Peptide inhibitors of APCCdc20 based on the Cdc20 Mad2-binding motif and human p31comet homolog in budding yeast can directly bind to the APC

• Main Authors: Yan Lun Liao, 廖彥綸
• Other Authors: S. C. Schuyler
• Format: Others
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/12204041943896104210

碩士 === 長庚大學 === 生物醫學研究所 === 103 === In mitosis, the spindle assembly checkpoint ensures genome stability by delaying the onset of anaphase until all kinetochores are properly attached to the mitotic spindle. A Previous study has shown that some proportion of tumor cells treated with anti-mitotic drugs resulted in abnormal mitotic exit, while after Cdc20 was knocked down by siRNA, it led to robust mitotic arrest followed by cell death. This report indicates that APCCdc20 inhibitor may be a potential role in development of cancer therapeutic agents. We have observed that DQ36, a peptide based on Mad2-binding motif in Cdc20, is sufficient to inhibit APCCdc20 activity in vitro. Here, I established a binding assay to verify that the peptide can directly bind to the APC in vitro. For the question that where does Cdc20 touch to the APC, we made a speculation on Apc9 subunit because of its conserved motifs. However, my result shows that Apc9 subunit is not required for APC-Cdc20 interaction. Nevertheless, we keep trying to find the answer by peptide cross-linking experiment. Another small peptide, Tyc1, is a novel tiny protein with unknown function identified from human p31comet BLAST in budding yeast genome. Amazingly, Tyc1 showed its inhibition activity in APC assay as well. Subsequently, binding tendency of Tyc1 to the APC was verified in binding assay and compared to DQ36. In addition, interaction of Tyc1 and Cdc20, a co-factor of the APC, was investigated by Co-IP. My result shows that Cdc20 is not the binding partner of Tyc1, indicating that Tyc1 peptide may disturb APCCdc20 activity by interacting with the APC. The mechanism of peptide inhibitors influencing APCCdc20 activity will be the major issue we focus on in the future.