Catalytic Assembly of the Mitotic Checkpoint Inhibitor BubR1-Cdc20 by a Mad2-Induced Functional Switch in Cdc20 (original) (raw)
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Checkpoint Signalling: Mad2 Conformers and Signal Propagation
Current Biology, 2005
The spindle checkpoint is one of the key self-monitoring systems of the eukaryotic cell cycle, acting to delay anaphase until all the sister chromatids are appropriately lined up so that the replicated genome is correctly segregated with one copy going to each daughter cell. Mad2 is a central player in the spindle checkpoint's regulation of anaphase onset [1,2]. Mad2 interacts with Mad1, which recruits it to unattached kinetochores [3], and with the spindle checkpoint effector Cdc20 [4,5]. The latter interaction can take place either in a Mad2-Cdc20 complex or in the mitotic checkpoint complex (MCC):Mad2-Mad3/BubR1-Bub3-Cdc20 [6,7]. There is much controversy over the site(s) of assembly of these complexes, their mode of action and their importance as in vivo 'anaphase inhibitors'. Mad2 multimers have been studied for some time, although their physiological relevance was uncertain [8,9]. NMR and crystal structures of Mad2 alone or with binding peptides from Mad1 or Cdc20 have shown how the respective complexes are formed [10-13]. A 'safety-belt' structure formed by the carboxy-terminal tail of Mad2 explains why they are so stable [13].
BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex
Journal of Biological Chemistry, 2011
The mitotic checkpoint maintains genomic stability by ensuring that chromosomes are accurately segregated during mitosis. When the checkpoint is activated, the mitotic checkpoint complex (MCC), assembled from BUBR1, BUB3, CDC20, and MAD2, directly binds and inhibits the anaphase-promoting complex/cyclosome (APC/C) until all chromosomes are properly attached and aligned. The mechanisms underlying MCC assembly and MCC-APC/C interaction are not well characterized. Here, we show that a novel interaction between BUBR1 and closed MAD2 (C-MAD2) is essential for MCC-mediated inhibition of APC/C. Intriguingly, Arg 133 and Gln 134 in C-MAD2 are required for BUBR1 interaction. The same residues are also critical for MAD2 dimerization and MAD2 binding to p31 comet , a mitotic checkpoint silencing protein. Along with previously characterized BUBR1-CDC20 and C-MAD2-CDC20 interactions, our results underscore the integrity of the MCC for its activity and suggest the fundamental importance of the MAD2 ␣C helix in modulating mitotic checkpoint activation and silencing.
The Mad1/Mad2 Complex as a Template for Mad2 Activation in the Spindle Assembly Checkpoint
Current Biology, 2005
The SAC monitors this process and delays anaphase until all chromosomes have attained bipolar attachment [1,. Spindle microtubules attach on kinetochores, prothe tension between sister chromatids building up during this process [1, 2]. European Institute of Oncology Via Ripamonti 435 The SAC is conserved in all eukaryotes and includes mitotic arrest deficient (MAD) and budding uninhibited 20141 Milano Italy by benzimidazole (BUB) genes [1, 2]. Their products temporarily sequester Cdc20, an activator of the ana-Hill phase-promoting complex/cyclosome, the E3 ubiquitin ligase targeting securin and cyclin B for proteasome-607 Fordham Hall Chapel Hill, North Carolina 27599 mediated degradation. Destruction of securin activates separase, which triggers anaphase by cleaving the complex linking the sister chromatids, named Cohesin [4, 5]. The sequestration of Cdc20 requires Mad2 and Summary BubR1 [1, 6]. Mad2 is a 002ف residue protein containing a Horma domain [7]. BubR1 consist of an N-terminal Background: The spindle assembly checkpoint (SAC) domain containing Bub3 and Cdc20 binding sites and a imparts fidelity to chromosome segregation by delaying C-terminal kinase domain (missing in the budding yeast anaphase until all sister chromatid pairs have become ortholog, Mad3) [1, 6]. Both Mad2 and BubR1 bind bipolarly attached. Mad2 is a component of the SAC Cdc20 tightly, and their effects are synergic [8-13]. Coneffector complex that sequesters Cdc20 to halt anasistently, Mad2, BubR1 (or Mad3), Bub3, and Cdc20 phase. In prometaphase, Mad2 is recruited to kinetoenter a single complex known as mitotic checkpoint chores with the help of Mad1, and it is activated to bind complex (MCC) [11, 14-16]. Cdc20. These events are linked to the existence of two Unattached kinetochores establish and maintain the distinct conformers of Mad2: a closed conformer bound SAC, and all SAC proteins show kinetochore localization to its kinetochore receptor Mad1 or its target in the in prometaphase [1, 3, 17]. Fluorescence recovery after checkpoint Cdc20 and an open conformer unbound to photobleaching (FRAP) revealed stable kinetochore resthese ligands.
The Mad2 spindle checkpoint protein has two distinct natively folded states
Nature Structural & Molecular Biology, 2004
The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/C Cdc20 . We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.
The Mad2 spindle checkpoint protein has two distinct natively folded statesNSMB
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2004
The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/CCdc20. We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.
Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint
PLoS ONE, 2007
Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C Cdc20 is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C Cdc20 inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.
EMBO reports, 2014
The spindle assembly checkpoint inhibits anaphase until all chromosomes have become attached to the mitotic spindle. A complex between the checkpoint proteins Mad1 and Mad2 provides a platform for Mad2:Mad2 dimerization at unattached kinetochores, which enables Mad2 to delay anaphase. Here, we show that mutations in Bub1 and within the Mad1 C-terminal domain impair the kinetochore localization of Mad1:Mad2 and abrogate checkpoint activity. Artificial kinetochore recruitment of Mad1 in these mutants co-recruits Mad2; however, the checkpoint remains non-functional. We identify specific mutations within the C-terminal head of Mad1 that impair checkpoint activity without affecting the kinetochore localization of Bub1, Mad1 or Mad2. Hence, Mad1 potentially in conjunction with Bub1 has a crucial role in checkpoint signalling in addition to presenting Mad2.
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An overview of the spindle assembly checkpoint status in oral cancer
BioMed research international, 2014
Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the cor...
Basis of catalytic assembly of the mitotic checkpoint complex
Nature, 2017
The crystal structure of MCC (Extended data Figure 1b) shows that each of the three interacting subunits (CDC20, MAD2, and BUBR1) has binding interfaces for the other two. One can therefore identify three different dissociation constants, one for each binary interaction, i.e. MAD2:CDC20, MAD2:BUBR1, and CDC20:BUBR1. We designate these dissociation constants as K d (MC), K d (MB), and K d (CB). K d (MC) is 150 nM (see Figure 1c). At the concentrations of MAD2 and BUBR1 used in our assays, we did not observe binding of MAD2 with BUBR1 in the absence of CDC20 (Extended data Figure 2d), indicating that K d (MB) is high (i.e. the affinity is
Chemical Structure-Biological Activity Models for Pharmacophores’ 3D-Interactions
International Journal of Molecular Sciences, 2016
Within medicinal chemistry nowadays, the so-called pharmaco-dynamics seeks for qualitative (for understanding) and quantitative (for predicting) mechanisms/models by which given chemical structure or series of congeners actively act on biological sites either by focused interaction/therapy or by diffuse/hazardous influence. To this aim, the present review exposes three of the fertile directions in approaching the biological activity by chemical structural causes: the special computing trace of the algebraic structure-activity relationship (SPECTRAL-SAR) offering the full analytical counterpart for multi-variate computational regression, the minimal topological difference (MTD) as the revived precursor for comparative molecular field analyses (CoMFA) and comparative molecular similarity indices analysis (CoMSIA); all of these methods and algorithms were presented, discussed and exemplified on relevant chemical medicinal systems as proton pump inhibitors belonging to the 4-indolyl,2-guanidinothiazole class of derivatives blocking the acid secretion from parietal cells in the stomach, the 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine congeners' (HEPT ligands) antiviral activity against Human Immunodeficiency Virus of first type (HIV-1) and new pharmacophores in treating severe genetic disorders (like depression and psychosis), respectively, all involving 3D pharmacophore interactions.
Genes & Nutrition, 2015
Celiac disease is an intestinal disease which shows different symptoms and clinical manifestations among pediatric and adult patients. These variations could be imputable to age-related changes in gut architecture and intestinal immune system, which could be characterized by gene expression differences possibly regulated by miR-NAs. We analyzed a panel of miRNAs and their target genes in duodenal biopsies of Marsh 3AB and 3C pediatric celiac patients, compared to controls. Moreover, to assess variation of expression in plasma samples, we evaluated circulating miRNA levels in controls and patients at diagnosis or on gluten-free diet. We detected a decreased miR-192-5p expression in celiac patients, but no variations in NOD2 and CXCL2, targets previously identified in adults. Conversely, we detected a significant increase in mRNA and protein levels of another target, MAD2L1, protein related to cell cycle control. miR-31-5p and miR-338-3p were down-regulated and their respective targets, FOXP3 and RUNX1, involved in Treg function, resulted up-regulated in celiac patients. Finally, we detected, in celiac patients, an increased expression of miR-21-5p, possibly caused by a regulatory loop with its putative target STAT3, which showed an increased activation in Marsh 3C patients. The analysis of plasma revealed a trend similar to that observed in biopsies, but in presence of gluten-free diet we could not detect circulating miRNAs values comparable to controls. miRNAs and their gene targets showed an altered expression in duodenal mucosa and plasma of celiac disease pediatric patients, and these alterations could be different from adult ones.
A dual molecular analogue tuner for dissecting protein function in mammalian cells
Nature communications, 2016
Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and particularly in stem cells, are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways, and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid, reversible, titratable and independent manner. By engineering molecular tuners for NANOG, CHK1, p53 and NOTCH1 in mammalian stem cells, we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes.
A Mathematical Framework for Kinetochore-Driven Activation Feedback in the Mitotic Checkpoint
Bulletin of Mathematical Biology, 2017
Proliferating cells properly divide into their daughter cells through a process that is mediated by kinetochores, protein-complexes that assemble at the centromere of each sister chromatid. Each kinetochore has to establish a tight bipolar attachment to the spindle apparatus before sister-chromatid separation is initiated. The Spindle Assembly Checkpoint (SAC) links the biophysical attachment status of the kinetochores to mitotic progression, and ensures that even a single misaligned kinetochore keeps the checkpoint active. The mechanism by which this is achieved is still elusive. Current computational models of the human SAC disregard important biochemical properties by omitting any kind of feedback loop, proper kinetochore signals, and other spatial properties such as the stability of the system and diffusion effects. To allow for more realistic in silico study of the dynamics of the SAC model, a minimal mathematical framework for SAC activation and silencing is introduced. A nonlinear ordinary differential equation model successfully reproduces bifurcation signaling switches with attachment of all 92 kinetochores and activation of APC/C by kinetochore-driven feedback. A partial differential equation model and mathematical linear stability analyses indicate the influence of diffusion and system stability. The conclusion is that quantitative models of the human SAC should account for the positive feedback on APC/C activation driven by the kinetochores which is essential for SAC silencing. Experimental diffusion coefficients for MCC sub-complexes are found to be insufficient for rapid APC/C inhibition. The presented analysis allows for systems-level understanding of mitotic control and the minimal new model can function as a basis for developing further quantitative-integrative models of the cell division cycle.
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Journal of Biological Chemistry, 2008
Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.
Biophysical Chemistry, 2008
For successful mitosis, metaphase has to be arrested until all centromeres are properly attached. The onset of anaphase, which is initiated by activating the APC, is controlled by the spindle assembly checkpoint M SAC. Mad2, which is a constitutive member of the M SAC, is supposed to inhibit the activity of the APC by sequestering away its co-activator Cdc20. Mad1 recruits Mad2 to unattached kinetochores and is compulsory for the establishment of the Mad2 and Cdc20 complexes. Recently, based on results from in vivo and in vitro studies, two biochemical models were proposed: the Template and the Exchange model. Here, we derive a mathematical description to compare the dynamical behaviour of the two models. Our simulation analysis supports the Template model. Using experimentally determined values for the model parameters, the Cdc20 concentration is reduced down to only about half. Thus, although the Template model displays good metaphase-to-anaphase switching behaviour, it is not able to completely describe M SAC regulation. This situation is neither improved by amplification nor by p31 comet inhibition. We speculate that either additional reaction partners are required for total inhibition of Cdc20 or an extended mechanism has to be introduced for M SAC regulation.
Structure of the Mad2 spindle assembly checkpoint protein and its interaction with Cdc20
Nature structural biology, 2000
The checkpoint protein Mad2 inhibits the activity of the anaphase promoting complex by sequestering Cdc20 until all chromosomes are aligned at the metaphase plate. We report the solution structure of human Mad2 and its interaction with Cdc20. Mad2 possesses a novel three-layered alpha/beta fold with three alpha-helices packed between two beta-sheets. Using deletion mutants we identified the minimal Mad2-binding region of human Cdc20 as a 40-residue segment immediately N-terminal to the WD40 repeats. Mutagenesis and NMR titration experiments show that a C-terminal flexible region of Mad2 is required for binding to Cdc20. Mad2 and Cdc20 form a tight 1:1 heterodimeric complex in which the C-terminal segment of Mad2 becomes folded. These results provide the first structural insight into mechanisms of the spindle assembly checkpoint.
The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex
Nature Communications, 2023
The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly. During mitosis, a eukaryotic cell divides into two genetically identical daughter cells. To achieve this, the duplicated chromosomes in the parent cell must be equally distributed into the daughter cells. The spindle assembly checkpoint (SAC) serves as a surveillance mechanism to ensure that duplicated chromosomes are stably attached to spindle microtubules through an adapter structure named the kinetochore. Kinetochores lacking end-on microtubule attachment activate the SAC to prevent premature anaphase onset and avoid chromosome missegregation. The effector molecule generated upon SAC activation is the Mitotic Checkpoint Complex (MCC). The MCC consists of two subcomplexes: BUBR1:BUB3 and CDC20:MAD2 1,2. It inhibits the E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) 3-5. APC/C ubiquitinates Cyclin B1, a key mitosis regulator, thereby targeting it for proteasome-mediated degradation 6-8. Inhibition of the APC/C suppresses the degradation of Cyclin B1, which in turn delays anaphase onset. The formation of the CDC20:MAD2 complex has been identified as the rate-limiting step in the assembly of the MCC 9,10. Other checkpoint proteins, including the MAD1:MAD2 complex and the BUB1:BUB3 complex, catalyze this reaction, by recruiting the MCC subunits at
F1000 - Post-publication peer review of the biomedical literature, 2000
Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules emanating from opposite spindle poles. The spindle assembly checkpoint is a conserved surveillance mechanism in eukaryotes that inhibits anaphase onset until all chromosomes are bioriented1, 2, 3. In current models, the recruitment of Mad2, via Mad1, to improperly attached kinetochores is a key step needed to stop cell cycle progression3, 4, 5, 6. However, it is not known if the localization of Mad1-Mad2 to kinetochores is sufficient to block anaphase. Furthermore, it is unclear if other signalling proteins (e.g. Aurora kinases7) that regulate chromosome biorientation have checkpoint functions downstream of Mad1-Mad2 recruitment to kinetochores or if they act upstream to merely quench the primary error signal8. Here, to address both these issues, we engineered a Mad1 construct which, unlike endogenous Mad1, localizes to kinetochores that are bioriented. We show that Mad1's constitutive localization at kinetochores is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora kinase, Mps1 and BubR1, but not Polo-like kinase, are needed to maintain the checkpoint arrest even when Mad1 is present on bioriented kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2's templated activation dynamics, but also through the activity of widely-conserved kinases, to ensure the fidelity of cell division. RESULTS The spindle assembly checkpoint, which can block anaphase when even a single chromosome is improperly attached to spindle microtubules, depends on Mad1 and Mad2 (ref. 3). In current models of checkpoint signalling, a key step is the recruitment of Mad1 and Mad2 to kinetochores that lack proper microtubule attachments. Mad1 forms a homodimer that binds two Mad2 molecules9, forming a "core tetramer", which "templates" the conversion of cytosolic Mad2 from an inactive "open" conformation to a "closed" form10 (Fig. 1a). A diffusible cytosolic complex, which includes closed-Mad2, blocks anaphase progression by inhibiting the activation of APC/C, the E3 ubiquitin ligase required Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
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