A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.     

Functional analysis of Cullin 3 E3 ligases in tumorigenesis

Cullin 3-RING ligases (CRL3) play pivotal roles in the regulation of various physiological and pathological processes, including neoplastic events. The substrate adaptors of CRL3 typically contain a BTB domain that mediates the interaction between Cullin 3 and target substrates to promote their ubiquitination and subsequent degradation. The biological implications of CRL3 adaptor proteins have been well described where they have been found to play a role as either an oncogene, tumor suppressor, or can mediate either of these effects in a context-dependent manner. Among the extensively studied CRL3-based E3 ligases, the role of the adaptor protein SPOP (speckle type BTB/POZ protein) in tumorigenesis appears to be tissue or cellular context dependent. Specifically, SPOP acts as a tumor suppressor via destabilizing downstream oncoproteins in many malignancies, especially in prostate cancer. However, SPOP has largely an oncogenic role in kidney cancer. Keap1, another well-characterized CRL3 adaptor protein, likely serves as a tumor suppressor within diverse malignancies, mainly due to its specific turnover of its downstream oncogenic substrate, NRF2 (nuclear factor erythroid 2-related factor 2). In accordance with the physiological role the various CRL3 adaptors exhibit, several pharmacological agents have been developed to disrupt its E3 ligase activity, therefore blocking its potential oncogenic activity to mitigate tumorigenesis.     

The Arabidopsis Anaphase Promoting Complex (APC): Regulation through Subunit Availability in Plant Tissues

Sister-chromatid separation and exit from mitosis require ubiquitin-mediated proteolysis of cellcycle regulators such as cyclin B and securin. The specificity of the reaction is controlled by anubiquitin-ligase multiprotein complex known as APC (Anaphase Promoting Complex).Comparison of the coding sequences of Arabidopsis genes with the Genbank database revealsextensive homology of the predicted ORFs with the corresponding proteins of other eukaryotes,indicating that the APC is well conserved in plants. However, different from other eukaryotes, theArabidopsis genes have some particular characteristics, such as the presence of two copies of theCDC27 gene. Furthermore, expression analyses of the AtAPC genes disclose complex profilesthat differ, depending on the tissue examined. In actively dividing cell suspensions there is adirect correspondence between the rates of proliferation and mRNA levels from the AtAPCcomponents. On the other hand, in plant organs, dark-grown seedlings and during leaf growth,this correlation is lost and the AtAPC genes are highly expressed in tissues with low overall celldivision. Moreover, expression patterns diverge between the subunit genes, raising the possibilitythat there could be more than one form of the APC, which would execute distinct functionsduring plant development. The results suggest that an important layer of regulation of APC/C inplants could operate through subunit availability in specific tissues and/or cellular compartments.     

Cullin-containing E3 ubiquitin ligases in plant development

In eukaryotes, the ubiquitin-proteasome system participates in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their poly-ubiquitylation, a prerequisite for their degradation by the 26S proteasome. On the basis of the analysis of the Arabidopsis genome sequence, it is predicted that there are more than 1000 E3 ubiquitin ligases in plants. Several types of E3 ubiquitin ligases have already been characterized in eukaryotes. Recently, some of these E3 enzymes have been implicated in specific plant signaling pathways.     

Regulated Separation of Sister Centromeres depends on the Spindle Assembly Checkpoint but not on the Anaphase Promoting Complex/Cyclosome

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.

Supplemental Figures     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

Glucose and ras activity influence the ubiquitin ligases APC/C and SCF in Saccharomyces cerevisiae

In budding yeast, the Ras/cAMP pathway is involved in the coordination of cell growth and cell division. Glucose-rich medium stimulates Ras/cAMP signaling, which causes an increase in the critical cell size for cell cycle entry. Here we show that glucose and activated Ras proteins also influence the function of the anaphase-promoting complex (APC/C), a ubiquitin-protein ligase required for sister chromatid separation and mitotic exit. We found that apc10-22 and other mutants defective in the APC/C are suppressed by reduced Ras signaling activity, by a deletion of the RAS2 gene, by a cdc25 mutation, by elevated levels of PDE2, or by growth without glucose. Viability of these mutants is also enhanced by decreased Cdk1 activity. In contrast, a constitutively activated RAS2(Val19) allele or shifts to glucose medium are deleterious to apc10-22 mutants. Remarkably, cdc34-2 mutants, which are impaired in SCF function, are differently affected with respect to Ras activity. Viability of cdc34-2 mutants at elevated temperatures is dependent on glucose and the RAS2 gene. We conclude that glucose and Ras proteins influence the APC/C and the SCF complex in an opposite manner. These ubiquitin ligases might represent novel targets for modulating cell division in response to growth conditions.     

A novel family of membrane-bound E3 ubiquitin ligases

A novel E3 ubiquitin ligase family that consists of viral E3 ubiquitin ligases (E3s) and their mammalian homologues was recently discovered. These novel E3s are membrane-bound molecules that share the secondary structure and catalytic domain for E3 activity. All family members have two transmembrane regions at the center and a RING-CH domain at the amino terminus. Forced expression of these novel E3s has been shown to reduce the surface expression of various membrane proteins through ubiquitination of target molecules. Initial examples of viral E3s were identified in Kaposi's sarcoma associated herpesvirus (KSHV) and murine gamma-herpesvirus 68 (MHV-68) and have been designated as modulator of immune recognition (MIR) 1, 2 and mK3, respectively. MIR 1, 2 and mK3 are able to down-regulate MHC class I molecule expression, and mK3 is required to establish an effective latent viral infection in vivo. The first characterized mammalian homologue to MIR 1, 2 and mK3 is c-MIR/MARCH VIII. Forced expression of c-MIR/MARCH VIII down-regulates B7-2, a co-stimulatory molecule important for antigen presentation. Subsequently, several mammalian molecules related to c-MIR/MARCH VIII have been characterized and named as membrane associated RING-CH (MARCH) family. However, the precise physiological function of MARCH family members remains as yet unknown.     

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.     

Cdh1, a Substrate-recruiting Component of Anaphase-promoting Complex/Cyclosome (APC/C) Ubiquitin E3 Ligase,Specifically Interacts with Phosphatase and Tensin Homolog (PTEN) and Promotes Its Removal from Chromatin

A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on Ser-380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains, and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on Ser-380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, the PTEN interaction with Cdh1 was phosphatase-independent, and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.     

E3 ubiquitin ligases and their control of T cell autoreactivity

A loss of T cell tolerance underlies the development of most autoimmune diseases. The design of therapeutic strategies to reinstitute immune tolerance, however, is hampered by uncertainty regarding the molecular mechanisms involved in the inactivation of potentially autoreactive T cells. Recently, E3 ubiquitin ligases have been shown to mediate the development of a durable state of unresponsiveness in T cells called clonal anergy. In this review, we will discuss the mechanisms used by E3 ligases to control the activation of T cells and prevent the development of autoimmunity.