DOCSLIB.ORG

Structural Analysis of Human Cdc20 Supports Multisite Degron Recognition by APC/C

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structural Analysis of Human Cdc20 Supports Multisite Degron Recognition by APC/C Structural analysis of human Cdc20 supports multisite degron recognition by APC/C Wei Tiana,1, Bing Lia,b,1, Ross Warringtona,b, Diana R. Tomchickc, Hongtao Yua,b,2, and Xuelian Luoa,2 aDepartment of Pharmacology, bHoward Hughes Medical Institute, and cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by Stephen C. Harrison, Howard Hughes Medical Institute and Children’s Hospital, Harvard Medical School, Boston, MA, and approved September 24, 2012 (received for review August 3, 2012) The anaphase-promoting complex/cyclosome (APC/C) promotes suggested to inhibit APC/C by acting as a pseudosubstrate, with anaphase onset and mitotic exit through ubiquitinating securin its KEN boxes competing against those of the substrates for and cyclin B1. The mitotic APC/C activator, the cell division cycle 20 Cdc20 binding (22, 23). (Cdc20) protein, directly interacts with APC/C degrons––the de- To better understand how APC/C recognizes its substrates, struction (D) and KEN boxes. APC/CCdc20 is the target of the spindle we determined the crystal structures of human Cdc20 alone or checkpoint. Checkpoint inhibition of APC/CCdc20 requires the bind- bound to the KEN1 box of BubR1, revealing a preexisting KEN- box-binding site at the top of the WD40 β propeller. Structure- ing of a BubR1 KEN box to Cdc20. How APC/C recognizes sub- based mutagenesis then defined a second conserved surface on strates is not understood. We report the crystal structures of the side of the β propeller as a D-box-contacting site. Surpris- human Cdc20 alone or bound to a BubR1 KEN box. Cdc20 has ingly, despite binding to distinct sites on Cdc20, the KEN and D a disordered N-terminal region and a C-terminal WD40 β propeller boxes of securin do not apparently interact with APC/C in a with a preformed KEN-box-binding site at its top face. We identify highly cooperative manner. The KEN box is largely dispensable a second conserved surface at the side of the Cdc20 β propeller as for securin ubiquitination by APC/CCdc20. This lack of strong a D-box-binding site. The D box of securin, but not its KEN box, is cooperativity between the two degrons also applies to APC/ critical for securin ubiquitination by APC/CCdc20. Although both CCdh1, but the KEN and D boxes have comparable contributions motifs contribute to securin ubiquitination by APC/CCdh1, securin in this case. D-box peptides inhibit APC/C-mediated ubiquiti- mutants lacking either motif are efficiently ubiquitinated. Further- nation of KEN-box substrates, suggesting competition between BIOPHYSICS AND the two motifs. Our results indicate that the multiple APC/C more, D-box peptides diminish the ubiquitination of KEN-box sub- COMPUTATIONAL BIOLOGY strates by APC/CCdh1, suggesting possible competition between degrons of securin do not engage APC/C in a highly cooperative manner. A single functional APC/C degron within a substrate the two motifs. Our results indicate the lack of strong positive Cdh1 cooperativity between the two degrons of securin. We propose supports APC/C -dependent ubiquitination. We propose that that low-cooperativity, multisite target recognition enables APC/C this low-cooperativity, multisite substrate recognition mechanism expands the substrate spectrum of APC/C, allowing it to atten- to robustly ubiquitinate diverse substrates and helps to drive cell uate the cellular concentrations of myriad substrates with one or cycle oscillations. more functional degrons. mitosis | crystallography | multivalency | molecular recognition Results and Discussion Structures of Human Cdc20 by Itself or in Complex with BubR1 KEN1. he anaphase-promoting complex/cyclosome (APC/C) is a To determine the structure of human Cdc20, we expressed and Tmultisubunit ubiquitin ligase that mediates the ubiquitination purified from Sf9 insect cells a Cdc20 truncation mutant with its Δ Δ of a multitude of substrates to drive cell cycle progression (1–3). N-terminal 60 residues deleted (Cdc20 N60). Cdc20 N60 con- At the metaphase–anaphase transition, APC/C in complex with tained all known functional motifs of Cdc20 (3), including the C its mitotic activator Cdc20 (APC/CCdc20) ubiquitinates securin box, the KEN box, and the Mad2-interacting motif (MIM), and and cyclin B1, triggering their destruction to allow separase ac- actively supported APC/C- and D-box-dependent ubiquitination tivation and cohesin cleavage. In anaphase, APC/C bound to the of cyclin B1 (Fig. 1A and Fig. S1). We obtained crystals of Cdh1 Δ Cdc20 homolog Cdh1 (APC/C ) further mediate the ubiq- Cdc20 N60 that diffracted to about 3 Å, but could not determine uitination of cyclin B1 and other mitotic regulators, resulting in its structure using molecular replacement. We thus made addi- Δ Δ their degradation and mitotic exit. tional Cdc20 truncation mutants Cdc20 N70 and Cdc20 N80, Δ Cdc20 and Cdh1 activate APC/C by contributing to substrate obtained crystals of Cdc20 N80 that diffracted to 2.0 Å, and recognition, among other mechanisms (1–3). All APC/C substrates determined its structure using molecular replacement with the contain one or more of several types of short peptide motifs with coordinates of Wdr5 as the search model (Table S1). Only the Δ highly degenerate sequences, referred to as APC/C degrons. For WD40 domain of Cdc20 N80 was well defined in the structure. example, cyclin B1 contains a destruction (D) box with the con- Both the N-terminal region (residues 81–164) and the C-terminal ΔN80 sensus of RXXLX4–5N (X, any residue), whereas securin contains region (residues 477–499; including the IR motif) of Cdc20 both a D box and a KEN box (4-6). Biochemical and biophysical were disordered and not visible in the structure. We then obtained Δ studies have established that Cdh1 and the APC/C subunit Apc10 an unrefined structure model of Cdc20 N60 using molecular serve as coreceptors for the D box, with the D box bridging an interaction between the two (7–11). APC/CCdc20 is the molecular target of the spindle checkpoint, which prevents premature sister-chromatid separation and an- Author contributions: W.T., B.L., R.W., and X.L. performed research; D.R.T., H.Y., and X.L. – analyzed data; H.Y. and X.L. designed research; and H.Y. and X.L. wrote the paper. euploidy (3, 12 15). In response to kinetochores not properly fl attached to the spindle microtubules, the spindle checkpoint The authors declare no con ict of interest. inhibits APC/CCdc20 using multiple mechanisms. Among these This article is a PNAS Direct Submission. mechanisms, the mitotic checkpoint complex (MCC; consisting Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, of BubR1/Mad3, Bub3, Mad2, and Cdc20) anchors Cdc20 at a www.pdb.org (PDB ID codes 4GGA, 4GGC, and 4GGD). site on APC/C that is different from the site bound by free 1W.T. and B.L. contributed equally to this work. Cdc20, presumably blocking the productive engagement of the D 2To whom correspondence may be addressed. E-mail: [email protected] or box with Cdc20 and Apc10 (16-21). BubR1/Mad3 contains two [email protected]. KEN boxes (KEN1 and KEN2), both of which are required for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the spindle checkpoint (22–25). BubR1/Mad3 has thus been 1073/pnas.1213438109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1213438109 PNAS | November 6, 2012 | vol. 109 | no. 45 | 18419–18424 Downloaded by guest on September 26, 2021 Fig. 1. Human Cdc20 has a preformed KEN-box- binding site at the top face of its WD40 β propeller. (A) Domains and motifs of human Cdc20. MIM, Mad2 Interacting Motif. (B) Ribbon diagram of hu- man Cdc20. The N and C termini and the seven blades of the WD40 β propeller are labeled. The DYY insert in the loop connecting blades 1 and 7 is colored salmon. All structure figures in this report are generated with PyMOL (www.pymol.org). (C) Ribbon diagram of Wdr5 (PDB ID code 2GNQ). (D) Surface drawing of the Cdc20ΔN70–BubR1 KEN1 complex with BubR1 KEN1 shown as sticks. The DYY insert is colored salmon. (E) Surface drawing of the Wdr5–histone H3 complex (PDB ID code 2H9O), with the bound H3 peptide shown as sticks. (F) A cross- sectional view of the surface drawing of Cdc20– BubR1 KEN1 in D.(G) A cross-sectional view of the surface drawing of Wdr5–H3 in E. Δ replacement with Cdc20 N80 as the search model, but could not of human BubR1. The structures of free and KEN-bound Cdc20 refine the structure due to poor quality diffraction data. were virtually identical, indicating that KEN-box binding did not Nonetheless, it was clear that the N- and C-terminal regions of induce conformational changes of Cdc20. Only eight residues of Δ the functional Cdc20 N60 (residues 61–164 and 477–499) were the KEN1 peptide (from E23 to Q30), including the KEN motif, also disordered. were visible in the structure (Fig. 1D). The KEN motif (residues The WD40 domain of Cdc20 expectedly folds into a canonical 26–28) folds into an irregular 310 helix that binds at the top face seven-bladed β propeller. Unlike Wdr5 and other WD40 domain of the propeller. A highly conserved DYY motif of the afore- structures (26), Cdc20 does not have a deep ligand-binding mentioned insert of Cdc20 establishes the base of the KEN-box- pocket at the top face of its β propeller (Fig. 1 B–G). Instead, an binding site, contacting the KEN backbone and the side chains of insert (containing residues 183–188) in the loop connecting the E27 and N28 (Figs.
Load more

Recommended publications
  • The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
    International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells.
    [Show full text]
  • The APC/C in Female Mammalian Meiosis I
    REPRODUCTIONREVIEW The APC/C in female mammalian meiosis I Hayden Homer1,2 1Mammalian Oocyte and Embryo Research Laboratory, Cell and Developmental Biology, UCL, London WC1E 6BT, UK and 2Reproductive Medicine Unit, Institute for Women’s Health, UCLH Elizabeth Garrett Anderson Wing, London NW1 2BU, UK Correspondence should be addressed to H Homer; Email: [email protected] Abstract 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. Reproduction (2013) 146 R61–R71 Introduction associated somatic follicular compartment at the time of ovulation. Significantly, although primordial germ cells Meiosis is the unique cell division that halves the in the ovary commit to meiosis during fetal life, it is chromosome compliment by coupling two successive not until postnatal adulthood that mature oocytes (or nuclear divisions with a single round of DNA replication.
    [Show full text]
  • 1 Spindle Assembly Checkpoint Is Sufficient for Complete Cdc20
    Spindle assembly checkpoint is sufficient for complete Cdc20 sequestering in mitotic control Bashar Ibrahim Bio System Analysis Group, Friedrich-Schiller-University Jena, and Jena Centre for Bioinformatics (JCB), 07743 Jena, Germany Email: [email protected] Abstract The spindle checkpoint assembly (SAC) ensures genome fidelity by temporarily delaying anaphase onset, until all chromosomes are properly attached to the mitotic spindle. The SAC delays mitotic progression by preventing activation of the ubiquitin ligase anaphase-promoting complex (APC/C) or cyclosome; whose activation by Cdc20 is required for sister-chromatid separation marking the transition into anaphase. The mitotic checkpoint complex (MCC), which contains Cdc20 as a subunit, binds stably to the APC/C. Compelling evidence by Izawa and Pines (Nature 2014; 10.1038/nature13911) indicates that the MCC can inhibit a second Cdc20 that has already bound and activated the APC/C. Whether or not MCC per se is sufficient to fully sequester Cdc20 and inhibit APC/C remains unclear. Here, a dynamic model for SAC regulation in which the MCC binds a second Cdc20 was constructed. This model is compared to the MCC, and the MCC-and-BubR1 (dual inhibition of APC) core model variants and subsequently validated with experimental data from the literature. By using ordinary nonlinear differential equations and spatial simulations, it is shown that the SAC works sufficiently to fully sequester Cdc20 and completely inhibit APC/C activity. This study highlights the principle that a systems biology approach is vital for molecular biology and could also be used for creating hypotheses to design future experiments. Keywords: Mathematical biology, Spindle assembly checkpoint; anaphase promoting complex, MCC, Cdc20, systems biology 1 Introduction Faithful DNA segregation, prior to cell division at mitosis, is vital for maintaining genomic integrity.
    [Show full text]
  • Bub1 Positions Mad1 Close to KNL1 MELT Repeats to Promote Checkpoint Signalling
    ARTICLE Received 14 Dec 2016 | Accepted 3 May 2017 | Published 12 June 2017 DOI: 10.1038/ncomms15822 OPEN Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling Gang Zhang1, Thomas Kruse1, Blanca Lo´pez-Me´ndez1, Kathrine Beck Sylvestersen1, Dimitriya H. Garvanska1, Simone Schopper1, Michael Lund Nielsen1 & Jakob Nilsson1 Proper segregation of chromosomes depends on a functional spindle assembly checkpoint (SAC) and requires kinetochore localization of the Bub1 and Mad1/Mad2 checkpoint proteins. Several aspects of Mad1/Mad2 kinetochore recruitment in human cells are unclear and in particular the underlying direct interactions. Here we show that conserved domain 1 (CD1) in human Bub1 binds directly to Mad1 and a phosphorylation site exists in CD1 that stimulates Mad1 binding and SAC signalling. Importantly, fusion of minimal kinetochore-targeting Bub1 fragments to Mad1 bypasses the need for CD1, revealing that the main function of Bub1 is to position Mad1 close to KNL1 MELTrepeats. Furthermore, we identify residues in Mad1 that are critical for Mad1 functionality, but not Bub1 binding, arguing for a direct role of Mad1 in the checkpoint. This work dissects functionally relevant molecular interactions required for spindle assembly checkpoint signalling at kinetochores in human cells. 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Correspondence and requests for materials should be addressed to G.Z.
    [Show full text]
  • Identification of Novel Genes in BRCA1-Regulated Pathways
    Identification of Novel Genes in BRCA1-Regulated Pathways DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shweta Kotian, M.S. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2013 Dissertation Committee: Professor Jeffrey D. Parvin, Advisor Professor Joanna L. Groden Professor Denis C. Guttridge Professor Mark R. Parthun Copyright by Shweta Kotian 2013 Abstract BRCA1 is an important breast- and ovarian-specific tumor suppressor gene. It is important in various cellular functions in the body including transcriptional regulation, cell cycle checkpoint activation, DNA damage response, and maintenance of genomic stability. Approximately 40% of hereditary breast cancers have mutations in BRCA1 or BRCA2, and it is unclear how the remaining cases are caused, presumably by mutations or in the alteration of expression of unknown genes. We hypothesize that these unknown genes or ‘missing BRCAs’ can be identified by bio-informatics methods. Performing gene co-expression analysis, we have compared the expression profiles of hundreds of genes across publicly available breast cancer microarray datasets, with those of BRCA1 and BRCA2. We propose that the genes, whose expression is highly correlated with BRCA1 and BRCA2, might function together in the same pathways. They could also potentially interact with each other. The main aim of this study is to identify these genes, and then biologically validate them in functional BRCA1-regulated pathways including homologous recombination and centrosome duplication. We also hope to find any mechanistic ii associations between these genes and BRCA1. Eventually, we hope to evaluate if these genes contribute to the process of carcinogenesis.
    [Show full text]
  • A Novel Function for HSF1-Induced Mitotic Exit Failure and Genomic Instability Through Direct Interaction Between HSF1 and Cdc20
    Oncogene (2008) 27, 2999–3009 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE A novel function for HSF1-induced mitotic exit failure and genomic instability through direct interaction between HSF1 and Cdc20 YJ Lee1, HJ Lee1, JS Lee2, D Jeoung3, CM Kang1, S Bae1, SJ Lee2, SH Kwon4, D Kang4 and YS Lee1 1Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea; 2Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea; 3College of Natural Sciences, Kangwon National University, Chunchon, Korea and 4Korea Basic Science Institute, Chunchon Center, Chunchon, Korea Although heat-shock factor (HSF) 1 is a known highly aneuploid, however, the molecular mechanisms transcriptional factor of heat-shock proteins, other path- underlying the development of aneuploidy have not wayslike production of aneuploidy and increasedprotein been fully defined, although mutations in mitotic stability of cyclin B1 have been proposed. In the present checkpoint genes have been identified in a subset of study, the regulatory domain of HSF1 (amino-acid human cancers and cell lines (Lengauer et al., 1997; sequence 212–380) was found to interact directly with Cahill et al., 1998). the amino-acid sequence 106–171 of Cdc20. The associa- Enhanced heat-shock protein (HSP) expression in tion between HSF1 and Cdc20 inhibited the interaction response to various stimuli is regulated by heat-shock between Cdc27 and Cdc20, the phosphorylation of Cdc27 factors (HSFs), the functional relevance of which is now and the ubiquitination activity of anaphase-promoting emerging.
    [Show full text]
  • An Architectural Map of the Anaphase-Promoting Complex
    Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press An architectural map of the anaphase-promoting complex Brian R. Thornton,1,3 Tessie M. Ng,1,3 Mary E. Matyskiela,2 Christopher W. Carroll,2 David O. Morgan,2 and David P. Toczyski1,4 1Cancer Research Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94115, USA; 2Department of Physiology, University of California, San Francisco, California 94143-2200, USA The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. We analyzed the architecture of the APC using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. We found that the largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). We found that the three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27⌬ complexes retain detectable activity in the presence of Cdh1.
    [Show full text]
  • Disruption of the Anaphase-Promoting Complex Confers Resistance to TTK Inhibitors in Triple-Negative Breast Cancer
    Disruption of the anaphase-promoting complex confers resistance to TTK inhibitors in triple-negative breast cancer K. L. Thua,b, J. Silvestera,b, M. J. Elliotta,b, W. Ba-alawib,c, M. H. Duncana,b, A. C. Eliaa,b, A. S. Merb, P. Smirnovb,c, Z. Safikhanib, B. Haibe-Kainsb,c,d,e, T. W. Maka,b,c,1, and D. W. Cescona,b,f,1 aCampbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 1L7; bPrincess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 1L7; cDepartment of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7; dDepartment of Computer Science, University of Toronto, Toronto, ON, Canada M5G 1L7; eOntario Institute for Cancer Research, Toronto, ON, Canada M5G 0A3; and fDepartment of Medicine, University of Toronto, Toronto, ON, Canada M5G 1L7 Contributed by T. W. Mak, December 27, 2017 (sent for review November 9, 2017; reviewed by Mark E. Burkard and Sabine Elowe) TTK protein kinase (TTK), also known as Monopolar spindle 1 (MPS1), ator of the spindle assembly checkpoint (SAC), which delays is a key regulator of the spindle assembly checkpoint (SAC), which anaphase until all chromosomes are properly attached to the functions to maintain genomic integrity. TTK has emerged as a mitotic spindle, TTK has an integral role in maintaining genomic promising therapeutic target in human cancers, including triple- integrity (6). Because most cancer cells are aneuploid, they are negative breast cancer (TNBC). Several TTK inhibitors (TTKis) are heavily reliant on the SAC to adequately segregate their abnormal being evaluated in clinical trials, and an understanding of karyotypes during mitosis.
    [Show full text]
  • The Internal Cdc20 Binding Site in Bubr1 Facilitates Both Spindle Assembly Checkpoint Signalling and Silencing
    ARTICLE Received 6 Aug 2014 | Accepted 13 Oct 2014 | Published 8 Dec 2014 DOI: 10.1038/ncomms6563 The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing Tiziana Lischetti1, Gang Zhang1, Garry G. Sedgwick1, Victor M. Bolanos-Garcia2 & Jakob Nilsson1 Improperly attached kinetochores activate the spindle assembly checkpoint (SAC) and by an unknown mechanism catalyse the binding of two checkpoint proteins, Mad2 and BubR1, to Cdc20 forming the mitotic checkpoint complex (MCC). Here, to address the functional role of Cdc20 kinetochore localization in the SAC, we delineate the molecular details of its interaction with kinetochores. We find that BubR1 recruits the bulk of Cdc20 to kinetochores through its internal Cdc20 binding domain (IC20BD). We show that preventing Cdc20 kinetochore localization by removal of the IC20BD has a limited effect on the SAC because the IC20BD is also required for efficient SAC silencing. Indeed, the IC20BD can disrupt the MCC providing a mechanism for its role in SAC silencing. We thus uncover an unexpected dual function of the second Cdc20 binding site in BubR1 in promoting both efficient SAC signalling and SAC silencing. 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, Copenhagen 2200, Denmark. 2 Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford OX3 0BP, UK. Correspondence and requests for materials should be addressed to J.N. (email: [email protected]). NATURE COMMUNICATIONS | 5:5563 | DOI: 10.1038/ncomms6563 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited.
    [Show full text]
  • Supplementary Table S5
    Supplementary Table S5 Predicted impact on structural MutatorAssessor MutatorAssessor Polyphen-2 Polyphen-2 Gene Mutation In structure? Surface/buried Implication/comment integrity Func. Impact FI score Prediction score ANAPC1 E139Q No low 1.5 benign 0.308 ANAPC1 F211V Yes Buried Destabilising Mild/Strong medium 2.83 probably damaging 0.992 ANAPC1 G602D Yes Surface Allows bend at loop; G>D destabilising Strong medium 2.02 probably damaging 0.973 ANAPC1 H1661Y Yes Surface Destabilising; H situates in hydrophilic environment Mild/Strong medium 2.155 possibly damaging 0.826 ANAPC1 H27R Yes Buried Destabilising Strong low 0.895 probably damaging 0.981 ANAPC1 M525V No low 1.32 benign 0.001 ANAPC1 N280K Yes Buried Destabilising Strong medium 2.015 benign 0.18 ANAPC1 P314S No medium 2.32 probably damaging 0.993 ANAPC1 P558L No low 1.575 benign 0.001 ANAPC1 Q846P Yes Buried Destabilising Strong medium 1.95 benign 0.261 ANAPC1 R1726T No low 1.69 benign 0.442 ANAPC1 R453L Yes Partially buried Destabilising Strong medium 2.36 probably damaging 0.993 ANAPC1 R871K Yes Surface Solvent exposed None neutral -0.88 benign 0 ANAPC1 Y1552C Yes Buried Destabilising Strong medium 3.41 probably damaging 0.999 ANAPC1 L1770F Yes Buried Part of interface with Apc5; end of helical repeat Mild. Similar side chain property. neutral -0.08 benign 0.003 ANAPC1 L230I No Surface On a loop of Apc1 WD40 domain N/A neutral 0.69 benign 0 ANAPC1 D568N No Surface Surface extended loop; not in sturcture N/A neutral 0.585 possibly damaging 0.455 Surface on beta jelly roll domain.
    [Show full text]
  • A Meta-Learning Approach for Genomic Survival Analysis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.053918; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A meta-learning approach for genomic survival analysis Yeping Lina Qiu1;2, Hong Zheng2, Arnout Devos3, Olivier Gevaert2;4;∗ 1Department of Electrical Engineering, Stanford University 2Stanford Center for Biomedical Informatics Research, Department of Medicine, Stanford University 3School of Computer and Communication Sciences, Swiss Federal Institute of Technology Lausanne (EPFL) 4Department of Biomedical Data Science, Stanford University ∗To whom correspondence should be addressed: [email protected] Abstract RNA sequencing has emerged as a promising approach in cancer prognosis as sequencing data becomes more easily and affordably accessible. However, it remains challenging to build good predictive models especially when the sample size is limited and the number of features is high, which is a common situation in biomedical settings. To address these limitations, we propose a meta-learning framework based on neural networks for survival analysis and evaluate it in a genomic cancer research setting. We demonstrate that, compared to regular transfer- learning, meta-learning is a significantly more effective paradigm to leverage high-dimensional data that is relevant but not directly related to the problem of interest. Specifically, meta-learning explicitly constructs a model, from abundant data of relevant tasks, to learn a new task with few samples effectively. For the application of predicting cancer survival outcome, we also show that the meta- learning framework with a few samples is able to achieve competitive performance with learning from scratch with a significantly larger number of samples.
    [Show full text]
  • Cell Cycle-Regulated Oscillator Coordinates Core Histone Gene Transcription Through Histone Acetylation
    Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation Christoph F. Kurata,1, Jean-Philippe Lambertb, Julia Petschnigga, Helena Friesena, Tony Pawsonb,c, Adam Rosebrocka, Anne-Claude Gingrasb,c, Jeffrey Fillinghamd, and Brenda Andrewsa,c,2 aThe Donnelly Center, University of Toronto, Toronto, ON, Canada M5S 3E1; bLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3E1; and dDepartment of Chemistry and Biology, Ryerson University, Toronto, ON, Canada M5B 2K3 Edited by Jasper Rine, University of California, Berkeley, CA, and approved August 21, 2014 (received for review July 25, 2014) DNA replication occurs during the synthetic (S) phase of the eukary- Although the roster of histone chaperones and chromatin otic cell cycle and features a dramatic induction of histone gene remodelers involved in histone gene regulation is impressive, the expression for concomitant chromatin assembly. Ectopic production cell cycle oscillator responsible for restricting histone gene acti- of core histones outside of S phase is toxic, underscoring the critical vation to S phase has remained elusive. In this context, we de- importance of regulatory pathways that ensure proper expression cided to revisit the functions of two poorly understood proteins of histone genes. Several regulators of histone gene expression in that physically interact and are involved in histone gene regula- the budding yeast Saccharomyces cerevisiae are known, yet the key tion, suppressor of Ty (Spt)10 and Spt21 (15–21). Spt10 is a DNA- oscillator responsible for restricting gene expression to S phase has binding protein that localizes to the upstream-activation sequences remained elusive.
    [Show full text]