12 results on '"William C. H. Chao"'
Search Results
2. APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability
- Author
-
Laurent Sansregret, Carlos López-García, David J. Barry, Charles Swanton, Mark Petronczki, Martin R. Singleton, René H. Medema, William C. H. Chao, Andre Koch, Stuart Horswell, Michael Howell, James O. Patterson, Andrew Rowan, Rachael Instrell, Sally M. Dewhurst, Michael Way, Paul Nurse, and Nicholas McGranahan
- Subjects
0301 basic medicine ,Genome instability ,Genetics ,Nonsense mutation ,Cancer ,Biology ,medicine.disease ,Chromosome segregation ,03 medical and health sciences ,Spindle checkpoint ,030104 developmental biology ,Oncology ,Chromosome instability ,Cancer cell ,Cancer research ,medicine ,Mitosis - Abstract
Intercellular heterogeneity, exacerbated by chromosomal instability (CIN), fosters tumor heterogeneity and drug resistance. However, extreme CIN correlates with improved cancer outcome, suggesting that karyotypic diversity required to adapt to selection pressures might be balanced in tumors against the risk of excessive instability. Here, we used a functional genomics screen, genome editing, and pharmacologic approaches to identify CIN-survival factors in diploid cells. We find partial anaphase-promoting complex/cyclosome (APC/C) dysfunction lengthens mitosis, suppresses pharmacologically induced chromosome segregation errors, and reduces naturally occurring lagging chromosomes in cancer cell lines or following tetraploidization. APC/C impairment caused adaptation to MPS1 inhibitors, revealing a likely resistance mechanism to therapies targeting the spindle assembly checkpoint. Finally, CRISPR-mediated introduction of cancer somatic mutations in the APC/C subunit cancer driver gene CDC27 reduces chromosome segregation errors, whereas reversal of an APC/C subunit nonsense mutation increases CIN. Subtle variations in mitotic duration, determined by APC/C activity, influence the extent of CIN, allowing cancer cells to dynamically optimize fitness during tumor evolution. Significance: We report a mechanism whereby cancers balance the evolutionary advantages associated with CIN against the fitness costs caused by excessive genome instability, providing insight into the consequence of CDC27 APC/C subunit driver mutations in cancer. Lengthening of mitosis through APC/C modulation may be a common mechanism of resistance to cancer therapeutics that increase chromosome segregation errors. Cancer Discov; 7(2); 218–33. ©2017 AACR. See related commentary by Burkard and Weaver, p. 134. This article is highlighted in the In This Issue feature, p. 115
- Published
- 2017
3. Erratum: Structural Basis of Eco1-Mediated Cohesin Acetylation
- Author
-
Andrew W. Jones, Benjamin O. Wade, Nicola O’Reilly, Stefania Federico, Andrew Purkiss, William C. H. Chao, Martin R. Singleton, Frank Uhlmann, Céline Bouchoux, and Ambrosius P. Snijders
- Subjects
Multidisciplinary ,Information retrieval ,Cohesin ,Section (archaeology) ,Atomic coordinates ,Basis (universal algebra) ,computer.file_format ,Protein Data Bank ,computer ,Accession ,Article ,Mathematics - Abstract
Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.
- Published
- 2017
4. Insights into Degron Recognition by APC/C Coactivators from the Structure of an Acm1-Cdh1 Complex
- Author
-
Ziguo Zhang, Jun He, Nora Cronin, David Barford, William C. H. Chao, and Jing Yang
- Subjects
Genetics ,Protein structure ,Coactivator ,Cell Biology ,CDC20 ,Binding site ,Degron ,Biology ,Molecular Biology ,Sister chromatid segregation ,Cdh1 Proteins ,APC/C activator protein CDH1 ,Cell biology - Abstract
The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs—predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.
- Published
- 2013
5. Structural Basis of Eco1-Mediated Cohesin Acetylation
- Author
-
Benjamin O. Wade, Frank Uhlmann, Céline Bouchoux, Stefania Federico, Andrew W. Jones, Nicola O’Reilly, Ambrosius P. Snijders, William C. H. Chao, Andrew Purkiss, and Martin R. Singleton
- Subjects
0301 basic medicine ,Models, Molecular ,Protein Conformation, alpha-Helical ,Saccharomyces cerevisiae Proteins ,Chromosomal Proteins, Non-Histone ,Protein subunit ,Beta sheet ,Gene Expression ,Cell Cycle Proteins ,Plasma protein binding ,Saccharomyces cerevisiae ,Xenopus Proteins ,Crystallography, X-Ray ,Substrate Specificity ,Chromosome segregation ,03 medical and health sciences ,Xenopus laevis ,Protein structure ,Acetyltransferases ,Chromosome Segregation ,Animals ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Binding site ,Multidisciplinary ,Binding Sites ,Cohesin ,Sequence Homology, Amino Acid ,Chemistry ,Nuclear Proteins ,Acetylation ,Recombinant Proteins ,030104 developmental biology ,Biophysics ,Protein Conformation, beta-Strand ,Erratum ,Sequence Alignment ,Protein Binding - Abstract
Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.
- Published
- 2016
6. Structure of the cohesin loader Scc2
- Author
-
Martin R. Singleton, Benjamin O. Wade, Andrew Purkiss, Yasuto Murayama, Xiao-Wen Hu, Ambrosius P. Snijders, William C. H. Chao, Andrew W. Jones, Sofía Muñoz, Frank Uhlmann, and Aaron J. Borg
- Subjects
0301 basic medicine ,Models, Molecular ,Saccharomyces cerevisiae Proteins ,Chromosomal Proteins, Non-Histone ,Protein subunit ,Science ,Saccharomyces cerevisiae ,General Physics and Astronomy ,Cell Cycle Proteins ,Plasma protein binding ,General Biochemistry, Genetics and Molecular Biology ,Article ,Conserved sequence ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Conserved Sequence ,Cohesin loading ,Multidisciplinary ,Cohesin ,biology ,Chemistry ,General Chemistry ,biology.organism_classification ,Chromatin ,Cell biology ,Protein Subunits ,030104 developmental biology ,biological phenomena, cell phenomena, and immunity ,030217 neurology & neurosurgery ,DNA ,Protein Binding - Abstract
The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2–Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large ‘hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2–Scc4 complex. Finally, using recombinantly purified Scc2–Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2–Scc4 uses its modular structure to make multiple contacts with cohesin., The cohesin complex maintains genome integrity by ensuring correct sister-chromatid segregation during mitosis and meiosis. Here, Chao et al. present a pseudo-atomic model of the full-length Scc2–Scc4 cohesin loader complex and reveal key Scc2 surfaces crucial for cohesin loading.
- Published
- 2016
7. Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader
- Author
-
Frank Uhlmann, Yasuto Murayama, Sofía Muñoz, Alessandro Costa, William C. H. Chao, and Martin R. Singleton
- Subjects
Cohesin loading ,Genetics ,Cohesin ,Cohesin complex ,Superhelix ,Chromosomal Proteins, Non-Histone ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Protein Structure, Secondary ,Chromatin ,Protein Structure, Tertiary ,Fungal Proteins ,Tetratricopeptide ,Protein structure ,lcsh:Biology (General) ,Ascomycota ,Biophysics ,Sister chromatids ,biological phenomena, cell phenomena, and immunity ,Protein Structure, Quaternary ,lcsh:QH301-705.5 - Abstract
SummaryThe remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.
- Published
- 2015
8. APC/C is an essential regulator of centrosome clustering
- Author
-
Konstantinos Drosopoulos, Chan Tang, Spiros Linardopoulos, and William C. H. Chao
- Subjects
Small interfering RNA ,Genes, APC ,Molecular Sequence Data ,Regulator ,General Physics and Astronomy ,Kinesins ,Centrosome cycle ,Biology ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Animals ,Humans ,Amino Acid Sequence ,RNA, Small Interfering ,Centrosome ,Multidisciplinary ,Sequence Homology, Amino Acid ,Protein Stability ,RNA ,Thiones ,General Chemistry ,Cell biology ,Monastrol ,Pyrimidines ,chemistry ,Kinesin ,Cellular model - Abstract
Centrosome amplification has been extensively associated with cancer. Cancer cells with extra centrosomes have the ability to cluster the extra centrosomes and divide in a bipolar fashion. Although a number of proteins have been shown to be involved in centrosome clustering, a mechanistic understanding of how this process is coordinated is not yet well defined. Here, to reveal regulators of centrosome clustering, we perform small interfering RNA (siRNA) screens with multiple assay readouts in a human isogenic cellular model. We find that APC/C activity is essential for centrosome clustering. We show that the motor kinesin Eg5 is a substrate of APC/C-CDH1, and that inhibition of APC/C results in stabilization of Eg5. Increased Eg5 protein levels disturb the balance of forces on the spindle and prevent centrosome clustering. This process is completely reversed after a short treatment with the Eg5 inhibitor, monastrol. These data advance our understanding of the regulation of centrosome clustering.
- Published
- 2013
9. The anaphase promoting complex contributes to the degradation of the S. cerevisiae telomerase recruitment subunit Est1p
- Author
-
Katherine L. Friedman, Jenifer L. Ferguson, Ethan Lee, and William C. H. Chao
- Subjects
Proteomics ,Telomerase ,lcsh:Medicine ,Yeast and Fungal Models ,Cdh1 Proteins ,S Phase ,0302 clinical medicine ,Molecular Cell Biology ,lcsh:Science ,0303 health sciences ,Multidisciplinary ,biology ,Protein Stability ,Chromosome Biology ,Ubiquitin-Protein Ligase Complexes ,Cell cycle ,Recombinant Proteins ,Cell biology ,Telomeres ,Cell Division ,Research Article ,Saccharomyces cerevisiae Proteins ,Protein subunit ,Saccharomyces cerevisiae ,Mitosis ,Anaphase-Promoting Complex-Cyclosome ,03 medical and health sciences ,Telomerase RNA component ,Model Organisms ,Genetic Mutation ,Cyclins ,Genetics ,Animals ,Amino Acid Sequence ,Biology ,030304 developmental biology ,lcsh:R ,G1 Phase ,Ubiquitination ,biology.organism_classification ,Molecular biology ,Telomere ,Mutagenesis ,Mutation ,Proteolysis ,lcsh:Q ,Anaphase-promoting complex ,030217 neurology & neurosurgery ,Protein Abundance - Abstract
Telomerase is a multi-subunit enzyme that reverse transcribes telomere repeats onto the ends of linear eukaryotic chromosomes and is therefore critical for genome stability. S. cerevisiae telomerase activity is cell-cycle regulated; telomeres are not elongated during G1 phase. Previous work has shown that Est1 protein levels are low during G1 phase, preventing telomerase complex assembly. However, the pathway targeting Est1p for degradation remained uncharacterized. Here, we show that Est1p stability through the cell cycle mirrors that of Clb2p, a known target of the Anaphase Promoting Complex (APC). Indeed, Est1p is stabilized by mutations in both essential and non-essential components of the APC. Mutations of putative Destruction boxes (D-boxes), regions shown to be important for recognition of known APC substrates, stabilize Est1p, suggesting that Est1p is likely to be targeted for degradation directly by the APC. However, we do not detect degradation or ubiquitination of recombinant Est1p by the APC in vitro, suggesting either that the recombinant protein lacks necessary post-translational modification and/or conformation, or that the APC affects Est1p degradation by an indirect mechanism. Together, these studies shed light on the regulation of yeast telomerase assembly and demonstrate a new connection between telomere maintenance and cell cycle regulation pathways.
- Published
- 2012
10. Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)
- Author
-
Jing Yang, Paula C. A. da Fonseca, Ziguo Zhang, William C. H. Chao, Edward P. Morris, Eric H. Kong, and David Barford
- Subjects
Models, Molecular ,Protein subunit ,Ubiquitin-Protein Ligases ,Saccharomyces cerevisiae ,Cell Cycle Proteins ,Biochemistry ,Anaphase-Promoting Complex-Cyclosome ,APC/C activator protein CDH1 ,law.invention ,Cell Line ,Substrate Specificity ,Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome ,CDC27 ,law ,Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome ,Animals ,Humans ,Protein Structure, Quaternary ,Molecular Biology ,biology ,Ubiquitination ,Ubiquitin-Protein Ligase Complexes ,Cell Biology ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Ubiquitin ligase ,Tetratricopeptide ,Microscopy, Electron ,Protein Subunits ,Multiprotein Complexes ,biology.protein ,Recombinant DNA ,Anaphase-promoting complex ,Protein Multimerization - Abstract
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.
- Published
- 2012
11. Structure of the mitotic checkpoint complex
- Author
-
David Barford, Ziguo Zhang, Eric H. Kong, William C. H. Chao, and Kiran Kulkarni
- Subjects
Models, Molecular ,Mad2 ,Saccharomyces cerevisiae Proteins ,Cdc20 Proteins ,Amino Acid Motifs ,Cell Cycle Proteins ,Spindle Apparatus ,Biology ,Crystallography, X-Ray ,Anaphase-Promoting Complex-Cyclosome ,Cdh1 Proteins ,APC/C activator protein CDH1 ,Substrate Specificity ,Structure-Activity Relationship ,Mad2 Proteins ,Schizosaccharomyces ,Humans ,Protein Structure, Quaternary ,Conserved Sequence ,Multidisciplinary ,food and beverages ,Mitotic checkpoint complex ,Nuclear Proteins ,Ubiquitin-Protein Ligase Complexes ,Cell biology ,Spindle apparatus ,Protein Structure, Tertiary ,Spindle checkpoint ,Mitotic spindle assembly checkpoint ,Multiprotein Complexes ,M Phase Cell Cycle Checkpoints ,Schizosaccharomyces pombe Proteins ,Anaphase-promoting complex - Abstract
In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.
- Published
- 2011
12. A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK
- Author
-
Alma L. Burlingame, Nicholas T. Hertz, Damian Brennan, Kevan M. Shokat, William C. H. Chao, David Barford, and Arvin C. Dar
- Subjects
Scaffold protein ,Models, Molecular ,Proto-Oncogene Proteins B-raf ,Allosteric regulation ,MAP Kinase Kinase 1 ,Biology ,Protein Serine-Threonine Kinases ,Crystallography, X-Ray ,Adenosine Triphosphate ,Allosteric Regulation ,Catalytic Domain ,Animals ,Humans ,Kinase activity ,Phosphorylation ,Protein kinase A ,Extracellular Signal-Regulated MAP Kinases ,Protein Structure, Quaternary ,Multidisciplinary ,Kinase ,Cell biology ,Enzyme Activation ,Biochemistry ,Protein kinase domain ,Biocatalysis ,Rabbits ,Signal transduction ,Protein Multimerization ,Signal Transduction - Abstract
The RAS–RAF–MEK–ERK signalling pathway is important in the regulation of cell proliferation and is often aberrantly activated in cancer. KSR (kinase suppressor of RAS) is essential for RAS signaling, but its mechanism of action has been unclear. It is a member of the protein kinase family but was thought to be a pseudokinase with only scaffold functions. Structural and biochemical studies now reveal that KSR can function as a kinase, and by forming a heterodimer with BRAF, it relays an activating signal to MEK. This work points to KSR as a potential drug target in the RAS–ERK signalling pathway. In metazoans, the Ras–Raf–MEK (mitogen-activated protein-kinase kinase)–ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR)1,2,3 functions as an essential scaffolding protein to coordinate the assembly of Raf–MEK–ERK complexes4,5. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling1,2,3. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)–MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2–BRAF heterodimer. Furthermore, KSR2–BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK’s phosphorylation by a separate catalytic Raf molecule in trans.
- Published
- 2010
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.