Identification of cytokinetic APC/C substrates

Cytokinesis is the last step of the cell cycle in which the cytoplasmic bridge between two daughter cells is broken and its regulation is key to the fidelity of cell division. Defects at this stage result in aneuploidy causing chromosomal aberration-induced developmental defects or cancer. Despite its importance, regulatory mechanisms of cytokinesis have been poorly studied because tools to examine the biochemistry of this phase of cell division have only recently been developed. Key events in cytokinesis are regulated by ubiquitin-dependent proteolysis that is mediated by the anaphase promoting complex/cyclosome (APC/C), which is active at this stage. Thus, APC/C is crucial for cell division and has to be investigated to get insight into regulatory mechanisms of cytokinesis. In the course of the proposed project a novel methodology, including quantitative and dynamic proteomics,will be developed andapplied to identify cytokinetic APC/Csubstrates. Pharmacological synchronization of HeLa S3 cell populations will be used to obtain samples at different time points along the cell cycle with the main focus on cytokinesis. Isobaric tagging-based quantification in combination with state-of-the-art LC/MS analysis will be applied for proteomic analysis. Quantitative data from multiplexed proteomics experiments will be analyzed using a novel Protein Profile Similarity Screening methodology (PPSS). Within this procedure model protein profiles will be defined on the basis of identified known cytokinesis related APC/C substrates. A high-quality shortlist of putative APC/C substrates will then be generated based on protein profile similarities. Finally, these substrate candidates will be validated using in vitro degradation assays. The knowledge of new APC/C substrates in cytokinesis and their timing of degradation during cell division will allow the ascertaining of proteins that are misregulated in cytokinesis during disease (e.g. overexpression in cancer). Hence, these investigations will contribute to the identification of important targets for the development of therapeutic approaches, similar to the identification of the APC/C substrate KIFC1, whose overexpression has recently been shown to cause resistance to anti-mitotic drugs.

Cytokinesis is the last step of the cell cycle in which the cytoplasmic bridge between two daughter cells is broken and its regulation is key to the fidelity of cell division. Defects at this stage result in aneuploidy causing developmental defects or cancer. Despite its importance, regulatory mechanisms of cytokinesis have been poorly studied because tools to examine the biochemistry of this phase of cell division have only recently been developed. Key events in cytokinesis are regulated by ubiquitin-dependent proteolysis that is mediated by the anaphase promoting complex/cyclosome (APC/C), which is active at this stage. Thus, APC/C is crucial for cell division and has to be investigated to get insight into regulatory mechanisms of cytokinesis. During this project a novel methodology, including quantitative and dynamic proteomics, was developed and applied to identify cytokinetic APC/C substrates. Pharmacological synchronization of HeLa S3 cell populations was used to obtain samples at different time points along the cell cycle with emphasis on cytokinesis. Isobaric tagging-based quantification in combination with state-of-the-art LC/MS analysis was applied for proteomic analysis. Quantitative data from multiplexed proteomics experiments were analyzed using a novel Protein Profile Similarity Screening methodology (PPSS). Within this procedure model protein profiles were defined on the basis of identified known cytokinesis related APC/C substrates. A high-quality shortlist of putative APC/C substrates was then generated based on protein profile similarities. These substrate candidates should be validated using in vitro degradation assays. Finally, the posttranslational modification of those substrates should be investigated with a new established method in the Steen lab. The knowledge of new APC/C substrates in cytokinesis and their timing of degradation during cell division will allow the ascertaining of proteins that are misregulated in cytokinesis during disease (e.g. overexpression in cancer). Hence, these investigations will contribute to the identification of important targets for the development of therapeutic approaches, similar to the identification of the APC/C substrate KIFC1, whose overexpression has recently been shown to cause resistance to anti-mitotic drugs.