolecules could be considered as a milestone in the history of life. Nucleic acids are the long stretches of nucleotides that contain all the possible codes and information of life. On the other hand, proteins are their actual translated outcomes, or reflections of modifications in their structure that have occurred at a slow, but steady rate over a very long period of evolution. Over the years of research, biophysicists, biochemists, molecular and structural biologists have unfurled several layers of the structural convolutions in these chemical molecules; however evolutionists look over their structures through a different prism, which may or may not coincide with others. There remains a need to outline several well-known, but less discussed features of protein structures, like intrinsically disordered states, degron signals and different types of ubiquitin chains providing degradation signals, which help the cellular proteolytic machinery to identify and target the proteins towards degradation pathways. There are several important factors, which are critical for folding of proteins into their native three-dimensional conformations by the cytoplasmic chaperones; but in real time how the chaperones fold the newly synthesized polypeptide sequences into a particular three-dimensional shape within a fraction of second is still a mystery for biologists as well as mathematicians. Multiple similar unsolved or unaddressed questions need to be addressed in detail so that future line of research can dig deeper into the finer details of these structures of the proteins.
Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6‐deficient Tregs were dysfunctional in vivo; mice with Treg‐restricted deletion of TRAF6 were resistant to implanted tumors and displayed enhanced anti‐tumor immunity. We further determined that FOXP3 undergoes K63‐linked ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. In the absence of TRAF6 activity or upon mutation of the ubiquitination site, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63‐linked ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene‐regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg‐stabilizing regulator that may be targeted in novel tolerance‐breaking therapies.
Da aktuelle Daten eine reduzierte postoperative Quadrizepsmuskelkraft nach Verwendung einer Blutsperre im Rahmen einer Knie-TEP-Implantation suggerieren, und das klinisch-funktionelle Outcome nach Knie-TEP unmittelbar mit der Masse und Kraft der kniegelenksübergreifenden Muskulatur zusammenhängt, stellte sich die Frage nach den molekularen Effekten der häufig angewandten intraoperativen Blutsperre / Ischämie auf den Proteinstoffwechsel der Skelettmuskulatur, die zum Zeitpunkt der Studienplanung unbekannt waren. Das Ubiquitin-Proteasomen-System (UPS) repräsentiert eine der Hauptstoffwechselwege zur Muskelproteindegradation. Die Hypothese der vorliegenden Studie beinhaltet, dass die Blutsperre im Rahmen der Knie-TEP-Implantation zu Veränderungen im UPS der Skelettmuskelzellen führt, vereinbar mit einer vermehrten Proteindegradation und dadurch folgender Muskelatrophie. Hierfür wurden die Auswirkungen der Blutsperre auf die Konzentration von freiem und konjugiertem Ubiquitin (Ub), die Ubiquitinierungsrate und die Proteasomen-abhängigen und -unabhängigen Peptidaseaktivitäten in Zellen des Musculus vastus medialis analysiert. 30 elektive Knie-TEP Patienten wurden in eine randomisierte, kontrollierte und monozentrische Studie eingeschlossen und mit schriftlicher Einwilligung randomisiert der Gruppe A (OP mit Blutsperre, n = 15) oder der Gruppe B (OP ohne Blutsperre, n = 15) zugeteilt. Muskelbiopsien (5x5x5 mm) wurden aus dem Musculus vastus medialis unmittelbar nach Anlage des chirurgischen Zugangs und exakt 60 min nach Entnahme der ersten Muskelprobe gewonnen und sofort mittels Flüssigstickstoff konserviert. Es erfolgte die Bestimmung der cytosolischen Konzentrationen von freiem und konjugiertem Ub mittels Western-Blot-Analysen. Unter Verwendung einer Enzymkinetik wurde die Ubiquitinierungsrate (Ubiquitin-Protein konjugierende Aktivität) ermittelt. Die Quantifizierung der Proteasomen-abhängigen und -unabhängigen Peptidaseaktivitäten erfolgte mittels Peptidase-Assay. Die Konzentrationen von freiem und konjugiertem Ub sowie die Ubiquitinierungsraten wurden durch die Blutsperre nicht beeinflusst. Dieses Ergebnis stützt die in der Literatur beschriebene These, dass eine strenge Regulation der intrazellulären Ub-Konzentrationen essentiell für den Zellstoffwechsel ist. Die Blutsperre induziert eine signifikante Steigerung der Proteasomen-abhängigen und -unabhängigen Peptidaseaktivitäten, mit möglicher konsekutiver Muskelprotein-degradation und postoperativer Muskelatrophie.
As a virus-encoded actin nucleation promoting factor (NPF), P78/83 induces actin polymerization to assist in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) propagation. According to our previous study, although P78/83 actively undergoes ubiquitin-independent proteasomal degradation, AcMNPV encodes budded virus/occlusion derived virus (BV/ODV)-C42 (C42), which allows P78/83 to function as a stable NPF by inhibiting its degradation during viral infection. However, whether there are other viral proteins involved in regulating P78/83-induced actin polymerization has yet to be determined. In this study, we found that Ac102, an essential viral gene product previously reported to play a key role in mediating the nuclear accumulation of actin during AcMNPV infection, is a novel regulator of P78/83-induced actin polymerization. By characterizing an ac102 knockout bacmid, we demonstrated that Ac102 participates in regulating nuclear actin polymerization as well as the morphogenesis and distribution of capsid structures in the nucleus. These regulatory effects are heavily dependent on an interaction between Ac102 and C42. Further investigation revealed that Ac102 binds to C42 to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable NPF to induce actin polymerization. Thus, Ac102 and C42 form a regulatory cascade to control viral NPF activity, representing a sophisticated mechanism for AcMNPV to orchestrate actin polymerization in both a ubiquitin-dependent and ubiquitin-independent manner.
IMPORTANCE
Actin is one of the most functionally important proteins in eukaryotic cells. Morphologically, actin can be found in two forms: a monomeric form called globular actin (G-actin) and a polymeric form called filamentous actin (F-actin). G-actin can polymerize to form F-actin, and nucleation promoting factor (NPF) is the initiator of this process. Many viral pathogens harness the host actin polymerization machinery to assist in virus propagation. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) induces actin polymerization in host cells. P78/83, a viral NPF, is responsible for this process. Previously, we identified that BV/ODV-C42 (C42) binds to P78/83 and protects it from degradation. In this report, we determined that another viral protein, Ac102, is involved in modulating C42 ubiquitination and, consequently, ensures P78/83 activity as an NPF to initiate actin polymerization. This regulatory cascade represents a novel mechanism by which a virus can harness the cellular actin cytoskeleton to assist in viral propagation.
Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5hours, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.
Sepsis, the systemic inflammatory response syndrome (SIRS) with infection is one of the leading causes of death in critically ill patients in the developed world due to the lack of effective antisepsis treatments. This study examined the efficacy of dietary dipeptide gamma-l-glutamyl-l-valine (γ-EV), which was characterized previously as an anti-inflammatory peptide, in an LPS-induced mouse model of sepsis. BALB/c mice were administered γ-EV via oral gavage followed by an intraperitoneal injection of LPS to induce sepsis. The γ-EV exhibited antisepsis activity by reducing the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in plasma and small intestine. γ-EV also reduced the phosphorylation of the signaling proteins JNK and IκBα. We concluded that γ-EV could possess an antisepsis effect against bacterial infection in intestine. This study proposes a signaling mechanism whereby the calcium-sensing receptor (CaSR) allosterically activated by γ-EV stimulates the interaction of β-arrestin2 with the TIR(TLR/IL-1R) signaling proteins TRAF6, TAB1, and IκBα to suppress inflammatory signaling.
Mammalian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is rapidly degraded in cells, an attribute important to the regulation of its activity. Mutant and chimeric ODCs were created to determine the structural requirements for two modes of proteolysis. Constitutive degradation requires the carboxy terminus and is independent of intracellular polyamines. Truncation of five or more carboxy-terminal amino acids prevents this mode of degradation, as do several internal deletions within the 37 carboxy-most amino acids that spare the last five residues. Polyamine-dependent degradation of ODC requires a distinct region outside the carboxy terminus. The ODC of a parasite, Trypanosoma brucei, is structurally very similar to mouse ODC but lacks the carboxy-terminal domain; it is not a substrate for either pathway. The regulatory properties of enzymatically active chimeric proteins incorporating regions of the two ODCs support the conclusion that distinct domains of mouse ODC confer constitutive degradation and polyamine-mediated regulation. Mouse ODC contains two PEST regions. The first was not required for either form of degradation; major deletions within the second ablated constitutive degradation. When mouse and T. brucei ODC RNAs were translated in vitro in a reticulocyte lysate system, the effects of polyamine concentration on ODC protein production and activity were similar for the two mRNAs, which contradicts claims that this system accurately reflects the in vivo effects of polyamines on responsive ODCs.
Red blood cell-mediated microinjection was used to introduce radioiodinated ubiquitin into ts85 cells, a mouse cell line that contains a thermolabile ubiquitin-activating enzyme (E1). The proportion of ubiquitin present as histone conjugates, high molecular weight conjugates, and free molecules was then determined by gel electrophoresis and autoradiography. When ts85 cells were incubated at the nonpermissive temperature, 39.5 degrees C, high molecular weight conjugates accumulated. This unexpected result was confirmed by Western blot analyses. To determine whether ubiquitin conjugates formed under nonpermissive conditions or merely persisted after the temperature increase, ts85 cells were incubated at 39.5 degrees C to generate large amounts of conjugates and then shifted to 42 degrees C. The higher temperature resulted in a 25% reduction in conjugates, but upon return to 39.5 degrees C, the ubiquitin conjugates were restored to pre-42 degrees C amounts. Since all changes in ubiquitin conjugate levels occurred above 39.5 degrees C, ts85 cells can couple ubiquitin to cellular proteins even after prolonged culture at nonpermissive temperatures. Western blot analyses showed that less than 10% of the E1 molecules present in ts85 cells at 31 degrees C remained after 2 h at 39.5 degrees C. However, when 125I-ubiquitin was added to extracts from heated ts85 cells an apparent high molecular weight form of E1 and thiol ester adducts between ubiquitin and the E2 carrier proteins were detected by electrophoresis at 4 degrees C. Considering both in vivo and in vitro demonstrations that heated ts85 cells retain the ability to conjugate ubiquitin to endogenous proteins, considerable caution must be exercised in the design and interpretation of proteolysis experiments using this mutant cell line.
Only monoubiquitinated (and to a much lesser extent diubiquitinated) 125I-alpha globin is observed as a conjugated intermediate in the ATP- and ubiquitin (Ub)-dependent proteolysis of 125I-alpha globin catalyzed by an unfractionated reticulocyte lysate (Shaeffer, J.R. (1994) J. Biol. Chem. 269, 22205-22210). A monoubiquitinated 125I-alpha globin (Ub1-alpha) fraction was isolated and incubated with a dilute acid reagent to selectively cleave the 125I-alpha globin moiety between residues 94 (Asp) and 95 (Pro). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the resulting polypeptides showed that the Ub1-alpha conjugate consisted of a mixture of molecules in which 57% had Ub attached to the amino-terminal two-thirds and 43% had Ub attached to the carboxyl-terminal one-third of the 125I-alpha globin monomer. This amino- to carboxyl-terminal region ubiquitination ratio was essentially unchanged when a Ub1-alpha conjugate intermediate, pulse-labeled for 15 min in the presence of ubiquitin aldehyde to inhibit disassembly, was analyzed after (a) a 2-h chase incubation with excess nonradioactive alpha globin or (b) isolation and incubation separately as a proteolysis substrate. Similar analysis of the diubiquitinated 125I-alpha globin (Ub2-alpha) conjugate molecules showed that, after pulse labeling, 58% were diubiquitinated within the amino-terminal two-thirds and 42% were monoubiquitinated within both amino- and carboxyl-terminal regions of the 125I-alpha globin moiety (a small amount of molecules diubiquitinated within the carboxyl-terminal region may also be present) and that the relative amounts of these molecular types changed little, if any, during the chase incubation. This invariance in the amino- to carboxyl-terminal region ubiquitination ratio during degradation of the Ub1-alpha and Ub2-alpha conjugates suggests that 125I-alpha globin molecules ubiquitinated within either the amino- or carboxyl-terminal regions were both intermediates in the proteolysis of the unconjugated substrate. This heterogeneous pattern of Ub conjugation may also occur during the proteolysis of other long-lived intracellular proteins.
The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination. Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor. We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity. Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct. Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F. Sprague, Jr., 1993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J. Rohrer, F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor. In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions. Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis. Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system. In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cell. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis. To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.
The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.
Ornithine decarboxylase (ODC) was converted from a protein with a short intracellular half-life in mammalian cells to a stable protein by truncating 37 residues at its carboxyl terminus. Cells expressing wild-type protein lost ODC activity with a half-life of approximately 1 hour. Cells expressing the truncated protein, however, retained full activity for at least 4 hours. Pulse-chase experiments in which immunoprecipitation and gel electrophoresis were used confirmed the stabilizing effect of the truncation. Thus, a carboxyl-terminal domain is responsible for the rapid intracellular degradation of murine ODC.
The covalent attachment of ubiquitin (Ub) to short-lived or damaged proteins is believed to be the signal that initiates their
selective degradation. In several cases, it has been shown that the proteolytic signal takes the form of a multi-Ub chain
in which successive Ub molecules are linked tandemly at lysine 48 (K-48). Here we show that Ub molecules can be linked together
in vivo at two other lysine positions, lysine 29 (K-29) and lysine 63 (K-63). The formation of these alternative linkages
is strongly dependent on the presence of the stress-related Ub conjugating enzymes UBC4 and UBC5. Furthermore, expression
of Ub carrying a K-63 to arginine 63 substitution in a strain of Saccharomyces cerevisiae that is missing the poly-Ub gene,
UBI4, fails to compensate for the stress defects associated with these cells. Taken together, these results suggest that the
formation of multi-Ub chains involving K-63 linkages plays an important role in the yeast stress response. In broader terms,
these results also suggest that Ub is a versatile signal in which different Ub chain configurations are used for different
functions.
S5a is a subunit of the 26S protease that binds and presumably selects multiubiquitinated proteins for destruction. We recently identified an Arabidopsis protein, MBP1, that is physically, immunologically and biochemically similar to S5a from the human erythrocyte 26S protease. Based upon the MBP1 cDNA sequence we have now isolated a HeLa cell cDNA coding for human S5a. The HeLa cDNA sequence is highly similar to MBP1 and it encodes peptides obtained directly from human erythrocyte S5a. Moreover, expression of the isolated cDNA in E. coli results in a recombinant protein with an apparent molecular mass and multiubiquitin binding properties that match those of human S5a obtained from the purified 26S enzyme.
In 1986, we proposed that polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction. For much of the past decade there were only sporadic experimental tests of the hypothesis. This situation changed markedly during the past two years with a number of papers providing strong evidence that PEST regions do, in fact, serve as proteolytic signals. Here, we briefly review the properties of PEST regions and some interesting examples of the conditional nature of such signals. Most of the article, however, focuses on recent experimental support for the hypothesis and on mechanisms responsible for the rapid degradation of proteins that contain PEST regions.
Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopysuppressors of ninl-1 Inhibition of ubiquitin-mediated proteoly-sis by the Arabidopsis 26S protease subunit S5a
- K Kominami
- N Okura
- M Kawamura
- G N Demartino
- C A Slaughter
- N Shimbara
- C H Chung
- M Fujimuro
- H Ye-Kosawa
- V Shimizu
- N Tanahashi
- K Tanaka
- A Toh-E
Kominami, K, Okura, N., Kawamura, M., DeMartino, G. N., Slaughter, C. A., Shimbara, N., Chung, C. H., Fujimuro, M., Ye-kosawa, H., Shimizu, V., Tanahashi, N, Tanaka, K, and Toh-e, A. (1997) Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopysuppressors of ninl-1. Mol. Biol. Cell8, 171-187 61. Deveraux, Q., van Nocker, S., Mahaffey, D., Vierstra, R., and Re-chsteiner, M. (1995) Inhibition of ubiquitin-mediated proteoly-sis by the Arabidopsis 26S protease subunit S5a.j Biol. Chem. 270, 29660-29663
The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein deg-radation in vivo Ninip, a regulatorysubunit of the 26S proteasome, isnecessary foractivation of Cdc28-kinase of Saccharomyces cerevisiae
- D J Demarini
- F R Papa
- S Swaminathan
- D Ursic
- T P Rasmus-Sen
- M R Culbertson
- M Hochstrasser
DeMarini, D.J., Papa, F. R., Swaminathan, S., Ursic, D., Rasmus-sen, T. P., Culbertson, M. R., and Hochstrasser, M. (1995) The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein deg-radation in vivo. Mol. Cell. Biol. 15, 6311-6321 63. Kominami, K., DeMartino, B. N., Moomaw, C. R., Slaughter, C. A., Shimbara, N., Fujimuro, M., Yokosawa, H., Hisamatsu, H., Tanahashi, N., Shimizu, Y., Tanaka, K., and Toh-e, A. (1995) Ninip, a regulatorysubunit of the 26S proteasome, isnecessary foractivation of Cdc28-kinase of Saccharomyces cerevisiae. EMBOJ. 14, 3105-3115
The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants) NPII, an essential yeast gene involved in induced degradation of Gapi and Fur4 permeases, encodes the RspS ubiquitin-protein ligase
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