Anaphase-promoting Complex Research Articles

Fzr1 Preserves Hematopoietic Stem Cell Quiescence through Inhibiting Runx1

Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. FZR1, as a regulatory subunit of anaphase promoting complex/cyclosome (APC), is a master regulator of cell cycle, but little is known with regard to its role in HSC quiescence. Thus, we examined the function of Fzr1 in HSCs during steady-state hematopoiesis and under stress. We demonstrate that Fzr1 deletion led to perturbed hematopoiesis with an approximately 2-fold decrease in HSC pool size, as consequence of Fzr1 loss driving HSC from quiescence into rapid cycling (~70.1% to ~28.4%), elevating proliferation (~4.58% to ~18.5%) and apoptosis (~0.23% to ~2.86%) in HSC. As shown by serial bone marrow transplantation and competitive repopulation assays, self-renewal capacity and regenerative capacity were impaired, but differentiation of HSCs was not affected post Fzr1 deletion. Mechanistically, Fzr1 loss led to upregulation of Runx1 expression, in line with the protein expression, the ubiquitin sites of the gene was also obviouly decreased, knockdown Runx1 can rescue the Fzr1-deficient phenotype in HSC. Together, our data therefore support that Fzr1 acts a positive regulator of HSC quiescence and self-renewal capacity through inhibiting Runx1 expression. Disclosures No relevant conflicts of interest to declare.

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis.

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.

Quantitative Proteomic Analysis of Porcine Intestinal Epithelial Cells Infected with Porcine Deltacoronavirus Using iTRAQ-Coupled LC-MS/MS.

Porcine deltacoronavirus (PDCoV) is an emergent enteropathogenic coronavirus associated with swine diarrhea. Porcine small intestinal epithelial cells (IPEC) are the primary target cells of PDCoV infection in vivo. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled to liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used to quantitatively identify differentially expressed proteins (DEPs) in PDCoV-infected IPEC-J2 cells. A total of 78 DEPs, including 23 upregulated and 55 downregulated proteins, were identified at 24 h postinfection. The data are available via ProteomeXchange with identifier PXD019975. To ensure reliability of the proteomics data, two randomly selected DEPs, the downregulated anaphase-promoting complex subunit 7 (ANAPC7) and upregulated interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), were verified by real-time PCR and Western blot, and the results of which indicate that the proteomics data were reliable and valid. Bioinformatics analyses, including GO, COG, KEGG, and STRING, further demonstrated that a majority of the DEPs are involved in numerous crucial biological processes and signaling pathways, such as immune system, digestive system, signal transduction, RIG-I-like receptor, mTOR, PI3K-AKT, autophagy, and cell cycle signaling pathways. Altogether, this is the first study on proteomes of PDCoV-infected host cells, which shall provide valuable clues for further investigation of PDCoV pathogenesis.

MDA-MB-157 Cell Line Presents High Levels of MAD2L2 and Dysregulated Mitosis.

Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this work, we focused on MAD2L2 (REV7), a central translesion (TLS) protein, which also functions as a mitotic regulator by inhibiting APC/C in prometaphase. Using bioinformatics analysis, live cell imaging and APC/C protein binding and degradation assays, we explored the influence of MAD2L2 over-expression in breast cancer. A significant over-expression of MAD2L2 was found in triple negative breast cancers (TNBC), compared to other breast cancers, correlating to poor patient prognosis. We also identified significant over-expression of MAD2L2 in the MDA-MB-157 triple negative (TN) cell line. A high percentage of MDA-MB-157 cells failed to complete mitosis and died during mitosis or shortly after. In addition, these cells completed mitosis at a significantly slower rate than control cells. MDA-MB-157 cells present high levels of mitotic slippage upon nocodazole treatment and acute dysregulation in APC/C function and substrate degradation. Moreover, silencing of MAD2L2 in the MDA-MB-157 cell line improved mitotic phenotypes. MAD2L2 over-expression supports the carcinogenic phenotype of MDA-MB-157 cells by promoting uncontrolled mitosis.

APC/CCdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit.

ABSTRACTDuring mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/CCdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/CCdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.

The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1 Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.

MAD2B contributes to parietal epithelial cell activation and crescentic glomerulonephritis via Skp2.

Mitotic spindle assembly checkpoint protein 2 (MAD2B), a well-known anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase-ζ, is critical for mitotic control and DNA repair. Previously, we detected a strong increase of MAD2B in the glomeruli from patients with crescentic glomerulonephritis and anti-glomerular basement membrane (anti-GBM) rats, which predominantly originated from activated parietal epithelial cells (PECs). Consistently, in vitro MAD2B was increased in TNF-α-treated PECs, along with cell activation and proliferation, as well as extracellular matrix accumulation, which could be reversed by MAD2B genetic depletion. Furthermore, we found that expression of S phase kinase-associated protein 2 (Skp2), an APC/CCDH1 substrate, was increased in the glomeruli of anti-GBM rats, and TNF-α-stimulated PECs and could be suppressed by MAD2B depletion. Additionally, genetic deletion of Skp2 inhibited TNF-α-induced PEC activation and dysfunction. Finally, TNF-α blockade or glucocorticoid therapy administered to anti-GBM rats could ameliorate MAD2B and Skp2 accumulation as well as weaken PEC activation. Collectively, our data suggest that MAD2B has a pivotal role in the pathogenesis of glomerular PEC activation and crescent formation through induction of Skp2 expression.

The role of Anaphase Promoting Complex activation, inhibition and substrates in cancer development and progression.

The Anaphase Promoting Complex (APC), a multi-subunit ubiquitin ligase, facilitates mitotic and G1 progression, and is now recognized to play a role in maintaining genomic stability. Many APC substrates have been observed overexpressed in multiple cancer types, such as CDC20, the Aurora A and B kinases, and Forkhead box M1 (FOXM1), suggesting APC activity is important for cell health. We performed BioGRID analyses of the APC coactivators CDC20 and CDH1, which revealed that at least 69 proteins serve as APC substrates, with 60 of them identified as playing a role in tumor promotion and 9 involved in tumor suppression. While these substrates and their association with malignancies have been studied in isolation, the possibility exists that generalized APC dysfunction could result in the inappropriate stabilization of multiple APC targets, thereby changing tumor behavior and treatment responsiveness. It is also possible that the APC itself plays a crucial role in tumorigenesis through its regulation of mitotic progression. In this review the connections between APC activity and dysregulation will be discussed with regards to cell cycle dysfunction and chromosome instability in cancer, along with the individual roles that the accumulation of various APC substrates may play in cancer progression.

CDH1 binds MAD2L2 in a Rev1-like pattern

MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a ‘closed’ active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an ‘open’ inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus β-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2’s C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2’s preferential complex formation.

USP13 controls the stability of Aurora B impacting progression through the cell cycle.

Aurora B kinase plays essential roles in mitosis. Its protein levels increase before the onset of mitosis and sharply decrease during mitosis exit. The latter decrease is due to a balance between the actions of the E3 ubiquitin ligase anaphase-promoting complex or cyclosome (activated by the Cdh1 adapter), and the deubiquitinating enzyme USP35. Aurora B also executes important functions in interphase. Abnormal modulation of Aurora B in interphase leads to cell cycle defects often linked to aberrant chromosomal condensation and segregation. Very little is however known about how Aurora B levels are regulated in interphase. Here we found that USP13-associates with and stabilizes Aurora B in cells, especially before their entry into mitosis. In order for USP13 to exert its stabilizing effect on Aurora B, their association is promoted by the Aurora B-mediated phosphorylation of USP13 at Serine 114. We also present evidence that USP13 instigates Aurora B deubiquitination and/or protect it from degradation in a non-catalytic manner. In addition, we report that genetic or chemical modulation of the cellular levels/activity of USP13 affects unperturbed cell-cycle progression. Overall our study unveils the molecular and cellular connections of the USP13-Aurora B axis, which potentially participates in the rewiring of the cell cycle happening in cancer cells.

Modeling the Control of Meiotic Cell Divisions: Entry, Progression, and Exit

Upon nitrogen starvation, Schizosaccharomyces pombe exit the mitotic cell cycle and become irreversibly committed to the completion of meiosis program. Meiotic cell divisions are coordinated with sporulation events to produce haploid spores. In the last few decades, experiments on fission yeast have revealed different molecular players involved in two meiotic cell divisions, meiosis I (MI) and meiosis II (MII). How the MI entry, MI-to-MII transition, and MII exit occur because of the dynamics of the regulatory network is not well understood. In this work, we developed a comprehensive mathematical model of the network that describes the temporal dynamics of meiotic progression. The model accounts for the phenotypes of several experimental data (single and multiple mutations). We demonstrate the control strategy involving multiple feedback loops to yield two successive division cycles. The differential regulation of anaphase-promoting complex/cyclosome (APC/C) coactivators and its inhibitors is crucial for the dynamics of both MI-to-MII transition and MII exit. This model generates mechanistic insights that help in further experiments and modeling.

The Cyclin CYCA3;4 Is a Postprophase Target of the APC/CCCS52A2 E3-Ligase Controlling Formative Cell Divisions in Arabidopsis.

The anaphase promoting complex/cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CELL DIVISION PROTEIN20 (CDC20) and CELL CYCLE SWITCH52 (CCS52). Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis (Arabidopsis thaliana) CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhance the scored ccs52a2-1 phenotypes. Furthermore, whereas the CYCA3;4 protein is promptly broken down after prophase in wild-type plants, it remains present in later stages of mitosis in ccs52a2-1 mutant plants, marking it as a putative APC/CCCS52A2 substrate. Strikingly, increased CYCA3;4 levels result in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4 gain-of-function plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 contributes to disorganized formative divisions, possibly in part caused by the inactivation of RBR1.

APC2CDH1 negatively regulates agrin signaling by promoting the ubiquitination and proteolytic degradation of DOK7.

Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.

The Possible Role of Point Mutations and Activation of the CDC27 Gene in Progression of Multiple Myeloma

Multiple myeloma (MM) is characterized by malignancy of terminally differentiated B-lymphocytes. Identification of mutations in genes and pathways in MM is crucial to elucidate the pathophysiology of this cancer. Genomic profiling of malignant myeloma cells may give unprecedented results, which may eventually lead to the development of novel drugs. MM is a complex disease with a complex interactive system that consists of dysregulation of different pathways that require precise investigation for designing more effective drugs. The recent advances in high throughput sequencing (HTS) and a better understanding of myeloma's genetic architecture provide yet another approach to uncovering the driver mutations in myeloma. In the present study, we aimed to study whether cell division cycle 27 (CDC27) has a role in MM's malignancy and performed Whole Exome Sequencing (WES) in four clinical cases. Given the importance of the CDC27 gene in cell division, various studies demonstrate in different cancers that the CDC27 gene is associated with tumor proliferation and progression. Analysis of our data in the coding region of MM cases revealed that all four cases under study carrying point mutations in multiple sites of the CDC27 gene.Interestingly, CDC27 plays a pivotal role in the anaphase-promoting complex during cell division. Therefore, we opine that CDC27 may also have a role in MM. We concluded that the effective investigation of translational genomics of CDC27 should be screened from the gene level to the functional level of the protein (bench to bedside research) and studying its possible role in MM cell progression, which might be a novel therapeutic approach.

Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell

FZR1/CDH1 is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. Although the critical role of FZR1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of FZR1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of FZR1. Although ablation of FZR1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of FZR1 phosphorylation, male juvenile germ cells entered meiosis normally but failed to enter meiosis II or form differentiated spermatids. In aged testis, male mutant germ cells were overall abolished, showing Sertoli cell-only phenotype. In contrast, female mutants showed apparently normal progression of meiosis. The present study demonstrated that phosphorylation of FZR1 is required for temporal regulation of APC/C activity at meiosis II entry, and for maintenance of spermatogonia, which raised an insight into the sexual dimorphism of FZR1-regulation in germ cells.

Complex Cartography: Regulation of E2F Transcription Factors by Cyclin F and Ubiquitin.

The E2F family of transcriptional regulators sits at the center of cell cycle gene expression and plays vital roles in normal and cancer cell cycles. Whereas control of E2Fs by the retinoblastoma family of proteins is well established, much less is known about their regulation by ubiquitin pathways. Recent studies placed the Skp1-Cul1-F-box-protein (SCF) family of E3 ubiquitin ligases with the F-box protein Cyclin F at the center of E2F regulation, demonstrating temporal proteolysis of both activator and atypical repressor E2Fs. Importantly, these E2F members, in particular activator E2F1 and repressors E2F7 and E2F8, form a feedback circuit at the crossroads of cell cycle and cell death. Moreover, Cyclin F functions in a reciprocal circuit with the cell cycle E3 ligase anaphase-promoting complex/cyclosome (APC/C), which also controls E2F7 and E2F8. This review focuses on the complex contours of feedback within this circuit, highlighting the deep crosstalk between E2F, SCF-Cyclin F, and APC/C in regulating the oscillator underlying human cell cycles.

Cell division cycle 23 is required for mouse oocyte meiotic maturation.

Precise regulation of chromosome segregation during oocyte meiosis is of vital importance to mammalian reproduction. Anaphase promoting complex/cyclosome (APC/C) is reported to play an important role in metaphase-to-anaphase transition. Here we report that cell division cycle 23 (Cdc23, also known as APC8) plays a critical role in regulating the oocyte chromosome separation. Cdc23 localized on the meiotic spindle, and microinjection of Cdc23 siRNA caused decreased ratios of metaphase-to-anaphase transition. Loss of Cdc23 resulted in abnormal spindles, misaligned chromosomes, errors of homologous chromosome segregation, and production of aneuploid oocytes. Further study showed that inactivation of spindle assembly checkpoint and degradation of Cyclin B1 and securin were disturbed after Cdc23 knockdown. Furthermore, we found that inhibiting spindle assembly checkpoint protein Msp1 partly rescued the decreased polar body extrusion and reduced the accumulation of securin in Cdc23 knockdown oocytes. Taken together, our data demonstrate that Cdc23 is required for the chromosome segregation through regulating the spindle assembly checkpoint activity, and cyclin B1 and securin degradation in meiotic mouse oocytes.

Boosting the apoptotic response of high-grade serous ovarian cancers with CCNE1-amplification to paclitaxel by targeting APC/C and the pro-survival protein MCL-1.

e18065 Background: Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. Methods: To do this, we used a specific anaphase-promoting complex/ cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the anti-apoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells with CCNE1-amplification that are challenged with clinically relevant dose of paclitaxel. Results: Using time-laps microscopy we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cells and restores death in mitosis (DIM). Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient´s survival. Conclusions: Our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel efficacy in this highly lethal gynecological disease.

Dun1, a Chk2-related kinase, is the central regulator of securin-separase dynamics during DNA damage signaling.

The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1′s role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.

GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system

BackgroundRecombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as “GoldenBac”.ResultsThis method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2–15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein.ConclusionsThis robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.