Control the Host Cell Cycle: Viral Regulation of the Anaphase-Promoting Complex

Viruses commonly manipulate cell cycle progression to create cellular conditions that are most beneficial to their replication. To accomplish this feat, viruses often target critical cell cycle regulators in order to have maximal effect with minimal input. One such master regulator is the large, multisubunit E3 ubiquitin ligase anaphase-promoting complex (APC) that targets effector proteins for ubiquitination and proteasome degradation. The APC is essential for cells to progress through anaphase, exit from mitosis, and prevent a premature entry into S phase. These far-reaching effects of the APC on the cell cycle are through its ability to target a number of substrates, including securin, cyclin A, cyclin B, thymidine kinase, geminin, and many others. Recent studies have identified several proteins from a number of viruses that can modulate APC activity by different mechanisms, highlighting the potential of the APC in driving viral replication or pathogenesis. Most notably, human cytomegalovirus (HCMV) protein pUL21a was recently identified to disable the APC via a novel mechanism by targeting APC subunits for degradation, both during virus infection and in isolation. Importantly, HCMV lacking both viral APC regulators is significantly attenuated, demonstrating the impact of the APC on a virus infection. Work in this field will likely lead to novel insights into viral replication and pathogenesis and APC function and identify novel antiviral and anticancer targets. Here we review viral mechanisms to regulate the APC, speculate on their roles during infection, and identify questions to be addressed in future studies.     

病毒感染对细胞周期调控影响的研究进展

大量研究表明,病毒感染细胞时,病毒编码的蛋白或DNA可以扰乱细胞周期通路:促进细胞向S期转化或者使细胞静息于G2/M期。在细胞内,细胞周期的调控机制十分复杂,其包含了由DNA损伤导致的细胞通路活化及其他方式。关于病毒对细胞周期的调控方式及细胞周期的改变对于病毒感染的研究已取得一定进展。对于病毒的此类研究可以揭示细胞活动中的关键调控因子及细胞周期检查点的具体分子机理。对病毒调控宿主细胞周期以达到自身最大化复制的机理进行综述。     

Structure, function and mechanism of the anaphase promoting complex (APC/C)

The complex molecular events responsible for coordinating chromosome replication and segregation with cell division and growth are collectively known as the cell cycle. Progression through the cell cycle is orchestrated by the interplay between controlled protein synthesis and degradation and protein phosphorylation. Protein degradation is primarily regulated through the ubiquitin proteasome system, mediated by two related E3 protein ubiquitin ligases, the Skp1 cullin F-box (SCF) and the anaphase promoting complex (also known as the cyclosome) (APC/C). The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that regulates progression through the mitotic phase of the cell cycle and controls entry into S phase by catalysing the ubiquitylation of cyclins and other cell cycle regulatory 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. The aim of this article is to discuss the APC/C from a structural and mechanistic perspective. Although an atomic structure of the APC/C is still lacking, a combination of genetic, biochemical, electron microscopy studies of intact APC/C and crystallographic analysis of individual subunits, together with analogies to evolutionarily related E3 ligases of the RING family, has provided deep insights into the molecular mechanisms of catalysis and substrate recognition, and structural organisation of the APC/C.     

Cezanne/OTUD7B is a cell cycle‐regulated deubiquitinase that antagonizes the degradation of APC/C substrates

The anaphase‐promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and key regulator of cell cycle progression. Since APC/C promotes the degradation of mitotic cyclins, it controls cell cycle‐dependent oscillations in cyclin‐dependent kinase (CDK) activity. Both CDKs and APC/C control a large number of substrates and are regulated by analogous mechanisms, including cofactor‐dependent activation. However, whereas substrate dephosphorylation is known to counteract CDK, it remains largely unknown whether deubiquitinating enzymes (DUBs) antagonize APC/C substrate ubiquitination during mitosis. Here, we demonstrate that Cezanne/OTUD7B is a cell cycle‐regulated DUB that opposes the ubiquitination of APC/C targets. Cezanne is remarkably specific for K11‐linked ubiquitin chains, which are formed by APC/C in mitosis. Accordingly, Cezanne binds established APC/C substrates and reverses their APC/C‐mediated ubiquitination. Cezanne depletion accelerates APC/C substrate degradation and causes errors in mitotic progression and formation of micronuclei. These data highlight the importance of tempered APC/C substrate destruction in maintaining chromosome stability. Furthermore, Cezanne is recurrently amplified and overexpressed in numerous malignancies, suggesting a potential role in genome maintenance and cancer cell proliferation.     

The Role of the Anaphase-Promoting Complex/Cyclosome in Plant Growth

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that plays a major role in the progression of the eukaryotic cell cycle. This unusual protein complex targets key cell cycle regulators, such as mitotic cyclins and securins, for degradation via the 26S proteasome by ubiquitination, triggering the metaphase-to-anaphase transition and exit from mitosis. Because of its essential role in cell cycle regulation, the APC/C has been extensively studied in mammals and yeasts, but relatively less in plants. Evidence shows that, besides its well-known role in cell cycle regulation, the APC/C also has functions beyond the cell cycle. In metazoans, the APC/C has been implicated in cell differentiation, disease control, basic metabolism and neuronal survival. Recent studies also have shed light on specific functions of the APC/C during plant development. Plant APC/C subunits and activators have been reported to play a role in cellular differentiation, vascular development, shoot branching, female and male gametophyte development and embryogenesis. Here, we discuss our current understanding of the APC/C controlling plant growth.     

Who guards the guardian? Mechanisms that restrain APC/C during the cell cycle

The cell cycle is principally controlled by Cyclin Dependent Kinases (CDKs), whose oscillating activities are determined by binding to Cyclin coactivators. Cyclins exhibit dynamic changes in abundance as cells pass through the cell cycle. The sequential, timed accumulation and degradation of Cyclins, as well as many other proteins, imposes order on the cell cycle and contributes to genome maintenance. The destruction of many cell cycle regulated proteins, including Cyclins A and B, is controlled by a large, multi-subunit E3 ubiquitin ligase termed the Anaphase Promoting Complex/Cyclosome (APC/C). APC/C activity is tightly regulated during the cell cycle. Its activation state increases dramatically in mid-mitosis and it remains active until the end of G1 phase. Following its mandatory inactivation at the G1/S boundary, APC/C activity remains low until the subsequent mitosis. Due to its role in guarding against the inappropriate or untimely accumulation of Cyclins, the APC/C is a core component of the cell cycle oscillator. In addition to the regulation of Cyclins, APC/C controls the degradation of many other substrates. Therefore, it is vital that the activity of APC/C itself be tightly guarded. The APC/C is most well studied for its role and regulation during mitosis. However, the APC/C also plays a similarly important and conserved role in the maintenance of G1 phase. Here we review the diverse mechanisms counteracting APC/C activity throughout the cell cycle and the importance of their coordinated actions on cell growth, proliferation, and disease.     

Preventing Fatal Destruction: Inhibitors of the Anaphase-Promoting Complex in Meiosis

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitinligasewhose major functions in the cell cycle are the initiation of sister chromatidseparation and the inactivation of cyclin-dependent kinases. This complex is alsoessential for meiosis, a specialised form of the cell cycle characterised by twoconsecutive rounds of chromosome segregation. To ensure a proper meiotic cell cycle,the activity of APC/C needs to be tightly controlled. It is now evident that inhibitorsof APC/C play pivotal roles to avert its untimely activation. During prophase I, this ubiquitin-ligase must be kept inactive to prevent precocious sister chromatidseparation. Studies in yeast showed that this inhibition is mediated by a specificsubunit of the complex. Accurate chromosome segregation in meiosis I depends onspindle checkpoint proteins such as Mad2 which delay APC/C activation in responseto an erroneous spindle attachment of chromosomes. Additional APC/C antagonistsare known to block complete cyclin destruction between meiosis I and II, therebyensuring that cyclin dependent kinases remain active and that DNA replication doesnot occur. Inhibitors of APC/C also mediate the cytostatic factor induced metaphase IIarrest of oocytes. This review highlights the current knowledge about the role andrelevance of these diverse regulators of the meiotic APC/C.     

KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Here we show that Bub1 is degraded during mitotic exit and that degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 (APC/C(Cdh1)). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. We further identify two KEN-box motifs on Bub1 that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, our study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1.     

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.     

The ubiquitin proteasome system — Implications for cell cycle control and the targeted treatment of cancer

Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin–proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/C^Cdc20) and establishes a stable G1 phase (APC/C^Cdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

APC/C-Mediated Degradation in Early Mitosis: How to Avoid Spindle Assembly Checkpoint Inhibition

The APC/C is an E3 ubiquitin ligase that, by targeting substrates for proteasomal degradation, plays a major role in cell cycle control. In complex with one of two WD40 activator proteins, Cdc20 or Cdh1, the APC/C is active from early mitosis through to late G1 and during this time targets many critical regulators of the cell cycle for degradation. However, this destruction is carefully ordered to ensure that cell cycle events are executed in a timely fashion. Recent studies have begun to shed light on how the APC/C selects different substrates at different times in the cell cycle. One particular problem is how the APC/C recognizes its first set of substrates, Nek2A and cyclin A, in early mitosis when, at this time, the spindle assembly checkpoint (SAC) inhibits most APC/C-dependent degradation. The answer may lie in how substrates are recruited to the APC/C. While checkpoint-dependent substrates appear to require Cdc20 for recruitment, experiments on the early mitotic substrate Nek2A demonstrate that it can bind the APC/C in the absence of Cdc20. The direct interaction of substrates with core subunits of the APC/C could allow their degradation to proceed unhindered even when the SAC is active.     

The anaphase-promoting complex or cyclosome supports cell survival in response to endoplasmic reticulum stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/C(Cdh1) in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/C(Cdh1) (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/C(Cdh1) under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/C(Cdh1) maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/C(Cdh1) as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults.     

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.     

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

This study shows that the cell cycle E3 ubiquitin ligase APC/C is a regulator of several chromatin regulatory proteins, including the multivalent epigenetic reader and writer UHRF1. Perturbing UHRF1 ubiquitylation and degradation alters cell cycle and DNA methylation patterning, pointing to a key role for cell cycle degradation in shaping chromatin environments.     

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.     

The NOMEGA gene required for female gametophyte development encodes the putative APC6/CDC16 component of the Anaphase Promoting Complex in Arabidopsis

Development of the female gametophyte involves several rounds of nuclear divisions during which nuclei are rearranged and finally cellularized to form a mature seven-celled embryo sac. During these nuclear divisions, key proteins involved in the cell cycle need to be degraded quickly in order to facilitate both the metaphase-anaphase transition stage and late anaphase. Here, we report the characterization of an Arabidopsis mutant nomega, which results in arrest of the embryo sac development at the two-nucleate stage. The NOMEGA gene product shows high homology to the APC6/cell division cycle (CDC)16 subunit of the Anaphase Promoting Complex/Cyclosome (APC/C). The phenotype of the nomega mutant is quite different from that of the hobbit mutant, which had suggested a role for the plant APC/C in auxin signalling. We show that nomega mutant embryo sacs are unable to degrade Cyclin B, an important APC/C substrate, providing further evidence of a role for the NOMEGA gene product and the plant APC/C in cell cycle progression during gametophyte development.     

Defining the role of Emi1 in the DNA replication–segregation cycle

Ordered progression through the cell cycle is essential to maintain genomic stability, and fundamental to this is ubiquitin-mediated proteolysis. In particular, the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase destabilises specific regulators at defined times in the cycle to ensure that each round of DNA replication is followed by cell division. Thus, the proper regulation of the APC/C is crucial in each cell cycle. There are several APC/C regulators that restrict its activity to specific cell cycle phases, and amongst these the early mitotic inhibitor 1 (Emi1) protein has recently come to prominence. Emi1 has been proposed to control APC/C in early mitosis; however, recent evidence questions this role. In this review we discuss new evidence that indicates that Emi1 is essential to restrict APC/C activity in interphase and, by doing so, ensure the proper coordination between DNA replication and mitosis.