APC/C-Cdh1

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets various substrates for proteolysis inside and outside of cell cycle. The activation of APC/C is depended on two WD-40 domain proteins, Cdc20 and Cdh1. While APC/Cdc20 principally regulates mitotic progression, APC/Cdh1 shows a broad spectrum of substrates in and beyond cell cycle. In past several years, numerous biochemical and mouse genetic studies have greatly attracted our attention to the emerging role of APC/Cdh1 in genomic integrity, cellular differentiation and human diseases. This review will aim to summarize the recent expended understanding of APC/Cdh1 in regulating biological function and how its dysfunction may lead to diseases.     

APC/C-Cdh1: From cell cycle to cellular differentiation and genomic integrity

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets various substrates for proteolysis inside and outside of the cell cycle. The activation of APC/C is dependent on two WD-40 domain proteins, Cdc20 and Cdh1. While APC/Cdc20 principally regulates mitotic progression, APC/Cdh1 shows a broad spectrum of substrates in and beyond cell cycle. In the past several years, numerous biochemical and mouse genetic studies have greatly attracted our attention to the emerging role of APC/Cdh1 in genomic integrity, cellular differentiation and human diseases. This review will aim to summarize the recently expanded understanding of APC/Cdh1 in regulating biological function and how its dysfunction may lead to diseases.Key words: APC/C, Cdh1, proteolysis, genomic integrity, signal transduction, differentiation, tumorigenesis     

泛素连接酶APC/C参与的泛素化与细胞周期调节

后期促进复合物/细胞周期体（anaphase promoting complex/cyclosome,APC/C）是一个多功能的泛素连接酶,参与细胞周期、代谢、DNA损伤修复、细胞自噬、凋亡、衰老及肿瘤发生等多种生物学过程。泛素化作为一种重要的翻译后修饰,可通过泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）调控蛋白质的降解。APC/C的分子量巨大,由多个亚基组成,在细胞周期调控中具有重要地位,可以通过介导细胞周期相关蛋白质的泛素化降解从而精确调控细胞周期的转换,并受共激活分子CDC20或CDH1的调控。了解APC/C的结构和功能,对于研究细胞周期及蛋白质翻译后修饰等生物学事件至关重要。近年,对APC/C分子结构和组成的解析工作取得了极大的进展,其在肿瘤中的作用及潜在的治疗应用也受到了关注。本文将着重对APC/C的组成和结构、参与泛素化的具体过程、在细胞周期中的调控和被调控机制以及参与肿瘤生成的最新研究进展进行综述。     

The emerging role of APC/CCdh1 in development

The function of APC/C (anaphase-promoting complex/cyclosome) was initially implicated with the onset of anaphase during mitosis, where its association with Cdc20 targets securin for destruction, thereby allowing the separation of two duplicated daughter genomes. When combined with Cdh1, APC regulates G1/S transition and DNA replication during cell cycle. Beyond cell cycle control, results from recent biochemical and mouse genetic studies have attracted our attention to the unexpected impact of APC/C(Cdh1) in cellular differentiation, genomic integrity and pathogenesis of various diseases. This review will aim to summarize current understanding of APC/C(Cdh1) in regulating crucial events during development.     

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.     

Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that primarily governs cell cycle progression. APC/C is composed of at least 14 core subunits and recruits its substrates for ubiquitination via one of the two adaptor proteins, Cdc20 or Cdh1, in M or M/early G1 phase, respectively. Furthermore, recent studies have shed light on crucial functions for APC/C in maintaining genomic integrity, neuronal differentiation, cellular metabolism and tumorigenesis. To gain better insight into the in vivo physiological functions of APC/C in regulating various cellular processes, particularly development and tumorigenesis, a number of mouse models of APC/C core subunits, coactivators or inhibitors have been established and characterized. However, due to their essential role in cell cycle regulation, most of the germline knockout mice targeting the APC/C pathway are embryonic lethal, indicating the need for generating conditional knockout mouse models to assess the role in tumorigenesis for each APC/C signaling component in specific tissues. In this review, we will first provide a brief introduction of the ubiquitin-proteasome system (UPS) and the biochemical activities and cellular functions of the APC/C E3 ligase. We will then focus primarily on characterizing genetic mouse models used to understand the physiological roles of each APC/C signaling component in embryogenesis, cell proliferation, development and carcinogenesis. Finally, we discuss future research directions to further elucidate the physiological contributions of APC/C components during tumorigenesis and validate their potentials as a novel class of anti-cancer targets.     

Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology

The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons--a function that could become impaired in Alzheimer's and other neurodegenerative diseases.     

APC as a mobile scaffold: regulation and function at the nucleus, centrosomes, and mitochondria

Genetic mutations of adenomatous polyposis coli (APC) predispose to high risk of human colon cancer. APC is a large tumor suppressor protein and truncating mutations disrupt its normal roles in regulating cell migration, DNA replication/repair, mitosis, apoptosis, and turnover of oncogenic β-catenin. APC is targeted to multiple subcellular sites, and here we discuss recent evidence implicating novel protein interactions and functions of APC in the nucleus and at centrosomes and mitochondria. The ability of APC to shuttle between these and other cell locations is hypothesized to be integral to its cellular function.     

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.     

Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.     

The Arabidopsis anaphase-promoting complex or cyclosome: molecular and genetic characterization of the APC2 subunit

In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis. We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-beta-glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems. Our observations support a conserved function of the APC/C in plants but a different mode of regulation.     

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.     

Association of Hsp90 with p53 and Fizzy related homolog (Fzr) synchronizing Anaphase Promoting Complex (APC/C): An unexplored ally towards oncogenic pathway

The intricate molecular interactions leading to the oncogenic pathway are the consequence of cell cycle modification controlled by a bunch of cell cycle regulatory proteins. The tumor suppressor and cell cycle regulatory proteins work in coordination to maintain a healthy cellular environment. The integrity of this cellular protein pool is perpetuated by heat shock proteins/chaperones, which assist in proper protein folding during normal and cellular stress conditions. Among these versatile groups of chaperone proteins, Hsp90 is one of the significant ATP-dependent chaperones that aid in stabilizing many tumor suppressors and cell cycle regulator protein targets. Recently, studies have revealed that in cancerous cell lines, Hsp90 stabilizes mutant p53, ‘the guardian of the genome.’ Hsp90 also has a significant impact on Fzr, an essential regulator of the cell cycle having an important role in the developmental process of various organisms, including Drosophila, yeast, Caenorhabditis elegans, and plants. During cell cycle progression, p53 and Fzr coordinately regulate the Anaphase Promoting Complex (APC/C) from metaphase to anaphase transition up to cell cycle exit. APC/C mediates proper centrosome function in the dividing cell. The centrosome acts as the microtubule organizing center for the correct segregation of the sister chromatids to ensure perfect cell division. This review examines the structure of Hsp90 and its co-chaperones, which work in synergy to stabilize proteins such as p53 and Fizzy-related homolog (Fzr) to synchronize the Anaphase Promoting Complex (APC/C). Dysfunction of this process activates the oncogenic pathway leading to the development of cancer. Additionally, an overview of current drugs targeting Hsp90 at various phases of clinical trials has been included.     

Protective Effects of Non-Anticoagulant Activated Protein C Variant (D36A/L38D/A39V) in a Murine Model of Ischaemic Stroke

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t_½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t_½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.     

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C^Cdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC^Cdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C^Cdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.     

Differential CD91 dependence for calreticulin and Pseudomonas exotoxin-A endocytosis

Calreticulin (CRT) interaction with cell-surface receptors is integral to its function in escorting associated peptides into the antigen-presenting cell (APC) antigen presentation pathway. Additionally, extracellular CRT is proposed to be required for lung APC interaction with collectins. In both cases, CD91 has been proposed to act as the APC cell-surface receptor requisite for mediating these processes. However, the evidence for a CRT interaction with CD91 is indirect, predicated on partial competition of cellular binding by gp96, of which CD91 has been proposed as the unique endocytic receptor, and by the CD91 ligand alpha2-macroglobulin. Here, we directly investigate the function of CD91 in binding and trafficking CRT. We find that the ability of CRT to interact with APC does not correlate with cellular CD91 expression or function. Additionally, in the first genetic test of CD91 function regarding CRT, CD91 expression neither conferred CRT association nor did CD91-deficient (CD91-/-) and CD91-expressing cells differ in their ability to traffic CRT. Finally, cellular CRT trafficking did not parallel that of Pseudomonas exotoxin-A, an obligate CD91 ligand, by the criteria of CD91 dependence, cell-type specificity and endocytic itinerary. These data identify that CRT trafficking is not, as previously hypothesized, CD91 dependent and indicate usage of alternative cellular trafficking pathways.