Destruction Box Research Articles

The ubiquitin E3 ligase APC/CCdc20 mediates mitotic degradation of OGT

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/CCdc20 interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/CCdc20-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly, in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/CCdc20 partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.

Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/CCdh1 complex.

H3K4 trimethylation (H3K4me3) is a conserved histone modification catalyzed by histone methyltransferase Set1, and its dysregulation is associated with pathologies. Here, we show that Set1 is intrinsically unstable and elucidate how its protein levels are controlled within cell cycle and during gene transcription. Specifically, Set1 contains a destruction box (D-box) that is recognized by E3 ligase APC/CCdh1 and degraded by the ubiquitin-proteasome pathway. Cla4 phosphorylates serine 228 (S228) within Set1 D-box, which inhibits APC/CCdh1-mediated Set1 proteolysis. During gene transcription, PAF complex facilitates Cla4 to phosphorylate Set1-S228 and protect chromatin-bound Set1 from degradation. By modulating Set1 stability and its binding to chromatin, Cla4 and APC/CCdh1 control H3K4me3 levels, which then regulate gene transcription, cell cycle progression, and chronological aging. In addition, there are 141 proteins containing the D-box that can be potentially phosphorylated by Cla4 to prevent their degradation by APC/CCdh1. We addressed the long-standing question about how Set1 stability is controlled and uncovered a new mechanism to regulate protein stability.

RNAi Analysis of Potential Functions of Cyclin B3 in Reproduction of Male Oriental River Prawns (Macrobrachium nipponense).

Cyclin B3 (CycB3) is involved in the metabolic pathway of the cell cycle, playing essential roles in the regulation of cell proliferation and mitosis. CycB3 is also predicted to be involved in the reproduction of male oriental river prawns (Macrobrachium nipponense). In this study, the potential functions of CycB3 in M. nipponense were investigated using quantitative real-time PCR, RNA interference, and histological observations. The full-length DNA sequence of CycB3 in M. nipponense was 2147 base pairs (bp) long. An open reading frame of 1500 bp was found, encoding 499 amino acids. A highly conserved destruction box and two conserved cyclin motifs were found in the protein sequence of Mn-CycB3. Phylogenetic tree analysis revealed that this protein sequence was evolutionarily close to that of CycB3s of crustacean species. Quantitative real-time PCR analysis results suggested that CycB3 was involved in the process of spermiogenesis, oogenesis, and embryogenesis in M. nipponense. RNA interference analysis showed that CycB3 had a positive regulatory relationship with insulin-like androgenic gland hormone (IAG) in M. nipponense. In addition, sperm were rarely observed in the testis of double-stranded CycB3-injected prawns after 14 days of treatment, and sperm abundance was dramatically lower than that in the double-stranded GFP-injected prawns on the same day. This result indicated that CycB3 can regulate the testis reproduction in M. nipponense through inhibiting the IAG expressions. Overall, these results indicated that CycB3 plays essential roles in the regulation of male reproduction in M. nipponense, which may promote the studies of male reproduction in other crustacean species.

The inhibitor of κB kinase β (IKKβ) phosphorylates IκBα twice in a single binding event through a sequential mechanism

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32s-1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1-54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.

Oscillation of Cdc20–APC/C–mediated CAMDI stability is critical for cortical neuron migration

Radial migration during cortical development is required for formation of the six-layered structure of the mammalian cortex. Defective migration of neurons is linked to several developmental disorders such as autism and schizophrenia. A unique swollen structure called the dilation is formed in migrating neurons and is required for movement of the centrosome and nucleus. However, the detailed molecular mechanism by which this dilation forms is unclear. We report that CAMDI, a gene whose deletion is associated with psychiatric behavior, is degraded by cell division cycle protein 20 (Cdc20)–anaphase-promoting complex/cyclosome (APC/C) cell-cycle machinery after centrosome migration into the dilation in mouse brain development. We also show that CAMDI is restabilized in the dilation until the centrosome enters the dilation, at which point it is once again immediately destabilized. CAMDI degradation is carried out by binding to Cdc20–APC/C via the destruction box degron of CAMDI. CAMDI destruction box mutant overexpression inhibits dilation formation and neuronal cell migration via maintaining the stabilized state of CAMDI. These results indicate that CAMDI is a substrate of the Cdc20–APC/C system and that the oscillatory regulation of CAMDI protein correlates with dilation formation for proper cortical migration.

Nuclear translocation promotes proteasomal degradation of human Rad17 protein through the N-terminal destruction boxes

The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230–270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.

Yeast Phosphatidic Acid Phosphatase Pah1; Why is Tryptophan 637 Required for Cellular Function?

Pah1 in Saccharomyces cerevisiae is an Mg2+-dependent phosphatidic acid (PA) phosphatase that plays a major role in lipid synthesis by controlling the levels of triacylglycerol and membrane phospholipids. The enzyme contains two conserved domains (N-lip and HAD-like) required for catalytic activity, and non-conserved regions containing multiple phosphorylation sites involved in the regulation of subcellular localization, catalytic activity, and protein stability. In a previous study, we found that Trp-637 in a putative WW-binding domain and destruction box is essential for the function of Pah1 in vivo, but not for its catalytic activity in vitro. In this work, we examined the Pah1(W637A) for its localization, abundance, and phosphorylation state. Subcellular fractionation analysis showed that the membrane translocation of Pah1(W637A), like that of the WT enzyme, depends on the Nem1-Spo7 phosphatase function. The mutant enzyme showed a higher level of membrane association than the WT enzyme, but a significantly lower cytosolic level. The phosphorylation status of Pah1(W637A), as determined by electrophoretic mobility, showed that the mutant enzyme is less phosphorylated in vivo when compared with the WT enzyme. The Nem1-Spo7-dependent membrane translocation of Pah1(W637A) was also shown in vitro by fractionation of cytosolic Pah1 incubated with the membrane containing the phosphatase complex. These results suggest that Trp-637 is not required for the translocation of Pah1, but for its catalytic activity on PA in the cellular membrane.

ASSESSMENT OF STERILIZATION STANDARDS IN PUBLIC AND PRIVATE DENTAL SETTINGS OF PESHAWAR – A CROSS SECTIONAL SURVEY

Objective:
 To assess the sterilization standards of the dental setups both in public and private settings of Peshawar.
 Materials and Methods:
 This Cross Sectional Study was started from Nov 2017 to Apr 2018 in Khyber College of Dentistry, Sardar Begum Dental College Peshawar, Rehman Dental College Peshawar and Private Clinics in Board Bazar and University Town Peshawar which were included in the study through Convenient Sampling technique. Total 100 professionals were interviewed; 50 from public and private. A semi-structured questionnaire was used with open and close ended questions. Data was analyzed using SPSS version 20 and Microsoft Excel.
 Results:
 Of the 100 total professionals, dental surgeons, house officers, TMOs, Specialists and technicians were interviewed. All the private settings were using autoclave method of sterilization i.e 50 while the public sector was 46. Immediate sterilization of instruments after use public : private 27 : 33. Disinfection of operatory surfaces in between patients: public : private 33 : 25. Presence of destruction boxes for syringe disposal: public : private 13 : 44. Both these settings were almost identical in sterilization practices except use of destruction box.
 Conclusion:
 Sterilization practices needs to be improved both in public and private settings. Dental personnel need to be given proper training on regular interval.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Aberrant activation of cyclin A-CDK induces G2/M-phase checkpoint in human cells

ABSTRACT Cyclin A-cyclin dependent kinase (CDK) activity is regulated by cyclin A proteolysis and CDK inhibitors (CKIs) during M and G1 phases. Our previous work has shown that constitutive activation of cyclin A-CDK in mouse somatic cells, by ectopic expression of stabilized human cyclin A2 (lacking the destruction box: CycAΔ80) in triple CKI (p21, p27, and p107)-knocked-out mouse embryonic fibroblasts, induces rapid tetraploidization. However, effects of such cyclin A-CDK hyperactivation in human cells have been unknown. Here, we show hyperactivity of cyclin A-CDK induces G2/M-phase arrest in human cell lines with relatively low expression of p21 and p27. Moreover, adenovirus E1A protein promoted CycAΔ80-derived G2/M-phase arrest by increasing the amount of cyclin A and cyclin A-CDK2 complex. This response was suppressed by an addition of ATR or Chk1 inhibitor. The amount of repressive phosphorylation of CDK1 at tyrosine 15 (Y15) was decreased by Chk1 inhibitor treatment. Moreover, we observed that co-expressing CDK1AF mutant, which is resistant to the repressive phosphorylation at threonine 14 and Y15, or cdc25A, which dephosphorylates CDK1 at Y15, suppressed the G2/M-phase arrest by CycAΔ80 with E1A. These results suggest that G2/M-phase arrest in human cells by hyperactivity of cyclin A-CDK2 is caused by repression of CDK1 via the cell cycle checkpoint ATR-Chk1 pathway.

The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function

The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.

Light-inducible activation of cell cycle progression in Xenopus egg extracts under microfluidic confinement.

Cell-free Xenopus egg extract is a widely used and biochemically tractable model system that allows recapitulation and elucidation of fundamental cellular processes. Recently, the introduction of microfluidic extract manipulation has enabled compartmentalization of bulk extract and a newfound ability to study organelles on length scales that recapitulate key features of cellular morphology. While the microfluidic confinement of extracts has produced a compelling platform for the in vitro study of cell processes at physiologically-relevant length scales, it also imposes experimental limitations by restricting dynamic control over extract properties. Here, we introduce photodegradable polyethylene glycol (PEG) hydrogels as a vehicle to passively and selectively manipulate extract composition through the release of proteins encapsulated within the hydrogel matrix. Photopatterned PEG hydrogels, passive to both extract and encapsulated proteins, serve as protein depots within microfluidic channels, which are subsequently flooded with extract. Illumination by ultraviolet light (UV) degrades the hydrogel structures and releases encapsulated protein. We show that an engineered fluorescent protein with a nuclear localization signal (GST-GFP-NLS) retains its ability to localize within nearby nuclei following UV-induced release from hydrogel structures. When diffusion is considered, the kinetics of nuclear accumulation are similar to those in experiments utilizing conventional, bulk fluid handling. Similarly, the release of recombinant cyclin B Δ90, a mutant form of the master cell cycle regulator cyclin B which lacks the canonical destruction box, was able to induce the expected cell cycle transition from interphase to mitosis. This transition was confirmed by the observation of nuclear envelope breakdown (NEBD), a phenomenological hallmark of mitosis, and the induction of mitosis-specific biochemical markers. This approach to extract manipulation presents a versatile and customizable route to regulating the spatial and temporal dynamics of cellular events in microfluidically confined cell-free extracts.

Abstract 3341: Inhibiting the destruction of the oncogene ZNF217 promotes breast cancer metastasis to lung

Abstract The oncogene and transcription factor ZNF217 is overexpressed in 20-30% of breast cancers. Its overexpression correlates strongly with poor prognosis in patients and causes accelerated tumor progression, metastasis, and chemoresistance in vivo. While several studies have examined the regulation of ZNF217 at the gene and mRNA levels, little is known about how ZNF217 is regulated as a protein. In this study we identify the regions of ZNF217 that are required for protein turnover and other cancer phenotypes, including increased invasion and metastasis. Due to the high expression and aberrant localization of ZNF217 in some human breast tumors, both ZNF217 protein expression levels and localization of ZNF217 may be critical determinants of ZNF217's function in vivo. To investigate the role of ZNF217 protein on cancer progression, we first examined ZNF217 protein expression in human breast tumors by Western analysis. In addition to full-length ZNF217 protein, smaller ZNF217 proteins were detected and were even more prominent than full-length ZNF217 in both human breast tumors and cell lines, but the importance of these smaller isoforms remains unknown. To investigate the function of the smaller ZNF217 proteins and to determine the requirements of the regions of ZNF217 protein to promote breast cancer, we examined the consequences of removing regions of ZNF217 using truncation mutants overexpressed in human breast cancer cells. As a structure-function analysis, we tested both truncation mutants and full-length ZNF217 for their ability to promote phenotypes usually associated with ZNF217 overexpression, including protein turnover, increased branching of organoids, tumor burden, and metastasis. For protein turnover, these mutants were expressed in breast cancer cells and treated with cycloheximide to inhibit protein synthesis. This assay identified the regions of ZNF217 required for ZNF217 protein turnover. Further analysis of the sequence in this region identified a putative destruction box required for the protein turnover. We next determined which regions of ZNF217 are required for organoid branching and metastatic tumor burden. Both the N-terminus and C-terminus of ZNF217 were required for organoid branching. Interestingly, the same region identified to be required for ZNF217 protein turnover was not required for metastatic tumor burden. In fact, overexpression of breast cancer cells expressing stabilized ZNF217 truncation mutants significantly increased the metastatic tumor burden in vivo when assayed by tail vein injection. These results suggest that regulation of ZNF217 protein expression may be clinically valuable in generating a clinical assay used to generate personalized treatment strategies for patients with advanced metastatic breast cancer and high ZNF217 expression. Citation Format: Beth Facchine, Junmin Wu, Megan Fabry, Matt Messana, William Kaliney, Laurie Littlepage. Inhibiting the destruction of the oncogene ZNF217 promotes breast cancer metastasis to lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3341.

Cyclins in aspergilli: Phylogenetic and functional analyses of group I cyclins.

We have identified the cyclin domain-containing proteins encoded by the genomes of 17 species of Aspergillus as well as 15 members of other genera of filamentous ascomycetes. Phylogenetic analyses reveal that the cyclins fall into three groups, as in other eukaryotic phyla, and, more significantly, that they are remarkably conserved in these fungi. All 32 species examined, for example, have three group I cyclins, cyclins that are particularly important because they regulate the cell cycle, and these are highly conserved. Within the group I cyclins there are three distinct clades, and each fungus has a single member of each clade. These findings are in marked contrast to the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, which have more numerous group I cyclins. These results indicate that findings on cyclin function made with a model Aspergillus species, such as A. nidulans, are likely to apply to other Aspergilli and be informative for a broad range of filamentous ascomycetes. In this regard, we note that the functions of only one Aspergillus group I cyclin have been analysed (NimECyclin B of A. nidulans). We have consequently carried out an analysis of the members of the other two clades using A. nidulans as our model. We have found that one of these cyclins, PucA, is essential, but deletion of PucA in a strain carrying a deletion of CdhA, an activator of the anaphase promoting complex/cyclosome (APC/C), is not lethal. These data, coupled with data from heterokaryon rescue experiments, indicate that PucA is an essential G1/S cyclin that is required for the inactivation of the APC/C-CdhA, which, in turn, allows the initiation of the S phase of the cell cycle. Our data also reveal that PucA has additional, non-essential, roles in the cell cycle in interphase. The A. nidulans member of the third clade (AN2137) has not previously been named or analyzed. We designate this gene clbA. ClbA localizes to kinetochores from mid G2 until just prior to chromosomal condensation. Deletion of clbA does not affect viability. However, by using a regulatable promoter system new to Aspergillus, we have found that expression of a version of ClbA in which the destruction box sequences have been removed is lethal and causes a mitotic arrest and a high frequency of non-disjunction. Thus, although ClbA is not essential, its timely destruction is essential for viability, chromosomal disjunction, and successful completion of mitosis.

Rev7, the regulatory subunit of Polζ, undergoes UV-induced and Cul4-dependent degradation.

In eukaryotic cells, Rev7 interacts with Rev3 and functions as a regulatory subunit of Polζ, a translesion DNA synthesis (TLS) polymerase. In addition to its role in TLS, mammalian Rev7, also known as Mad2B/Mad2L2, participates in multiple cellular activities including cell cycle progression and double-strand break repair through its interaction with several proteins. Here we show that in mammalian cells, Rev7 undergoes ubiquitin/proteasome-mediated degradation upon UV irradiation in a time-dependent manner. We identified the Rev7 N-terminal destruction box as the degron and Cul4A/B as putative E3 ligases in this process. We also show that the nucleotide excision repair (NER) pathway protein HR23B physically interacts and colocalizes with Rev7 in the nuclear foci after UV irradiation and protects Rev7 from accelerated degradation. Furthermore, a similar Rev7 degradation profile was observed in cells treated with the UV-mimetic agent 4-nitroquinoline 1-oxide but not with cisplatin or camptothecin, suggesting a role of the NER pathway protein(s) in UV-induced Rev7 degradation. These data and the observation that cells deficient in Rev7 are sensitized to UV irradiation while excessive Rev7 protects cells from UV-induced DNA damage provide a new insight into the potential interplay between TLS and NER.

Proper division plane orientation and mitotic progression together allow normal growth of maize

How growth, microtubule dynamics, and cell-cycle progression are coordinated is one of the unsolved mysteries of cell biology. A maize mutant, tangled1, with known defects in growth and proper division plane orientation, and a recently characterized cell-cycle delay identified by time-lapse imaging, was used to clarify the relationship between growth, cell cycle, and proper division plane orientation. The tangled1 mutant was fully rescued by introduction of cortical division site localized TANGLED1-YFP. A CYCLIN1B destruction box was fused to TANGLED1-YFP to generate a line that mostly rescued the division plane defect but still showed cell-cycle delays when expressed in the tangled1 mutant. Although an intermediate growth phenotype between wild-type and the tangled1 mutant was expected, these partially rescued plants grew as well as wild-type siblings, indicating that mitotic progression delays alone do not alter overall growth. These data indicate that division plane orientation, together with proper cell-cycle progression, is critical for plant growth.

Degradation of the Mitotic Cyclin Clb3 Is not Required for Mitotic Exit but Is Necessary for G1 Cyclin Control of the Succeeding Cell Cycle.

B-type cyclins promote mitotic entry and inhibit mitotic exit. In Saccharomyces cerevisiae, four B-type cyclins, Clb1-4, carry out essential mitotic roles, with substantial but incomplete overlap of function among them. Previous work in many organisms has indicated that B-type cyclin-dependent inhibition of mitotic exit imposes a requirement for mitotic destruction of B-type cyclins. For instance, precise genomic removal of the Clb2 destruction box (D box) prevents mitotic proteolysis of Clb2, and blocks mitotic exit. Here, we show that, despite significant functional overlap between Clb2 and Clb3, D-box-dependent Clb3 proteolysis is completely dispensable for mitotic exit. Removal of the Clb3 D box results in abundant Clb3 protein and associated kinase throughout the cell cycle, but mitotic exit occurs with close to normal timing. Clb3 degradation is required for pre-Start G1 control in the succeeding cell cycle. Deleting the CLB3 D box essentially eliminates all time delay before cell cycle Start following division, even in very small newborn cells. CLB3∆db cells show no cell cycle arrest response to mating pheromone, and CLB3∆db completely bypasses the requirement for CLN G1 cyclins, even in the absence of the early expressed B-type cyclins CLB5,6 Thus, regulated mitotic proteolysis of Clb3 is specifically required to make passage of Start in the succeeding cell cycle "memoryless"-dependent on conditions within that cycle, and independent of events such as B-type cyclin accumulation that occurred in the preceding cycle.

The destruction box is involved in the degradation of the NTE family proteins by the proteasome.

Neuropathy target esterase (NTE) and NTE-related esterase (NRE) are endoplasmic reticulum (ER) membrane-anchored proteins belonging to the NTE protein family. NTE and NRE are degraded by macroautophagy and by the ubiquitin-proteasome pathway. However, the regulation of NTE and NRE by proteasome has not been well understood. Western blotting showed that the deletion of the regulatory region of NTE and NRE led to protein accumulation compared with that of the corresponding wild-type proteins. Further, deletion and site-directed mutagenesis experiments demonstrated that the destruction (D) box was required for the proteasomal degradation of NTE and NRE. However, unlike the deletion of the regulatory region, the deletion of the D box did not affect the subcellular localisation of NTE or NRE or disrupt the ER. Moreover, the deletion of the D box or the regulatory region of NTE has similar inhibitory effects on cell growth, which are greater than those produced by the full-length NTE. Here, for the first time, we show that the D box is involved in the regulation of NTE family proteins by the proteasome but not in their subcellular localisation. In addition, these results suggest that the NTE overexpression-mediated inhibition of cell growth is related to active protein levels but not to its ER disruption effect.

Abstract 2737: Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression

Abstract Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). CIN is associated with poor prognosis in many cancer types, and targeting of CIN is an attractive strategy for anti-cancer therapeutics. The mechanisms causing CIN and its contributions to tumor initiation and growth are not well defined, partly because there is no straightforward, quantitative assays for CIN in human cells. To address this problem, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in mammalian cells, with which we can easily score the rates of CIN within one cell division under different experimental conditions. We have constructed a HAC encoding copies of enhanced green fluorescent protein (eGFP) fused to the destruction box (DB) of hSecurin, a substrate of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, which becomes active during anaphase to catalyze the proteolysis of critical mitotic target proteins. This HAC also contains tet operator (tetO) arrays and sequences encoding the tetracycline repressor fused to monomeric cherry fluorescent protein (tetR-mCherry). We have produced human U2OS cells (U2OS-Phoenix) carrying this HAC, in which we monitor HAC segregation in two ways: First, APC/C degrades the DB-eGFP fusion expressed from the HAC at anaphase onset, and DB-eGFP re-accumulates in the daughter cells after G1 phase, when APC/C becomes inactive. Daughter cells that do not obtain a copy of the HAC will thus be GFP negative in the subsequent interphase. Second, because tetR-mCherry binds to the tetO arrays, the HAC itself could be followed by live imaging. Following the HAC by live cell imaging experiments, we show that U2OS-Phoenix cells have low inherent levels of CIN, but HAC missegregation is markedly increased by treatment with Reversine, an inhibitor of Mps1, and microtubule agents Nocodazole and Paclitaxel. In summary, we have developed new assays to score CIN levels in human cells and have shown that CIN levels increase upon chemical disruption of mitotic progression, which makes our assays ideal for chemical screens. Citation Format: Sarine Markossian, Alexei Arnaoutov, Nakhle S. Saba, Vladimir Larionov, Mary Dasso. Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2737.

Substrate Recognition by the Cdh1 Destruction Box Receptor Is a General Requirement for APC/CCdh1-mediated Proteolysis

The anaphase-promoting complex, or cyclosome (APC/C), is a ubiquitin ligase that selectively targets proteins for degradation in mitosis and the G1 phase and is an important component of the eukaryotic cell cycle control system. How the APC/C specifically recognizes its substrates is not fully understood. Although well characterized degron motifs such as the destruction box (D-box) and KEN-box are commonly found in APC/C substrates, many substrates apparently lack these motifs. A variety of alternative APC/C degrons have been reported, suggesting either that multiple modes of substrate recognition are possible or that our definitions of degron structure are incomplete. We used an in vivo yeast assay to compare the G1 degradation rate of 15 known substrates of the APC/C co-activator Cdh1 under normal conditions and conditions that impair binding of D-box, KEN-box, and the recently identified ABBA motif degrons to Cdh1. The D-box receptor was required for efficient proteolysis of all Cdh1 substrates, despite the absence of canonical D-boxes in many. In contrast, the KEN-box receptor was only required for normal proteolysis of a subset of substrates and the ABBA motif receptor for a single substrate in our system. Our results suggest that binding to the D-box receptor may be a shared requirement for recognition and processing of all Cdh1 substrates.