scholarly journals Building a pseudo-atomic model of the anaphase-promoting complex

2013 ◽  
Vol 69 (11) ◽  
pp. 2236-2243 ◽  
Author(s):  
Kiran Kulkarni ◽  
Ziguo Zhang ◽  
Leifu Chang ◽  
Jing Yang ◽  
Paula C. A. da Fonseca ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Homology Modelling ◽  
Active Form ◽  
Protein Crystallography ◽  
Hybrid Approach ◽  
Atomic Model ◽  
Anaphase Promoting Complex ◽  
Atomic Models ◽  
Entire Complex ◽  
Cell Cycle Regulatory Proteins

The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14–15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.

scholarly journals Degradation of FBXO31 by APC/C is regulated by AKT- and ATM-mediated phosphorylation

2018 ◽  
Vol 115 (5) ◽  
pp. 998-1003 ◽  
Author(s):  
Srinadh Choppara ◽  
Sunil K. Malonia ◽  
Ganga Sankaran ◽  
Michael R. Green ◽  
Manas Kumar Santra
Keyword(s):  
Cell Cycle ◽  
Genotoxic Stress ◽  
Anaphase Promoting Complex ◽  
Physiological Regulation ◽  
Cell Cycle Regulatory Proteins ◽  
A Cell ◽  
Low Levels ◽  
Stressed Cells ◽  
F Box Protein ◽  
Prosurvival Kinase

The F-box protein FBXO31 is a tumor suppressor that is encoded in 16q24.3, for which there is loss of heterozygosity in various solid tumors. FBXO31 serves as the substrate-recognition component of the SKP/Cullin/F-box protein class of E3 ubiquitin ligases and has been shown to direct degradation of pivotal cell-cycle regulatory proteins including cyclin D1 and the p53 antagonist MDM2. FBXO31 levels are normally low but increase substantially following genotoxic stress through a mechanism that remains to be determined. Here we show that the low levels of FBXO31 are maintained through proteasomal degradation by anaphase-promoting complex/cyclosome (APC/C). We find that the APC/C coactivators CDH1 and CDC20 bind to a destruction-box (D-box) motif present in FBXO31 to promote its polyubiquitination and degradation in a cell-cycle–regulated manner, which requires phosphorylation of FBXO31 on serine-33 by the prosurvival kinase AKT. Following genotoxic stress, phosphorylation of FBXO31 on serine-278 by another kinase, the DNA damage kinase ATM, results in disruption of its interaction with CDH1 and CDC20, thereby preventing FBXO31 degradation. Collectively, our results reveal how alterations in FBXO31 phosphorylation, mediated by AKT and ATM, underlie physiological regulation of FBXO31 levels in unstressed and genotoxically stressed cells.

scholarly journals Understanding the structural basis for controlling chromosome division

2015 ◽  
Vol 373 (2036) ◽  
pp. 20130392 ◽  
Author(s):  
David Barford
Keyword(s):  
Regulatory Proteins ◽  
Structural Basis ◽  
Macromolecular Complex ◽  
Anaphase Promoting Complex ◽  
Atomic Models ◽  
Sequence Of Events ◽  
Cryo Electron Microscopy ◽  
Density Maps ◽  
Cell Cycle Regulatory Proteins ◽  
Functional States

The process of chromosome division, termed mitosis, involves a complex sequence of events that is tightly controlled to ensure that the faithful segregation of duplicated chromosomes is coordinated with each cell division cycle. The large macromolecular complex responsible for regulating this process is the anaphase-promoting complex or cyclosome (APC/C). In humans, the APC/C is assembled from 20 subunits derived from 15 different proteins. The APC/C functions to ubiquitinate cell cycle regulatory proteins, thereby targeting them for destruction by the proteasome. This review describes our research aimed at understanding the structure and mechanism of the APC/C. We have determined the crystal structures of individual subunits and subcomplexes that provide atomic models to interpret density maps of the whole complex derived from single particle cryo-electron microscopy. With this information, we are generating pseudo-atomic models of functional states of the APC/C that provide insights into its overall architecture and mechanisms of substrate recognition, catalysis and regulation by inhibitory complexes.

scholarly journals Structural insights into anaphase-promoting complex function and mechanism

2011 ◽  
Vol 366 (1584) ◽  
pp. 3605-3624 ◽  
Author(s):  
David Barford
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Substrate Recognition ◽  
Complex Function ◽  
Anaphase Promoting Complex ◽  
Temporal Regulation ◽  
Chromatid Segregation ◽  
Cell Cycle Regulatory Proteins ◽  
Structural Insights

The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C.

scholarly journals WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

2016 ◽  
Vol 113 (38) ◽  
pp. 10547-10552 ◽  
Author(s):  
Qiuhong Li ◽  
Leifu Chang ◽  
Shintaro Aibara ◽  
Jing Yang ◽  
Ziguo Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Catalytic Activity ◽  
Cell Cycle Progression ◽  
Ubiquitin Proteasome System ◽  
Chain Elongation ◽  
Mutant Form ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Chain ◽  
Cell Cycle Regulatory Proteins ◽  
Ubiquitin Proteasome

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.

scholarly journals Mechanisms for the temporal regulation of substrate ubiquitination by the anaphase-promoting complex/cyclosome

Cell Division ◽  
2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Shivangee Bansal ◽  
Swati Tiwari
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Anaphase Promoting Complex ◽  
Substrate Selection ◽  
Post Translational Modifications ◽  
Temporal Regulation ◽  
Numerous Cell ◽  
Cell Cycle Regulatory Proteins ◽  
C Complex ◽  
Temporal Degradation

AbstractThe anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit, multifunctional ubiquitin ligase that controls the temporal degradation of numerous cell cycle regulatory proteins to direct the unidirectional cell cycle phases. Several different mechanisms contribute to ensure the correct order of substrate modification by the APC/C complex. Recent advances in biochemical, biophysical and structural studies of APC/C have provided a deep mechanistic insight into the working of this complex ubiquitin ligase. This complex displays remarkable conformational flexibility in response to various binding partners and post-translational modifications, which together regulate substrate selection and catalysis of APC/C. Apart from this, various features and modifications of the substrates also influence their recognition and affinity to APC/C complex. Ultimately, temporal degradation of substrates depends on the kind of ubiquitin modification received, the processivity of APC/C, and other extrinsic mechanisms. This review discusses our current understanding of various intrinsic and extrinsic mechanisms responsible for ‘substrate ordering’ by the APC/C complex.

scholarly journals The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 489-503 ◽  
Author(s):  
Karen E Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Growth Defects ◽  
Genes Encoding ◽  
Specificity Factor

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

scholarly journals Structure-Based Virtual Screening and Biological Evaluation of Peptide Inhibitors for Polo-Box Domain

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 107 ◽  
Author(s):  
Fang Yan ◽  
Guangmei Liu ◽  
Tingting Chen ◽  
Xiaochen Fu ◽  
Miao-Miao Niu
Keyword(s):  
Cell Cycle ◽  
Virtual Screening ◽  
Fold Increase ◽  
Pharmacophore Modeling ◽  
Biological Evaluation ◽  
Peptide Inhibitors ◽  
Regulatory Proteins ◽  
Competitive Binding Assay ◽  
M Phase ◽  
Cell Cycle Regulatory Proteins

The polo-box domain of polo-like kinase 1 (PLK1-PBD) is proved to have crucial roles in cell proliferation. Designing PLK1-PBD inhibitors is challenging due to their poor cellular penetration. In this study, we applied a virtual screening workflow based on a combination of structure-based pharmacophore modeling with molecular docking screening techniques, so as to discover potent PLK1-PBD peptide inhibitors. The resulting 9 virtual screening peptides showed affinities for PLK1-PBD in a competitive binding assay. In particular, peptide 5 exhibited an approximately 100-fold increase in inhibitory activity (IC50 = 70 nM), as compared with the control poloboxtide. Moreover, cell cycle experiments indicated that peptide 5 effectively inhibited the expression of p-Cdc25C and cell cycle regulatory proteins by affecting the function of PLK1-PBD, thereby inducing mitotic arrest at the G2/M phase. Overall, peptide 5 can serve as a potent lead for further investigation as PLK1-PBD inhibitors.

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