Journal articles on the topic 'APC/C-Cdh1'
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Qiao, Xinxian, Liyong Zhang, Armin M. Gamper, Takeo Fujita, and Yong Wan. "APC/C-Cdh1." Cell Cycle 9, no. 19 (October 2010): 3904–12. http://dx.doi.org/10.4161/cc.9.19.13585.
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Höckner, Sebastian, Lea Neumann-Arnold, and Wolfgang Seufert. "Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1." Molecular Biology of the Cell 27, no. 14 (July 15, 2016): 2198–212. http://dx.doi.org/10.1091/mbc.e15-11-0787.
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The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.
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Listovsky, Tamar, and Julian E. Sale. "Sequestration of CDH1 by MAD2L2 prevents premature APC/C activation prior to anaphase onset." Journal of Cell Biology 203, no. 1 (October 7, 2013): 87–100. http://dx.doi.org/10.1083/jcb.201302060.
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The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.
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Arnold, Lea, Sebastian Höckner, and Wolfgang Seufert. "Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons." Molecular Biology of the Cell 26, no. 5 (March 2015): 843–58. http://dx.doi.org/10.1091/mbc.e14-09-1342.
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In vivo analysis in budding yeast identifies APC/C-Cdh1–specific minimal degrons carrying either a D or a KEN box and a nuclear localization sequence. APC/C-Cdh1 activity is restricted to the nucleus, maximal in the nucleoplasm, and absent from the cytoplasm, allowing for spatiotemporal control of Cdh1 substrate proteolysis.
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Martinez, Juan S., Dah-Eun Jeong, Eunyoung Choi, Brian M. Billings, and Mark C. Hall. "Acm1 Is a Negative Regulator of the Cdh1-Dependent Anaphase-Promoting Complex/Cyclosome in Budding Yeast." Molecular and Cellular Biology 26, no. 24 (October 9, 2006): 9162–76. http://dx.doi.org/10.1128/mcb.00603-06.
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ABSTRACT Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/C Cdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Δ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Δ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Δ and hsl1Δ resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.
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Bhattacharjee, Debanjan, Sreeram Kaveti, and Nishant Jain. "APC/C CDH1 ubiquitinates STAT3 in mitosis." International Journal of Biochemistry & Cell Biology 154 (January 2023): 106333. http://dx.doi.org/10.1016/j.biocel.2022.106333.
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Li, Min, J. Philippe York, and Pumin Zhang. "Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos." Molecular and Cellular Biology 27, no. 9 (February 26, 2007): 3481–88. http://dx.doi.org/10.1128/mcb.02088-06.
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ABSTRACT The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.
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Krohs, Julika, Dominik Schnerch, Marie Follo, Julia Felthaus, Monika Engelhardt, and Ralph M. Waesch. "The Tumor Suppressor APC/CCdh1 and Its Role In Replication Stress and The Origin Of Genomic Instability." Blood 122, no. 21 (November 15, 2013): 2489. http://dx.doi.org/10.1182/blood.v122.21.2489.2489.
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Abstract Introduction We have previously proposed that Cdh1 is a tumor suppressor by maintaining genomic stability. We also found Cdh1 downregulated in several tumor cell lines including AML (Oncogene 2008; 27:907-17). Heterozygous Cdh1 knockout mice develop epithelial tumors, myelodysplasia and plasma cell dyscrasias (Nat. Cell Biol. 2008;10:802-11). By analyzing primary AML samples from bone marrow (BM) or peripheral blood (PB) we detected downregulation of Cdh1 in the vast majority of samples when compared to normal CD34+ HSCs. Progression through the cell cycle is tightly regulated by different cyclin-dependent kinases (Cdks) and their activating cyclin subunits. Stage-specific proteolysis of cyclins and other cell cycle regulators is important for transition to the next cell cycle phase. The anaphase-promoting complex/cyclosome (APC/C) is an E3-ubiquitin ligase that controls mitosis and G1 through degradation of these proteins. Through its activating subunits Cdh1 and Cdc20 the APC/C ensures substrate-specifity. While Cdc20 regulates progression through mitosis, Cdh1 is activated in late mitosis to coordinate accurate entry into S-phase. Thereby, the APC/C is crucial for maintaining genomic stability during the cell cycle. Suppression of APC/C-Cdh1 can lead to unscheduled cyclin expression and Cdk activity, which can cause cell cycle defects leading to the accumulation of DNA alterations and further to malignant transformations. However, the exact nature of the origin of genomic instability upon downregulation of Cdh1 is unclear. Methods To investigate stability of cyclins in Cdh1-knockdown (kd) cells, origin loading and start of replication, cells were released from a mitotic block and samples were taken every 2 h until S-phase entry for FACS and immunoblotting. For live-cell imaging cells were seeded 24 h before imaging in chambered coverslips, after which progression through the cell cycle was analyzed by automated microscopy. Results Characterization of a Cdh1-kd revealed strong stabilization of the substrates cyclin A/B leading to diminished loading of mini-chromosome maintenance (MCM) proteins on replication origins in G1. Stabilization of cyclin A/B and unscheduled Cdk1/2 activity may cause the observed premature entry into S-phase, while the reduced loading of MCMs in G1 could be responsible for the prolonged replication in S-phase seen in Cdh1-kd cells. Accordingly, treatment with the Cdk1 inhibitor RO-3306 restored reduced MCM loading. Polo-like kinase 1 (Plk1) was stabilized in Cdh1-kd cells, which may cause bypass of the Cdc14B-Cdh1-Plk1 dependent DNA damage checkpoint. Indeed, potential replication stress in Cdh1-kd cells did not lead to G2/M arrest, but was enforced by inhibition of the Cdh1 substrate Plk1. Underreplicated DNA and replication intermediates in mitosis may be the reason for increased genomic instability, namely lagging chromosomes, anaphase bridges and micronuclei in Cdh1-kd cells detected by live-cell imaging. In addition, aberrant cytokinesis and the development of polyploid cells generated by misseparation of chromosomes during mitosis were enhanced in Cdh1-kd cells. Finally, monitoring of 53BP1, a DNA-repair marker, in living cells showed amplified DNA-damage through increased double-strand breaks in Cdh1-kd cells. Conclusions Downregulation of the tumor suppressor APC/C-Cdh1 leads to deregulation of DNA-replication by stabilizing cyclin A and B in G1 and reduced loading of replication origins with MCM proteins resulting in the accumulation of enhanced genomic instability and DNA damage. Disclosures: No relevant conflicts of interest to declare.
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Dohrn, Maike F., and Juan P. Bolaños. "Does APC/C CDH1 control the human brain size?" Journal of Neurochemistry 151, no. 1 (August 23, 2019): 8–10. http://dx.doi.org/10.1111/jnc.14835.
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Ahlskog, Johanna K., Johanna K. Björk, Alexandra N. Elsing, Camilla Aspelin, Marko Kallio, Pia Roos-Mattjus, and Lea Sistonen. "Anaphase-Promoting Complex/Cyclosome Participates in the Acute Response to Protein-Damaging Stress." Molecular and Cellular Biology 30, no. 24 (October 11, 2010): 5608–20. http://dx.doi.org/10.1128/mcb.01506-09.
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ABSTRACT The ubiquitin E3 ligase anaphase-promoting complex/cyclosome (APC/C) drives degradation of cell cycle regulators in cycling cells by associating with the coactivators Cdc20 and Cdh1. Although a plethora of APC/C substrates have been identified, only a few transcriptional regulators are described as direct targets of APC/C-dependent ubiquitination. Here we show that APC/C, through substrate recognition by both Cdc20 and Cdh1, mediates ubiquitination and degradation of heat shock factor 2 (HSF2), a transcription factor that binds to the Hsp70 promoter. The interaction between HSF2 and the APC/C subunit Cdc27 and coactivator Cdc20 is enhanced by moderate heat stress, and the degradation of HSF2 is induced during the acute phase of the heat shock response, leading to clearance of HSF2 from the Hsp70 promoter. Remarkably, Cdc20 and the proteasome 20S core α2 subunit are recruited to the Hsp70 promoter in a heat shock-inducible manner. Moreover, the heat shock-induced expression of Hsp70 is increased when Cdc20 is silenced by a specific small interfering RNA (siRNA). Our results provide the first evidence for participation of APC/C in the acute response to protein-damaging stress.
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Nagai, Masayoshi, Atsuko Shibata, and Takashi Ushimaru. "Cdh1 degradation is mediated by APC/C–Cdh1 and SCF–Cdc4 in budding yeast." Biochemical and Biophysical Research Communications 506, no. 4 (December 2018): 932–38. http://dx.doi.org/10.1016/j.bbrc.2018.10.179.
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Ke, Po-Yuan, and Zee-Fen Chang. "Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Pathway." Molecular and Cellular Biology 24, no. 2 (January 15, 2004): 514–26. http://dx.doi.org/10.1128/mcb.24.2.514-526.2004.
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ABSTRACT The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G1 phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G1 phase of the cell cycle in mammalian cells.
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Park, Hyun Jung, Robert H. Costa, Lester F. Lau, Angela L. Tyner, and Pradip Raychaudhuri. "Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Proteolysis of the Forkhead Box M1 Transcription Factor Is Critical for Regulated Entry into S Phase." Molecular and Cellular Biology 28, no. 17 (June 23, 2008): 5162–71. http://dx.doi.org/10.1128/mcb.00387-08.
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ABSTRACT The forkhead box M1 (FoxM1) transcription factor is overexpressed in many cancers, and in mouse models it is required for tumor progression. FoxM1 activates expression of the cell cycle genes required for both S and M phase progression. Here we demonstrate that FoxM1 is degraded in late mitosis and early G1 phase by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. FoxM1 interacts with the APC/C complex and its adaptor, Cdh1. Expression of Cdh1 stimulated degradation of the FoxM1 protein, and depletion of Cdh1 resulted in stabilization of the FoxM1 protein in late mitosis and in early G1 phase of the cell cycle. Cdh1 has been implicated in regulating S phase entry. We show that codepletion of FoxM1 inhibits early S phase entry observed in Cdh1-depleted cells. The N-terminal region of FoxM1 contains both destruction box (D box) and KEN box sequences that are required for targeting by Cdh1. Mutation of either the D box sequence or the KEN box sequence stabilized FoxM1 and blocked Cdh1-induced proteolysis. Cells expressing a nondegradable form of FoxM1 entered S phase rapidly following release from M phase arrest. Together, our observations show that FoxM1 is one of the targets of Cdh1 in late M or early G1 phase and that its proteolysis is important for regulated entry into S phase.
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Schmidts, Andrea, Dagmar Wider, Julia Felthaus, Manuel Hein, Dominik Schnerch, Monika Engelhardt, and Ralph M. Waesch. "APC/C-Cdh1 as a Novel Regulator of Hematopoietic Stem Cell Differentiation." Blood 116, no. 21 (November 19, 2010): 1563. http://dx.doi.org/10.1182/blood.v116.21.1563.1563.
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Abstract Abstract 1563 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by targeting various cell cycle regulators for proteasomal destruction. APC/C in conjunction with its adaptor protein Cdh1, both stabilizes G1-Phase (a pre-condition for an accurate cell cycle progression) and is involved in the induction of cell cycle arrest and differentiation. Further evidence suggests that Cdh1 is involved in the differentiation of a variety of cells such as neurons, myocytes, hepatocytes and lens epithelial cells. During differentiation Cdh1 interacts with the TGFb signaling pathway, targets Id2 for destruction and indirectly leads to accumulation of p27 by Skp2 degradation. We have been able to demonstrate that the expression of Cdh1 is decreased in both, AML cell lines and in primary blast samples carrying the translocation t(8;21), which consequently leads to an AML1/Eto fusion protein and is one of the most common chromosomal rearrangements in AML. Furthermore, we have data suggesting that APC/C-Cdh1 significantly influences the differentiation of malignant myeloid cells. Here, we have analyzed the expression of Cdh1, its target proteins and relevant cell cycle regulators during normal myeloid differentiation. Methods: A cytokine cocktail consisting of SCF (50ng/ml), IL-3 (5 ng/ml) and G-CSF (100 ng/ml) was used to induce differentiation of CD34+ cells into CD11b+ macrophage-like cells over seven days. Daily protein isolation, CD11b-FACS and FACS analysis of propidium iodide staining were performed to analyze Cdh1 status, differentiation kinetics and cell cycle distribution. In addition, we have established a Cdh1 knockdown in CD34+ cells by lentiviral vector mediated RNA interference. By means of GFP-cell-sorting the initially achieved transduction efficiency of 30% in CD34+ cells was increased to 70–80%. Results: The differentiation experiments carried out with normal CD34+ cells showed that after 7 days of stimulation the predominant majority of them had lost the CD34 marker and about 30% expressed CD11b on their surface confirming previous results. During the differentiation process an initial rise in Cdh1 levels, followed by a continuously high expression, was observed. Furthermore, we detected a downregulation of the Cdh1 target proteins Id2 and Skp2 and stable protein levels of p27. The cell cycle profile indicated an initial proliferation with an incremental G2/M-peak and at day 6 increasing apoptosis with a high Sub-G1-peak. In the transduced CD34+ cells we have been able to confirm by Western blotting analysis and RT-PCR that the CD34 positive cells harboring a Cdh1 shRNA had significantly decreased protein and RNA levels of Cdh1 compared to CD34 positive cells harboring a control shRNA against GFP. Analysis of the influence of Cdh1 knockdown on differentiation of CD34+ cells is ongoing and will be presented at the meeting. Conclusion: These results are consistent with the important role of Cdh1 in initiating differentiation and also show its sustained function in post mitotic myeloid cells. Studying the differentiation characteristics of CD34+ cells with a Cdh1 knockdown is likely to help to further determine its function. Disclosures: No relevant conflicts of interest to declare.
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Min, Mingwei, Tycho E. T. Mevissen, Maria De Luca, David Komander, and Catherine Lindon. "Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages." Molecular Biology of the Cell 26, no. 24 (December 2015): 4325–32. http://dx.doi.org/10.1091/mbc.e15-02-0102.
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The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.
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Aulia, Selina, and Bor Luen Tang. "Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer’s disease pathology." Biochemical and Biophysical Research Communications 339, no. 1 (January 2006): 1–6. http://dx.doi.org/10.1016/j.bbrc.2005.10.059.
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Tsunematsu, Takaaki, Rieko Arakaki, Hidehiko Kawai, Jan Ruppert, Koichi Tsuneyama, Naozumi Ishimaru, William C. Earnshaw, Michele Pagano, and Yasusei Kudo. "APC/CCdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit." Journal of Cell Science 133, no. 18 (September 15, 2020): jcs251314. http://dx.doi.org/10.1242/jcs.251314.
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ABSTRACTDuring mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/CCdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/CCdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.
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Manchado, Eusebio, Manuel Eguren, and Marcos Malumbres. "The anaphase-promoting complex/cyclosome (APC/C): cell-cycle-dependent and -independent functions." Biochemical Society Transactions 38, no. 1 (January 19, 2010): 65–71. http://dx.doi.org/10.1042/bst0380065.
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The APC/C (anaphase-promoting complex/cyclosome) is an E3 ubiquitin ligase that targets specific substrates for degradation by the 26S proteasome. APC/C activity depends on two cofactors, namely Cdc20 (cell division cycle 20) and Cdh1, which select the appropriate targets for ubiquitination. It is well established that APC/C is a target of the SAC (spindle assembly checkpoint) during mitosis and has critical roles in controlling the protein levels of major regulators of mitosis and DNA replication. In addition, recent studies have suggested new cell-cycle-independent functions of APC/C in non-mitotic cells and specifically in neuronal structure and function. Given the relevant functions of APC/C in cell proliferation and neuronal physiology, modulating APC/C activity may have beneficial effects in the clinic.
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Ewerth, Daniel, Stefanie Kreutmair, Andrea Schmidts, Marie Follo, Dagmar Wider, Julia B. Schueler, Julia Felthaus, et al. "The APC/C Coactivator Cdh1 Controls Self-Renewal and Differentiation of Human and Murine HSPCs." Blood 128, no. 22 (December 2, 2016): 2650. http://dx.doi.org/10.1182/blood.v128.22.2650.2650.
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Abstract Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo. Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice. Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation. Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.
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Schmidts, Andrea, Daniel Ewerth, Dagmar Wider, Birgit Kuegelgen, Monika Engelhardt, and Ralph Waesch. "APC/CCdh1 Regulates Self-Renewal and Differentiation of Hematopoietic Stem Cells." Blood 118, no. 21 (November 18, 2011): 2379. http://dx.doi.org/10.1182/blood.v118.21.2379.2379.
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Abstract Abstract 2379 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by ubiquitinylation of various cell cycle regulators to tag them for proteasomal degradation. APC/C in conjunction with its adaptor protein Cdh1 (APC/CCdh1), both stabilizes G1-phase and is involved in the induction of cell cycle arrest and differentiation. Here, we have analyzed the influence of APC/CCdh1 on self-renewal capacity and differentiation potential and kinetics of human hematopoietic stem cells (HSCs). Methods: In order to study the expression levels of Cdh1 among different hematopoietic lineages, we stained mononuclear cells from bone marrow of healthy donors with antibodies against the cell surface markers CD11b, Glycophorin A, CD41a, CD34, CD3 and CD19, and isolated these subsets via cell sorting. We extracted protein from these subsets and performed Western Blot analysis. We established a strong lentiviral Cdh1 knock down (kd) in CD34+ cells and performed colony forming cell (CFC) assays: 1×104 Cdh1-kd-CD34+ cells and CD34+ cells transduced with a control vector (ctrl-CD34+) were plated in MethoCult H4534 and MethoCult H4534 + EPO (1 IU/ml). At 14 days, CFU-GEMM, CFU-GM, CFU-G, CFU-M and BFU-E were scored. CD11b- and Glycophorin A-FACS, FACS analysis of propidium iodide staining and Pappenheim's staining were carried out on individually picked colonies in order to assess differentiation kinetics and cell cycle distribution. Self-renewal capacity of ctrl- and Cdh1-kd-CD34+ cells was examined by performing replating assays (secondary CFCs) with the obtained CFU-GEMM colonies as previously described (Katayama et al., BMT, 1999). Secondary colonies were analyzed after another 14 days of incubation. Results: We observed Cdh1 protein levels to significantly vary among hematopoietic cell subsets: The highest Cdh1 levels were detected in CD34+ cells, lower levels in cells of the lymphoid lineage (CD3+; CD19+) and only marginal expression levels in cells arising from myeloid progenitors (CD11b; Glycophorin A; CD41a). By correlating Cdh1 levels of the individual cell subsets with their cell cycle profiles, we were able to exclude the possibility that this was merely due to differences in cell cycle distribution. Analysis of the CFC assays performed with the lentiviraly infected CD34+ cells showed a considerable decrease of about 40% in the number of BFU-E and 35% in the number of CFU-G- and CFU-M-numbers, when Cdh1-kd-CD34+ cells were plated compared to ctrl-CD34+ cells. Furthermore, we observed an increase of CFU-GEMMs with Cdh1 depletion. The expression levels of the cell surface markers CD11b and Glycophorin A were 10–20% lower among the colonies arisen from Cdh1-kd-CD34+ cells vs. ctrl-CD34+ cells. Upon manual counting of Pappenheim stained preparations, we found the early stages of both erythroid and myeloid differentiation to be more prevalent in the Cdh1-kd colonies. When studying the replating capacity, we observed that Cdh1-depleted cells gave rise to almost twice as many secondary colonies as compared to ctrl-cells. There was no difference with regard to the relative proportions of the colony types. Interestingly, tracking of GFP, which had been used as a reporter-gene for the lentiviral transduction of the ctrl- and Cdh1-kd-cells, showed that it was enhanced in Cdh1-kd-secondary colonies compared to ctrl-secondary colonies. Conclusion: By analyzing human bone marrow subsets we observed that Cdh1 levels diminish from HSCs to mature lymphoid and, to an even greater extent, mature myeloid cells, suggesting that Cdh1 is important to induce differentiation but dispensable for maintaining the differentiated state. Our in vitro results are consistent with an important role of APC/CCdh1 in both myeloid and erythroid differentiation of HSCs. The data suggests that depletion of Cdh1 in HSC interferes with normal differentiation both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. HSCs deficient in Cdh1 seem to increasingly undergo self-renewal. The stronger the Cdh1-kd the more likely the generation of secondary colonies appeared to be. In vivo models may be particularly helpful to further elucidate these phenomena. Disclosures: No relevant conflicts of interest to declare.
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Bobo-Jiménez, Verónica, María Delgado-Esteban, Julie Angibaud, Irene Sánchez-Morán, Antonio de la Fuente, Javier Yajeya, U. Valentin Nägerl, José Castillo, Juan P. Bolaños, and Angeles Almeida. "APC/CCdh1-Rock2 pathway controls dendritic integrity and memory." Proceedings of the National Academy of Sciences 114, no. 17 (April 10, 2017): 4513–18. http://dx.doi.org/10.1073/pnas.1616024114.
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Disruption of neuronal morphology contributes to the pathology of neurodegenerative disorders such as Alzheimer’s disease (AD). However, the underlying molecular mechanisms are unknown. Here, we show that postnatal deletion of Cdh1, a cofactor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase in neurons [Cdh1 conditional knockout (cKO)], disrupts dendrite arborization and causes dendritic spine and synapse loss in the cortex and hippocampus, concomitant with memory impairment and neurodegeneration, in adult mice. We found that the dendrite destabilizer Rho protein kinase 2 (Rock2), which accumulates in the brain of AD patients, is an APC/CCdh1 substrate in vivo and that Rock2 protein and activity increased in the cortex and hippocampus of Cdh1 cKO mice. In these animals, inhibition of Rock activity, using the clinically approved drug fasudil, prevented dendritic network disorganization, memory loss, and neurodegeneration. Thus, APC/CCdh1-mediated degradation of Rock2 maintains the dendritic network, memory formation, and neuronal survival, suggesting that pharmacological inhibition of aberrantly accumulated Rock2 may be a suitable therapeutic strategy against neurodegeneration.
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Yamano, Hiroyuki. "APC/C: current understanding and future perspectives." F1000Research 8 (May 23, 2019): 725. http://dx.doi.org/10.12688/f1000research.18582.1.
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The separation of sister chromatids at anaphase, which is regulated by an E3 ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C), is arguably the most important irrevocable event during the cell cycle. The APC/C and cyclin-dependent kinase 1 (Cdk1) are just two of the many significant cell cycle regulators and exert control through ubiquitylation and phosphorylation, respectively. The temporal and spatial regulation of the APC/C is achieved by multiple mechanisms, including phosphorylation, interaction with the structurally related co-activators Cdc20 and Cdh1, loading of distinct E2 ubiquitin-conjugating enzymes, binding with inhibitors and differential affinities for various substrates. Since the discovery of APC/C 25 years ago, intensive studies have uncovered many aspects of APC/C regulation, but we are still far from a full understanding of this important cellular machinery. Recent high-resolution cryogenic electron microscopy analysis and reconstitution of the APC/C have greatly advanced our understanding of molecular mechanisms underpinning the enzymatic properties of APC/C. In this review, we will examine the historical background and current understanding of APC/C regulation.
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Tyagi, Apoorvi, Neha Sarodaya, Kamini Kaushal, Arun Pandian Chandrasekaran, Ainsley Mike Antao, Bharathi Suresh, Byung Ho Rhie, Kye Seong Kim, and Suresh Ramakrishna. "E3 Ubiquitin Ligase APC/CCdh1 Regulation of Phenylalanine Hydroxylase Stability and Function." International Journal of Molecular Sciences 21, no. 23 (November 28, 2020): 9076. http://dx.doi.org/10.3390/ijms21239076.
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Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of Cdh1 stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.
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Lindon, Catherine, and Jonathon Pines. "Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells." Journal of Cell Biology 164, no. 2 (January 19, 2004): 233–41. http://dx.doi.org/10.1083/jcb.200309035.
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We have found that key mitotic regulators show distinct patterns of degradation during exit from mitosis in human cells. Using a live-cell assay for proteolysis, we show that two of these regulators, polo-like kinase 1 (Plk1) and Aurora A, are degraded at different times after the anaphase-promoting complex/cyclosome (APC/C) switches from binding Cdc20 to Cdh1. Therefore, events in addition to the switch from Cdc20 to Cdh1 control the proteolysis of APC/CCdh1 substrates in vivo. We have identified a putative destruction box in Plk1 that is required for degradation of Plk1 in anaphase, and have examined the effect of nondegradable Plk1 on mitotic exit. Our results show that Plk1 proteolysis contributes to the inactivation of Plk1 in anaphase, and that this is required for the proper control of mitotic exit and cytokinesis. Our experiments reveal a role for APC/C-mediated proteolysis in exit from mitosis in human cells.
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Fanyi, Meng, Qiang Wang, Hongsheng Zhou, Mo Yang, and Liu Xiaoli. "Imatinib and Bortezomib Induce the Expression and Distribution of Anaphase-Promoting Complex Cdh1 in Blast Crisis of Chronic Myelogenous Leukemia." Blood 118, no. 21 (November 18, 2011): 4428. http://dx.doi.org/10.1182/blood.v118.21.4428.4428.
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Abstract Abstract 4428 Introduction: CML is well-known as the best understood human malignancy and a paradigm for cancer research from bench to bedside. However, treatment options for imatinib-resistant patients are still limited. Imatinib (IM) has already established as the standard first-line therapy for patients with chronic-phase CML. Bortezomib (BOR) not only prolonged life span for relapsed multiple myeloma(MM) patients, but also induced cell apoptosis in CML. However, the efficacy of TKIs and bortezomib on imatinib-resistant CML remains obscure. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. Cdh1, the adaptor protein of APC/C, in conjunction with APC/C in G1-phase, is required for preventing unscheduled proliferation and protecting primary mammalian cells from genomic instability. Cdh1-deficient cells showed a large variety of chromosomal aberrations. On the contrary, Cdc20, the substrate of Cdh1, could contribute to genetic aberration. However, it’s marginally addressed about the relationship between interaction of Cdh1-Cdc20 with IM resistance and genetic aberrations. Methods: We treated K562-WT and IM-resistant K562 cells with IM/nilotinib(AMN) and BOR, verified the effect of TKIs and BOR by FACS, and explored the change of the expression and the subcellular localization of Cdh1 by immunoblot and immunofluorescence microscopic analysis. Then, we enrolled ten patients with newly diagnosed CML-BC and dissected Cdh1-Skp2-p27 cascade in primary CML cells by immunoblot analysis. We explored the subcellular localization and interaction in space between Cdh1 and Skp2 by immunofluorescence in IM-sensitive or resistant primary cells and cell line. Moreover, the single siRNAs of Cdh1 and Skp2 were designed and were transiently transfected with HiPerFect Transfection Reagent. The expressions of Cdh1-Skp2-p27 pathway proteins were detected by immunoblot, the change of cell cycle and apoptosis were detected by flow cytometry. Result: We found that IM and BOR induced cell cycle quiescence and arrest, and changed the expression and nuclear relocation of Cdh1 in CML-BC cells. Moreover, AMN and BOR resulted in up-regulation of Cdh1 in IM-resistant cells. Our study revealed Cdh1 and Skp2 were co-localized more abundantly in the cytoplasm of IM-sensitive cells, but co-distributed to nuclears in IM-resistant cells. Furthermore, Cdh1 was lower level in IM-resistant CML-blast crisis (BC) than that of IM-sensitive patients. On the other side, Cdh1 silencing resulted in stabilization of Skp2 and Cdc20, subsequently promoting G1-S transition and formation of multinucleated cells. Conclusion: Expression and dynamic distribution of Cdh1, induced by IM and BOR, provide a novel interpretation of underlying mechanism to inhibit BCR-ABL downstream cascade via Cdh1-Skp2-p27 axis. Furthermore, we demonstrated BOR still exerted the regulation effect on expression and relocation of Cdh1 even in IM-resistant CML-BC cells, which provided evidences for synergy combination of TKI and proteasome inhibitor for overcoming IM-resistance. Besides, our results revealed Cdh1 tends to be related to genomic stability in CML-BC. Disclosures: No relevant conflicts of interest to declare. Footnotes:* Asterisk with author names denotes non-ASH members.
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Ondracka, Andrej, Jonathan A. Robbins, and Frederick R. Cross. "An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition." PLOS ONE 11, no. 7 (July 13, 2016): e0159166. http://dx.doi.org/10.1371/journal.pone.0159166.
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Homer, Hayden. "The APC/C in female mammalian meiosis I." REPRODUCTION 146, no. 2 (August 2013): R61—R71. http://dx.doi.org/10.1530/rep-13-0163.
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The anaphase-promoting complex or cyclosome (APC/C) orchestrates a meticulously controlled sequence of proteolytic events critical for proper cell cycle progression, the details of which have been most extensively elucidated during mitosis. It has become apparent, however, that the APC/C, particularly when acting in concert with its Cdh1 co-activator (APC/CCdh1), executes a staggeringly diverse repertoire of functions that extend its remit well outside the bounds of mitosis. Findings over the past decade have not only earmarked mammalian oocyte maturation as one such case in point but have also begun to reveal a complex pattern of APC/C regulation that underpins many of the oocyte's unique developmental attributes. This review will encompass the latest findings pertinent to the APC/C, especially APC/CCdh1, in mammalian oocytes and how its activity and substrates shape the stop–start tempo of female mammalian first meiotic division and the challenging requirement for assembling spindles in the absence of centrosomes.
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Lambhate, Surbhi, Debanjan Bhattacharjee, and Nishant Jain. "APC/C CDH1 ubiquitinates IDH2 contributing to ROS increase in mitosis." Cellular Signalling 86 (October 2021): 110087. http://dx.doi.org/10.1016/j.cellsig.2021.110087.
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Toda, Kazuhiro, Kayoko Naito, Satoru Mase, Masaru Ueno, Masahiro Uritani, Ayumu Yamamoto, and Takashi Ushimaru. "APC/C-Cdh1-dependent anaphase and telophase progression during mitotic slippage." Cell Division 7, no. 1 (2012): 4. http://dx.doi.org/10.1186/1747-1028-7-4.
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de Boer, H. Rudolf, S. Guerrero Llobet, and Marcel A. T. M. van Vugt. "Controlling the response to DNA damage by the APC/C-Cdh1." Cellular and Molecular Life Sciences 73, no. 5 (December 9, 2015): 949–60. http://dx.doi.org/10.1007/s00018-015-2096-7.
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Almeida, Angeles. "Regulation of APC/C-Cdh1 and Its Function in Neuronal Survival." Molecular Neurobiology 46, no. 3 (July 27, 2012): 547–54. http://dx.doi.org/10.1007/s12035-012-8309-2.
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García-Higuera, Irene, Eusebio Manchado, Pierre Dubus, Marta Cañamero, Juan Méndez, Sergio Moreno, and Marcos Malumbres. "Genomic stability and tumour suppression by the APC/C cofactor Cdh1." Nature Cell Biology 10, no. 7 (June 15, 2008): 802–11. http://dx.doi.org/10.1038/ncb1742.
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Zhang, Ziguo, Jing Yang, Eric H. Kong, William C. H. Chao, Edward P. Morris, Paula C. A. da Fonseca, and David Barford. "Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)." Biochemical Journal 449, no. 2 (December 14, 2012): 365–71. http://dx.doi.org/10.1042/bj20121374.
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Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.
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Kim, Wantae, Yong Suk Cho, Xiaohui Wang, Ogyi Park, Xueyan Ma, Hanjun Kim, Wenjian Gan, et al. "Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1." Proceedings of the National Academy of Sciences 116, no. 19 (April 18, 2019): 9423–32. http://dx.doi.org/10.1073/pnas.1821370116.
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The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.
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Castro, Anna, Suzanne Vigneron, Cyril Bernis, Jean-Claude Labbé, and Thierry Lorca. "Xkid Is Degraded in a D-Box, KEN-Box, and A-Box-Independent Pathway." Molecular and Cellular Biology 23, no. 12 (June 15, 2003): 4126–38. http://dx.doi.org/10.1128/mcb.23.12.4126-4138.2003.
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ABSTRACT During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.
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Fujita, T., G. Wu, R. D. Wood, and Y. Wan. "Clinical significance of Cdh1-Skp2 cascade in breast cancer." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 10609. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.10609.
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10609 Background: Skp2 (S-phase kinase-associated protein 2) is substrate-recognizing subunit of an ubiquitin E3 ligase SCF (Skp1, Cullin and F-box) complex and has a major role in p27 regulation. Overexpression of Skp2 is frequently seen in human tumors, often correlating with poor prognosis. Anaphase-promoting complex/cyclosome (APC/C) is another most prominent ubiquitin E3 ligase in cell cycle control, and its function is tightly controlled by activating subunit Cdh1 (Fizzy-related). APC-Cdh1 complex is supposed to be involved in Skp2 proteolysis and have essential role for S phase entry, however clinical significance of Cdh1 have not evaluated yet. Herein we examined the significance of Cdh1 in breast cancer. Methods: Firstly, MCF7 human breast cancer cells and MCF10A normal breast epithelial cells were analyzed in vitro and in vivo. Thereafter, using tissue microarray, we evaluate the expression profile of Cdh1, Skp2 and p27 in both breast cancer and normal breast epithelial tissue. Moreover, clinicopathological significance (age, tumor size, lymph node metastasis, distant metastasis, histological grade, and stage) of Cdh1 was analyzed from 126 breast cancer patients. Chi square-test, Fisher's exact test were used for statistical analysis of immunostaining results and clinicopathological data. p<0.05 was considered statistically significant. Results: Overexpression of Cdh1 induced attenuation of Skp2 and increased p27 protein expression, resulted in growth suppression. Moreover, knockdown of Cdh1 promoted higher Skp2 expression and S-phase population, reduced p27 and consequently induced cell transformation and proliferation in vitro and in vivo. Tissue microarray results appeared that positive Cdh1 and p27 was more frequently seen in normal breast tissue and statistically significant. On the other hand, Skp2 was less in normal tissue. Furthermore, Cdh1 positive breast cancer was more frequently seen in low histological grade tumors and statistically significant (p=0.04). Conclusions: Skp2 protein expression is regulated by Cdh1 in breast cancer. Cdh1 expression could be possible novel biomarker in patient with breast cancer. No significant financial relationships to disclose.
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Edgerton-Morgan, Heather, and Berl R. Oakley. "γ-Tubulin plays a key role in inactivating APC/CCdh1 at the G1–S boundary." Journal of Cell Biology 198, no. 5 (August 27, 2012): 785–91. http://dx.doi.org/10.1083/jcb.201203115.
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A γ-tubulin mutation in Aspergillus nidulans, mipA-D159, causes failure of inactivation of the anaphase-promoting complex/cyclosome (APC/C) in interphase, resulting in failure of cyclin B (CB) accumulation and removal of nuclei from the cell cycle. We have investigated the role of CdhA, the A. nidulans homologue of the APC/C activator protein Cdh1, in γ-tubulin–dependent inactivation of the APC/C. CdhA was not essential, but it targeted CB for destruction in G1, and APC/CCdhA had to be inactivated for the G1–S transition. mipA-D159 altered the localization pattern of CdhA, and deletion of the gene encoding CdhA allowed CB to accumulate in all nuclei in strains carrying mipA-D159. These data indicate that mipA-D159 causes a failure of inactivation of APC/CCdhA at G1–S, perhaps by altering its localization to the spindle pole body, and, thus, that γ-tubulin plays an important role in inactivating APC/CCdhA at this point in the cell cycle.
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Kaushal, Kamini, Sang Hyeon Woo, Apoorvi Tyagi, Dong Ha Kim, Bharathi Suresh, Kye-Seong Kim, and Suresh Ramakrishna. "E3 Ubiquitin Ligase APC/CCdh1 Negatively Regulates FAH Protein Stability by Promoting Its Polyubiquitination." International Journal of Molecular Sciences 21, no. 22 (November 18, 2020): 8719. http://dx.doi.org/10.3390/ijms21228719.
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Fumarylacetoacetate hydrolase (FAH) is the last enzyme in the degradation pathway of the amino acids tyrosine and phenylalanine in mammals that catalyzes the hydrolysis of 4-fumarylacetoacetate into acetoacetate and fumarate. Mutations of the FAH gene are associated with hereditary tyrosinemia type I (HT1), resulting in reduced protein stability, misfolding, accelerated degradation and deficiency in functional proteins. Identifying E3 ligases, which are necessary for FAH protein stability and degradation, is essential. In this study, we demonstrated that the FAH protein level is elevated in liver cancer tissues compared to that in normal tissues. Further, we showed that the FAH protein undergoes 26S proteasomal degradation and its protein turnover is regulated by the anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 E3 ubiquitin ligase complex. APC/CCdh1 acts as a negative stabilizer of FAH protein by promoting FAH polyubiquitination and decreases the half-life of FAH protein. Thus, we envision that Cdh1 might be a key factor in the maintenance of FAH protein level to regulate FAH-mediated physiological functions.
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Wan, Lixin, Weiguo Zou, Daming Gao, Hiroyuki Inuzuka, Hidefumi Fukushima, Anders H. Berg, Rebecca Drapp, et al. "Cdh1 Regulates Osteoblast Function through an APC/C-Independent Modulation of Smurf1." Molecular Cell 44, no. 5 (December 2011): 721–33. http://dx.doi.org/10.1016/j.molcel.2011.09.024.
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Ewerth, Daniel, Stefanie Kreutmair, Birgit Kügelgen, Dagmar Wider, Julia Felthaus, Julia Schüler, Andrea Schmidts, et al. "Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1." Blood 126, no. 23 (December 3, 2015): 2370. http://dx.doi.org/10.1182/blood.v126.23.2370.2370.
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Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.
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Hein, Manuel, Dominik Schnerch, Andrea Schmidts, Julia Felthaus, Dagmar Wider, Gabriele Ihorst, Monika Engelhardt, and Ralph M. Waesch. "Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML." Blood 114, no. 22 (November 20, 2009): 5045. http://dx.doi.org/10.1182/blood.v114.22.5045.5045.
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Abstract Abstract 5045 Introduction The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and Results We have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. Conclusion This is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. Disclosures No relevant conflicts of interest to declare.
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Huang, Nai-Jia, Liguo Zhang, Wanli Tang, Chen Chen, Chih-Sheng Yang, and Sally Kornbluth. "The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1." Journal of Cell Biology 197, no. 3 (April 23, 2012): 361–67. http://dx.doi.org/10.1083/jcb.201111141.
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Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/CCdh1) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/CCdh1-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid–mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.
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Schmidts, Andrea, Daniel Ewerth, Dominik Schnerch, Arunas Kvainickas, Dagmar Wider, Birgit Kügelgen, Heike L. Pahl, Monika Engelhardt, and Ralph Wäsch. "APC/CCdh1 As a Mediator Of Hematopoietic Differentiation, Self-Renewal and Malignant Transformation." Blood 122, no. 21 (November 15, 2013): 3676. http://dx.doi.org/10.1182/blood.v122.21.3676.3676.
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Abstract Introduction Cdh1 is an important activator of the anaphase-promoting complex/cyclosome (APC/C) and may play a major role in both the stabilization of G1-phase and the induction of cell cycle arrest and differentiation. Our work focuses on the function of APC/CCdh1in hematopoietic stem cells (HSCs) with regard to their potential for differentiation, self-renewal and malignant transformation. Methods Physiological expression levels of Cdh1 were studied among different human hematopoietic lineages (defined by staining for the cell surface markers CD11b, CD41a, CD34, CD3 and CD19) obtained from bone marrow (BM) of healthy donors and mobilized peripheral blood (PB). Next we established a strong lentiviral Cdh1 knock down (kd) in human CD34+ cells and performed colony forming cell (CFC) assays and replating assays. We also analyzed Cdh1-protein levels in 30 samples of BM or PB of patients first diagnosed with acute myeloid leukemia (AML). Finally experiments to further look into possible mechanisms of Cdh1 regulation during leukemogenesis were carried out. On the transcriptional level we reanalyzed published microarray data from CD34+-AML blasts and normal CD34+ cells (Leukemia 2011;25:1825-1833). On the post-transcriptional level we tested the hypothesis of Cdh1 degradation mediated by the ubiquitin-ligase SCF by expressing a dominant negative mutant of the core SCF subunit Cullin-1 (delta-Cul1) in the AML cell lines Kasumi-1 and HL-60. Results Western blot analysis of physiological Cdh1-distribution among the variable human hematopoietic lineages showed significant differences in Cdh1 protein levels. We saw diminishing levels of Cdh1 from HSCs to mature lymphoid and myeloid cells, suggesting that Cdh1 may be important to induce differentiation but dispensable for maintaining the differentiated state. In the Cdh1-kd-CFC assays a significant decrease of total colony numbers, CFU-Gs, CFU-GMs and BFU-Es >50% was observed. At morphological examination and FACS analysis these colonies proved to be more immature than the control colonies. Thus, depletion of Cdh1 in HSC hinders normal differentiation into the myeloid and erythroid lineage both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. Upon replating, we noticed a significant increase in the number of secondary colonies, with a doubling of total colony numbers, when using Cdh1 deficient HSC. This result indicates an advantage for self-renewal over differentiation in these cells, which seems to correlate with the intensity of the Cdh1-kd. Examination of Cdh1 protein levels in AML blasts revealed that basically all AML samples showed a strong down-regulation of Cdh1 protein levels compared to normal CD34+ cells, which may be a contributing factor to the differentiation block in leukemogenesis. Indeed, if we performed knockdown of Cdh1 in the HL-60 leukemia cell line they were in a less differentiated state as judged by CD11b expression. The evaluation of microarray data, in order to further address the mechanism of Cdh1 down-regulation in AML blasts, showed that Cdh1 transcription levels were not significantly different in CD34+ AML cells compared to normal CD34+ cells. This would be consistent with a posttranscriptional cause of decreased Cdh1-protein levels in AML blasts. Our ongoing work indicates SCF-dependent degradation of Cdh1, since inhibition of the SCF function (by expression of a dominant-negative form of the SCF subunit Cullin-1 (delta-Cul1)) in AML cell lines leads to a strong upregulation of Cdh1. Conclusions Our data establish Cdh1 as an important cell cycle regulator in the regulation of differentiation and self-renewal in HSCs. Its posttranscriptional downregulation by the SCF ubiquitin ligase may contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.
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Li, Qiuhong, Leifu Chang, Shintaro Aibara, Jing Yang, Ziguo Zhang, and David Barford. "WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity." Proceedings of the National Academy of Sciences 113, no. 38 (September 6, 2016): 10547–52. http://dx.doi.org/10.1073/pnas.1607147113.
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The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin–RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1WD40). To understand how Apc1WD40 contributes to APC/C activity, a mutant form of the APC/C with Apc1WD40 deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1WD40 abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C–Cdh1 complex with Apc1WD40 deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1WD40 is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.
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van Leuken, Renske, Linda Clijsters, Wouter van Zon, Dan Lim, XueBiao Yao, Rob M. F. Wolthuis, Michael B. Yaffe, René H. Medema, and Marcel A. T. M. van Vugt. "Polo-Like Kinase-1 Controls Aurora A Destruction by Activating APC/C-Cdh1." PLoS ONE 4, no. 4 (April 23, 2009): e5282. http://dx.doi.org/10.1371/journal.pone.0005282.
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Hall, Mark C., Erin N. Warren, and Christoph H. Borchers. "Multi-Kinase Phosphorylation of the APC/C Activator Cdh1 Revealed by Mass Spectrometry." Cell Cycle 3, no. 10 (October 12, 2004): 1278–84. http://dx.doi.org/10.4161/cc.3.10.1153.
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Wiebusch, Lüder, Miriam Bach, Ralf Uecker, and Christian Hagemeier. "Human Cytomegalovirus Inactivates the G0/G1-APC/C Ubiquitin Ligase by Cdh1 Dissociation." Cell Cycle 4, no. 10 (September 23, 2005): 1435–39. http://dx.doi.org/10.4161/cc.4.10.2077.
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de Boer, H. Rudolf, Sergi Guerrero Llobet, and Marcel A. T. M. van Vugt. "Erratum to: Controlling the response to DNA damage by the APC/C-Cdh1." Cellular and Molecular Life Sciences 73, no. 15 (June 1, 2016): 2985–98. http://dx.doi.org/10.1007/s00018-016-2279-x.
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Stewart, Scott, and Guowei Fang. "Anaphase-Promoting Complex/Cyclosome Controls the Stability of TPX2 during Mitotic Exit." Molecular and Cellular Biology 25, no. 23 (December 1, 2005): 10516–27. http://dx.doi.org/10.1128/mcb.25.23.10516-10527.2005.
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ABSTRACT TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/CCdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/CCdh1 in vitro and for its degradation in vivo. We conclude that APC/CCdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.
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Qin, Liang, Arda Mizrak, Dimitrius Santiago P. S. F. Guimarães, Hana M. Tambrin, David O. Morgan, and Mark C. Hall. "The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function." Journal of Biological Chemistry 294, no. 46 (September 27, 2019): 17249–61. http://dx.doi.org/10.1074/jbc.ra119.009468.
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The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif–dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.