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The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C–coactivator complexes with either Emi1 or a UbcH10–ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10–ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo . A cryo-electron microscopy determination of the atomic structures of anaphase-promoting complex (APC/C)–coactivator complexes with either Emi1 or a UbcH10–ubiquitin conjugate. APC/C ubiquitination mechanism This paper describes the atomic structures of the anaphase-promoting complex (APC/C) bound to the coactivator Cdh1 and the early mitotic inhibitor Emi1, or the E2 enzyme UbcH10 conjugated to ubiquitin. The (APC/C) is a multisubunit E3 ubiquitin ligase enzyme that controls chromosome segregation and the subsequent exit from mitotic cell division. The resolutions the authors achieved (3.6 Å and 4.1 Å) allow them to define the architecture of all APC/C subunits and inter-subunit interactions within the assembly and the position of the catalytic module. The structures explain how Emi1 mediates inhibition of not just UbcH10 but also another E2 enzyme, Ube2S. Among other insights gained is how the association of APC/C with the coactivator Cdh1 is antagonized by Cdh1 phosphorylation.

Phosphorylation of the anaphase-promoting complex (APC/C) allows for its control by the co-activator Cdc20; a mechanism that has relevance to understanding the control of other large multimeric complexes by phosphorylation. Coactivator Cdc20 control of APC/C The anaphase-promoting complex/cyclosome (APC/C) is a large E3 ubiquitin ligase that coordinates sister chromatid segregation, cytokinesis and the initiation of chromosome duplication. It is regulated by elaborate mechanisms including by its coactivator subunits (Cdc20 and Cdh1), reversible phosphorylation, and the spindle assembly checkpoint. Here using cryo-electron microscopy and biochemical analysis, David Barford and colleagues define how mitotic phosphorylation of APC/C allows for its control by Cdc20. Of almost 150 phospho-sites in mitotic APC/C, only a few directly regulate Cdc20 binding through displacement of a newly identified auto-inhibitory segment. This study has relevance to understanding the control of other large multimeric complexes by multi-site phosphorylation. In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase 1 , 2 . The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons 3 , and enhance the affinity of the APC/C for its cognate E2 (refs 4 , 5 , 6 ). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C 7 , 8 , 9 , 10 , 11 , 12 , whereas phosphorylation of Cdh1 prevents its association with the APC/C 9 , 13 , 14 . Since both coactivators associate with the APC/C through their common C-box 15 and Ile-Arg tail motifs 16 , 17 , the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk–cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl- l -arginine methyl ester, preferentially suppresses APC/C Cdc20 rather than APC/C Cdh1 , and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

Significance The anaphase-promoting complex/cyclosome (APC) is a multisubunit RING E3 ubiquitin (Ub) ligase that regulates mitosis, meiosis, and numerous facets of neurobiology by targeting key regulatory proteins for Ub-mediated degradation. Despite great importance, it remains unclear how APC, or most of the other 600 RING E3s in humans, targets Ub to lysines in disordered substrates. Here, we report the structural and molecular basis for substrate ubiquitination by APC and its partner E2, UBCH10. UBCH10 is recruited to APC, activated for ubiquitination, and positioned for substrate targeting through multisite interactions with the APC cullin–RING core. We propose that many RING E3–E2 assemblies work similarly, with multisite interactions establishing specificity, harnessing ubiquitination machineries to accelerate searching for target lysines, and facilitating regulation. For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2∼Ub catalytic modules such as APC11–UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC Cᴰᴴ¹ and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC Cᴰᴴ¹–UBCH10∼Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.

Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However, the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by CDK1 in complex with p9/Cks2 (a CDK regulatory subunit) controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognized subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target, and our findings may facilitate the development of specific inhibitors.

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

The corneal endothelium is vital for transparency and proper hydration of the cornea. Here, we conduct a genome-wide association study of corneal endothelial cell density (cells/mm 2 ), coefficient of cell size variation (CV), percentage of hexagonal cells (HEX) and central corneal thickness (CCT) in 6,125 Icelanders and find associations at 10 loci, including 7 novel. We assess the effects of these variants on various ocular biomechanics such as corneal hysteresis (CH), as well as eye diseases such as glaucoma and corneal dystrophies. Most notably, an intergenic variant close to ANAPC1 (rs78658973[A], frequency = 28.3%) strongly associates with decreased cell density and accounts for 24% of the population variance in cell density (β = −0.77 SD, P  = 1.8 × 10 −314 ) and associates with increased CH (β = 0.19 SD, P  = 2.6 × 10 −19 ) without affecting risk of corneal diseases and glaucoma. Our findings indicate that despite correlations between cell density and eye diseases, low cell density does not increase the risk of disease. The corneal endothelium is crucial for proper vision. Here, Ivarsdottir et al. perform genome-wide association studies for various corneal endothelial cell measurements and find that an intergenic variant near ANAPC1 explains 24% of the variance of endothelial cell density and associates with corneal hysteresis.

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).

Mitochondrial ATP-Mg/Pi carriers import adenine nucleotides into the mitochondrial matrix and export phosphate to the cytosol. They are calcium-regulated to control the size of the matrix adenine nucleotide pool in response to cellular energetic demands. They consist of three domains: an N-terminal regulatory domain containing four calcium-binding EF-hands, a linker loop domain with an amphipathic α-helix and a C-terminal mitochondrial carrier domain for the transport of substrates. Here, we use thermostability assays to demonstrate that the carrier is regulated by calcium via a locking pin mechanism involving the amphipathic α-helix. When calcium levels in the intermembrane space are high, the N-terminus of the amphipathic α-helix is bound to a cleft in the regulatory domain, leading to substrate transport by the carrier domain. When calcium levels drop, the cleft closes, and the amphipathic α-helix is released to bind to the carrier domain via its C-terminus, locking the carrier in an inhibited state.

The anaphase-promoting complex/cyclosome (APC/C) is key to cell cycle regulation and is an E3 ubiquitin ligase. The overall shape of the substrate-free complex has previously been determined in various species. Budding yeast and human APC/Cs are now analyzed by EM structural and biochemical approaches to assess the substrate-binding site and elucidate the relative positioning of Doc1, a factor known to promote processive substrate ubiquitylation. The anaphase-promoting complex/cyclosome (APC/C) is a 22S ubiquitin ligase complex that initiates chromosome segregation and mitotic exit. We have used biochemical and electron microscopic analyses of Saccharomyces cerevisiae and human APC/C to address how the APC/C subunit Doc1 contributes to recruitment and processive ubiquitylation of APC/C substrates, and to understand how APC/C monomers interact to form a 36S dimeric form. We show that Doc1 interacts with Cdc27, Cdc16 and Apc1 and is located in the vicinity of the cullin–RING module Apc2–Apc11 in the inner cavity of the APC/C. Substrate proteins also bind in the inner cavity, in close proximity to Doc1 and the coactivator Cdh1, and induce conformational changes in Apc2–Apc11. Our results suggest that substrates are recruited to the APC/C by binding to a bipartite substrate receptor composed of a coactivator protein and Doc1.

Delaying mitotic cell cycle progression has been proposed as a strategy to potentiate the effects of anti-mitotic anti-cancer drugs that induce multipolar mitotic spindles. Toward this end, we have performed an in silico docking screen targeting anaphase-promoting complex/cyclosome subunit 1 (APC1) at a conserved 10-amino acid surface site that was modeled to interact via a single hydrogen bond with the essential mitotic anaphase-promoting complex/cyclosome (APC/C) co-factor cell division cycle 20 (CDC20). Five molecules were identified after screening 15,000 small molecules. As a secondary in cellulo bioactivity screening, MDA-MB-231 genomically unstable aneuploid breast cancer cells were exposed to each compound in the absence and presence of 10 nM paclitaxel or 1 nM eribulin, the likely clinically relevant doses of these drugs in these cells. Two of the five compounds, which share a common 2-(trifluoromethyl)quinazolin-4-amine chemical structure, induced elevated levels of cell death in combination with paclitaxel, as observed by fluorescence-activated cell sorting (FACS). These two compounds will now serve as a starting point for further optimization and target validation experiments and for additional in silico screens in search of other chemically related small molecules that display more potent but specific anti-cancer cell effects.