Hücre bölünmesi sürecinde palmitolasyon dinamikleri

The cell cycle is mainly regulated by post-translational modifications. While phos- phorylation is well-established regulator of the cell cycle progression, the role of palmitoylation in this process has not been systematically characterized. This the- sis investigates the regulatory role of palmitoylation in the cell cycle. To profile dynamic palmitoylation changes this study combines SILAC-based metabolic label- ing with click chemistry. HeLa cells were labelled with heavy and light isotopes and synchronized to mitosis and interphase, respectively. After metabolic labeling with 17-ODYA, palmitoylated proteins are biotinylated by click chemistry and enriched using streptavidin pulldown. Quantitative analysis identified 3,239 proteins, 741 of which showed significant changes in palmitoylation between interphase and mitotic cells. Among these, 381 proteins are enriched as mitosis selective and 360 as inter- phase selective palmitoylated proteins. Furthermore, quantitative analysis identifies 2678 proteins in cells synchronized at mitosis and cytokinesis. Among these, palmi- toylation of 83 proteins are significant in mitosis while 109 proteins are significant in cytokinesis. The results suggest that palmitoylation is regulated in a cell cycle- dependent manner. RNAi-based functional screening identifies ZDHHC5 as a key regulator of cell division, with its depletion resulting in prolonged cytokinesis and an increase in multinucleated cell rate. A remarkable crosstalk between palmitoy- lation and phosphorylation is observed. The most significant interaction between palmitoylation and phosphorylation is observed in mitotic proteins. The mitosis- selective cluster (Cluster 4) is identified as the largest cluster in the phosphopro- teome dataset [Karayel et al., 2018]. Cluster 4 is most enriched in proteins that are likely co-regulated by both palmitoylation and phosphorylation. A CRISPR/Cas9 mediated ZDHHC5 knockout (KO) cell line was constructed to investigate its en- zymatic role during cytokinesis. Label-free quantitative proteomics analysis of the KO cells identified 210 potential candidate substrates of ZDHHC5 at cytokinesis. The findings indicate a potential role for lipid-based modifications in coordinating cell division mechanisms.