The APC/C E3 ligase subunit ANAPC11 mediates FOXO3 protein degradation to promote cell proliferation and lymph node metastasis in urothelial bladder cancer.

Urothelial bladder cancer (UBC) is one of the most prevalent malignancies worldwide, with striking tumor heterogeneity. Elucidating the molecular mechanisms that can be exploited for the treatment of aggressive UBC is a particularly relevant goal. Protein ubiquitination is a critical post-translational modification (PTM) that mediates the degradation of target protein via the proteasome. However, the roles of aberrant protein ubiquitination in UBC development and the underlying mechanisms by which it drives tumor progression remain unclear. In this study, taking advantage of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 technology, we identified the ubiquitin E3 ligase ANAPC11, a critical subunit of the anaphase-promoting complex/cyclosome (APC/C), as a potential oncogenic molecule in UBC cells. Our clinical analysis showed that elevated expression of ANAPC11 was significantly correlated with high T stage, positive lymph node (LN) metastasis, and poor outcomes in UBC patients. By employing a series of in vitro experiments, we demonstrated that ANAPC11 enhanced the proliferation and invasiveness of UBC cells, while knockout of ANAPC11 inhibited the growth and LN metastasis of UBC cells in vivo. By conducting immunoprecipitation coupled with mass spectrometry, we confirmed that ANAPC11 increased the ubiquitination level of the Forkhead transcription factor FOXO3. The resulting decrease in FOXO3 protein stability led to the downregulation of the cell cycle regulator p21 and decreased expression of GULP1, a downstream effector of androgen receptor signaling. Taken together, these findings indicated that ANAPC11 plays an oncogenic role in UBC by modulating FOXO3 protein degradation. The ANAPC11-FOXO3 regulatory axis might serve as a novel therapeutic target for UBC.

Zinc supplementation prevents mitotic accumulation in human keratinocyte cell lines upon environmentally relevant arsenic exposure.

Disrupted cell cycle progression underlies the molecular pathogenesis of multiple diseases. Chronic exposure to inorganic arsenic (iAs) is a global health issue leading to multi-organ cancerous and non-cancerous diseases. Exposure to supratherapeutic concentrations of iAs causes cellular accumulation in G2 or M phase of the cell cycle in multiple cell lines by inducing cyclin B1 expression. It is not clear if iAs exposure at doses corresponding to serum levels of chronically exposed populations (∼100 nM) has any effect on cell cycle distribution. In the present study we investigated if environmentally relevant iAs exposure induced cell cycle disruption and mechanisms thereof employing two human keratinocyte cell lines (HaCaT and Ker-CT), flow cytometry, immunoblots and quantitative real-time PCR (qRT-PCR). iAs exposure (100 nM; 24 h) led to mitotic accumulation of cells in both cell lines, along with the stabilization of ANAPC11 ubiquitination targets cyclin B1 and securin, without affecting their steady state mRNA levels. This result suggested that induction of cyclin B1 and securin is modulated at the level of protein degradation. Moreover, zinc supplementation successfully prevented iAs-induced mitotic accumulation and stabilization of cyclin B1 and securin without affecting their mRNA levels. Together, these data suggest that environmentally relevant iAs exposure leads to mitotic accumulation possibly by displacing zinc from the RING finger subunit of anaphase promoting complex/cyclosome (ANAPC11), the cell cycle regulating E3 ubiquitin ligase. This early cell cycle disruptive effect of environmentally relevant iAs concentration could underpin the molecular pathogenesis of multiple diseases associated with chronic iAs exposure.

LncRNA BCAR4 promotes liver cancer progression by upregulating ANAPC11 expression through sponging miR­1261.

Liver cancer is a malignant tumor that occurs in the liver and can be divided into primary and secondary liver cancer. Long non­coding RNA (lncRNA) breast cancer anti­estrogen resistance 4 (BCAR4) has been demonstrated to promote the development of various types of cancer. However, the function of lncRNA BCAR4 in liver cancer remains unclear. In the present study, the expression of lncRNA BCAR4 was notably elevated in liver cancer compared with adjacent non­tumor tissues. Functional in vitro assays demonstrated that knockdown of lncRNA BCAR4 inhibited the proliferation, migration and invasion of Huh­7 cells. In addition, lncRNA BCAR4 was demonstrated to directly bind to microRNA (miR)­1261, and miR­1261 expression negatively correlated with the expression of lncRNA BCAR4. Through bioinformatics analysis, lncRNA BCAR4 was predicted to target anaphase­promoting complex subunit 11 (ANAPC11) through miR­1261. In addition, the results demonstrated that lncRNA BCAR4 increased the expression of ANAPC11 by inhibiting miR­1261 expression. Consistently, overexpression of ANAPC11 or inhibition of miR­1261 significantly rescued liver cancer cell proliferation induced by knockdown of lncRNA BCAR4. Collectively, the results of the present study demonstrated that lncRNA BCAR4 may promote liver cancer development by directly binding to miR­1261 and targeting ANAPC11.

Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.

One of the challenges encountered by the protein quality control machinery is the need to ensure that members of multiprotein complexes are available in the correct proportions. In this study, we demonstrate that the ubiquitin proteasome system (UPS) mediates the degradation of Apc11, the catalytic core subunit of the anaphase promoting complex/cyclosome (APC/C). In vitro studies have shown that Apc11, together with its E2 enzyme, is sufficient to ubiquitinate substrates independently of the APC/C. Here, we establish that this can occur in living yeast cells. We show that the tight controls regulating the function of the fully assembled APC/C can be circumvented when its substrates are ubiquitinated by the excess levels of Apc11 independently of the assembled complex. We thus suggest that the UPS-mediated degradation of Apc11 is an overlooked mechanism ensuring that proper function of the APC/C is limited to suitably delimited holoenzymes and that an imbalance in protein expression may result in detrimental gain-of-function activity, rather than merely the disruption of protein complex stoichiometry.-Volpe, M., Levinton, N., Rosenstein, N., Prag, G., Ben-Aroya, S. Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.

Integrated analysis highlights APC11 protein expression as a likely new independent predictive marker for colorectal cancer.

After a diagnosis of colorectal cancer (CRC), approximately 50% of patients will present distant metastasis. Although significant progress has been made in treatments, most of them will die from the disease. We investigated the predictive and prognostic potential of APC11, the catalytic subunit of APC/C, which has never been examined in the context of CRC. The expression of APC11 was assessed in CRC cell lines, in tissue microarrays (TMAs) and in public datasets. Overexpression of APC11 mRNA was associated with chromosomal instability, lymphovascular invasion and residual tumor. Regression models accounting for the effects of well-known protein markers highlighted association of APC11 protein expression with residual tumor (odds ratio: OR = 6.51; 95% confidence intervals: CI = 1.54-27.59; P = 0.012) and metastasis at diagnosis (OR = 3.87; 95% CI = 1.20-2.45; P = 0.024). Overexpression of APC11 protein was also associated with worse distant relapse-free survival (hazard ratio: HR = 2.60; 95% CI = 1.26-5.37; P = 0.01) and worse overall survival (HR = 2.69; 95% CI = 1.31-5.51; P = 0.007). APC11 overexpression in primary CRC thus represents a potentially novel theranostic marker of metastatic CRC.

USP9X Limits Mitotic Checkpoint Complex Turnover to Strengthen the Spindle Assembly Checkpoint and Guard against Chromosomal Instability.

Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which delays progression through mitosis until every chromosome has stably attached to spindle microtubules via the kinetochore. We show here that the deubiquitinase USP9X strengthens the SAC by antagonizing the turnover of the mitotic checkpoint complex produced at unattached kinetochores. USP9X thereby opposes activation of anaphase-promoting complex/cyclosome (APC/C) and specifically inhibits the mitotic degradation of SAC-controlled APC/C substrates. We demonstrate that depletion or loss of USP9X reduces the effectiveness of the SAC, elevates chromosome segregation defects, and enhances chromosomal instability (CIN). These findings provide a rationale to explain why loss of USP9X could be either pro- or anti-tumorigenic depending on the existing level of CIN.

ANAPHASE PROMOTING COMPLEX/CYCLOSOME-mediated cyclin B1 degradation is critical for cell cycle synchronization in syncytial endosperms.

Although it is known that in most angiosperms mitosis in early endosperm development is syncytial and synchronized, it is unclear how the synchronization is regulated. We showed previously that APC11, also named ZYG1, in Arabidopsis activates zygote division by interaction and degradation of cyclin B1. Here, we report that the mutation in APC11/ZYG1 led to unsynchronized mitosis and over-accumulation of cyclin B1-GUS in the endosperm. Mutations in two other APC subunits showed similar defects. Transgenic expression of stable cyclin B1 in the endosperm also caused unsynchronized mitosis. Further, downregulation of APC11 generated multi-nucleate somatic cells with unsynchronized mitotic division. Together, our results suggest that APC/C-mediated cyclin B1 degradation is critical for cell cycle synchronization.

A single-nucleotide exon found in Arabidopsis.

The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future.

Atomic structure of the APC/C and its mechanism of protein ubiquitination.

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.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex.

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.

Ubiquitin chain elongation requires E3-dependent tracking of the emerging conjugate.

Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2.

Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly.

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

Knockdown expression of Apc11 leads to cell-cycle distribution reduction in G2/M phase.

Anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase in cell division, which catalyses ubiquitination of cell-cycle regulators. Studying this complex could reveal important information regarding its application in cancer research and therapy. In this study, 4 synthesized small interfering RNAs (siRNAs) were transfected into HEK293T cells to suppress messenger RNA (mRNA) of Apc11; 2 of these reduced the amount of Apc11 mRNA by over 50%. Further experiments showed that rather than causing apoptosis, siRNA transfection led to cell-cycle distributions characterized by less time spent in G2/M phase and more time spent in G1 phase. This phenomenon was specifically induced by Apc11 silencing, as co-transfection of siRNA and an Apc11 plasmid could reverse this distribution bias. Our results suggested that siRNA targeted against Apc11 could hamper entry into G2/M phase. Current efforts are focused on elucidating the function and utility of the APC complex for clinical applications.

Transcription-independent function of Polycomb group protein PSC in cell cycle control.

Polycomb group (PcG) proteins control development and cell proliferation through chromatin-mediated transcriptional repression. We describe a transcription-independent function for PcG protein Posterior sex combs (PSC) in regulating the destruction of cyclin B (CYC-B). A substantial portion of PSC was found outside canonical PcG complexes, instead associated with CYC-B and the anaphase-promoting complex (APC). Cell-based experiments and reconstituted reactions established that PSC and Lemming (LMG, also called APC11) associate and ubiquitylate CYC-B cooperatively, marking it for proteosomal degradation. Thus, PSC appears to mediate both developmental gene silencing and posttranslational control of mitosis. Direct regulation of cell cycle progression might be a crucial part of the PcG system's function in development and cancer.

Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1.

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.

Cell cycle deregulation by a poxvirus partial mimic of anaphase-promoting complex subunit 11.

The anaphase-promoting complex (APC), or cyclosome, is a ubiquitin ligase with major roles in cell cycle regulation. It is required for mitotic exit, but must be deactivated for the G(1)/S phase transition to occur. APC consists of at least 12 subunits with the catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. APC11 facilitates ubiquitin chain formation by recruiting ubiquitin-charged conjugating enzymes through its RING-H2 domain. We report that a small number of poxviruses encode RING-H2 proteins with sequence similarities to APC11. We show that a representative of these viral proteins mimics APC11 in its interactions with APC, but unlike APC11, the viral protein fails to promote ubiquitin chain formation. This absence of ubiquitin ligase activity is linked to a distinctive sequence variation within its RING-H2 domain. Expression of the viral protein led to cell cycle deregulation and the accumulation of APC substrates in a manner consistent with impaired APC function. Our data characterize this protein as a regulator of APC activity, and consequently, we have called it PACR (poxvirus APC/cyclosome regulator). Deletion of the PACR gene substantially reduced viral replication. Here, we report a viral mimic of an APC component and reveal an intriguing mechanism by which viruses can manipulate cell cycle progression and, thereby, promote their own replication.

Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex.

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC's RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC(Cdc20) and APC(Cdh1) complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.