The Preliminary Study Of The Ubiquitin-Proteasome System Related Turnover Of Sarcomere Z-line Protein Cypher

Maintenance of cellular integrity and homeostasis requires tight control of the balance of protein synthesis and degradation. This is of particular importance in heart for its permanent systolic and diastolic functions. Sarcomeres are the basic contractile units of cardiac myocytes. The normal self-renew of sarcomere proteins are important in maintaining the structure and function of sarcromeres. Some diseases may occur in association with accumulation of Z-line and cytoplasmic proteins (such as Myofibrillar myopathy). Such as in other tissues and organs, the turnover of sarcomere proteins in heart are closely related to the ubiquitin—proteasome system.The ubiquitin—proteasome system (UPS) is the major pathway for intracellular protein degradation in eukaryotic cells.The pathway includes two steps:protein ubiquitination and degradation by proteaosome. This pathway is of vital significance to regulatory of cellular activities.Cypher is identified as a striated muscle-specific PDZ/LIM domain-containing protein and plays an important role in maintaining the normal function and structure of Z-line. Cardiac-specific ablation of Cypher leads to severe form of dilated cardiomyopathy and cardiac dysfunction. Mutations of human orthologue of Cypher, Z-band alternatively spliced PDZ-motif protein (ZASP), can also lead to myocardial hypertrophy and dilated cardiomyopathy.Our aims were studying the UPS pathway related to sarcomere Z-line protein Cypher. First, we screened the UPS enzymes interacting with Cypher by yeast two-hybrid system. Through generating bait clone, the library screening and sequence alignment, we discoverd that the ZM motif of Cypher interacted with ubiquitin-conjugating enzyme E2N and ubiquitin specific peptidase48.The interaction between USP48and Cypher was tested in vitro. In the second part of our experiments,we studyed the impact of mutations of Cypher on ubiquitin degradation. We discovered that the mutation of206site (T206I), locating at the exon4of cypher gene caused the increase of degradation, which may be due to the impact on the interaction between Cypher and ubiquitin. At last, as a preliminary study, we explored the function of Cypher in the UPS related degradation of Stat3.