USP13 modulates the stability of the APC/C adaptor CDH1.

BACKGROUND: The cell division cycle is a process that is exquisitely controlled by a complex interplay between E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). We have previously reported that the DUB USP13 regulates Aurora B levels along the cell cycle. That observation prompted us to explore any possible connection between USP13 and the APC/CCDH1, the major E3 controlling Aurora B levels in cells. METHODS: We performed immunoprecipitation assays followed by western-blotting to assess the interaction between USP13 and CDH1. The cellular effects of USP13 gain or loss of function were analyzed by transfection of FLAG-tagged USP13 plasmid or small interfering RNAs and short hairpin RNAs directed against USP13. The levels of CDH1 and other proteins were quantified in cell extracts by western-blotting. RESULTS: We found that USP13 binds to the APC/C adaptor CDH1. In addition, we report for the first time that USP13 controls CDH1 protein levels in cells: overexpression of USP13 increased CDH1 levels, whereas depletion of USP13 decreased CDH1 levels. CONCLUSIONS: We unveil the existing interplay between USP13 and CDH1: USP13 is capable of stabilizing CDH1 levels. We previously reported that USP13 stabilizes Aurora B in cells, a known substrate of the APC/CCDH1 E3 ubiquitin ligase, before their entry into mitosis. Altogether, our data identify and establish the USP13-CDH1-Aurora B axis as a new regulatory module required for flawless cell cycle progression in mammalian cells, whose misfunction may be involved in the rewiring of cell cycle pathways linked to cancer development.

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.

Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.

The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.

Detection and Analysis of SUMOylation Substrates In Vitro and In Vivo.

SUMOylation is a widely used protein posttranslational mechanism capable of regulating substrates localization, stability, and/or activity. Identification and characterization of bona fide SUMO substrates is a laborious task but its discovery can shed light to exquisite and crucial regulatory signaling events occurring within the cell. Experiments performed in the SUMOylation field often demand a good understanding of the putative substrate's function and necessitate a solid knowledge regarding both in vitro and in vivo approaches. This contribution offers a simplified view into some of the most common experiments performed in biochemical and cell biological research of the SUMO pathway in mammalian systems. It also summarizes and updates well established protocols and tricks in order to improve the likelihood to obtain reliable and reproducible results.

Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): comparison with tobacco cigarette smoke extract.

CONTEXT: Electronic cigarettes (ECs) are used as alternatives to smoking; however, data on their cytotoxic potential are scarce. OBJECTIVE: To evaluate the cytotoxic potential of 21 EC liquids compared to the effects of cigarette smoke (CS). METHODS: Cytotoxicity was evaluated according to UNI EN ISO 10993-5 standard. By activating an EC device, 200 mg of liquid was evaporated and was extracted in 20 ml of culture medium. CS extract from one cigarette was also produced. The extracts, undiluted (100%) and in five dilutions (50%, 25%, 12.5%, 6.25% and 3.125%), were applied to cultured murine fibroblasts (3T3), and viability was measured after 24-hour incubation by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Viability of less than 70% was considered cytotoxic. RESULTS: CS extract showed cytotoxic effects at extract concentrations above 12.5% (viability: 89.1 ± 3.5% at 3.125%, 77.8 ± 1.8% at 6.25%, 72.8 ± 9.7% at 12.5%, 5.9 ± 0.9% at 25%, 9.4 ± 5.3% at 50% and 5.7 ± 0.7% at 100% extract concentration). Range of fibroblast viability for EC vapor extracts was 88.5-117.8% at 3.125%, 86.4-115.3% at 6.25%, 85.8-111.7% at 12.5%, 78.1-106.2% at 25%, 79.0-103.7% at 50% and 51.0-102.2% at 100% extract concentration. One vapor extract was cytotoxic at 100% extract concentration only (viability: 51.0 ± 2.6%). However, even for that liquid, viability was 795% higher relative to CS extract. CONCLUSIONS: This study indicates that EC vapor is significantly less cytotoxic compared tobacco CS. These results should be validated by clinical studies.