尋找與鑑定MGCRABGAP蛋白之受質於造精過程的角色
葉忠信1,2、汪雅雲3、吳盈羽3、江漢聲2,3、林盈宏2,3
1新光醫院 泌尿科 2輔仁大學 醫學院 3輔仁大學 基礎醫學研究所
Identification of MGCRAPGAP-interacted substrates during mammalian spmiogenesis
Chung-Hsin Yeh1,2、Ya-Yun Wan、Ying-Yu Wu、Han-Sun Chiang2,3、Ying-Hung Lin2,3
1Division of Urology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
2School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
3Graduate Institute of Basic Medicine, Fu-Jen Catholic University
Purpose: It is estimated that 10- 15% of couples are infertile and male factors account for about half of the cases. Approximately 25% of the causes of male infertility are still unknown and the majority of infertile cases have defects in spermatogenesis. But large of those spermatogenesis are still unknown. We have previously identified novel testis-specific genes using cDNA-microarray analysis of human testicular tissues. One of novel genes, Male Germ Cells Rab GTPase- Activating Proteins (MgcRabGAP), which is characterized by the conserved RabGAP catalytic domain, TBC (Tre2/Bub2/Cdc16) domain. RABGAPs are involved in various physiological processes (e.g., vesicular trafficking, cytoskeletal remodeling and cell migration) through inactivating RAB proteins. Next, we have found MgRabcGAP transcripts are mainly expressed in the mouse and human testes. And, the major of MGCRABGAP protein is expressed in the elongating and elongated spermatids. In clinical aspect, the amounts of MGCRABGAP transcript are reduced in the testicular tissues of men with various types of spermatogenic defect. The overall project is to determine the reproductive function of MGCRABGAP and its interacted- proteins (e.g., Rabs and small G proteins) during mammalian spermiogenesis.
Methods and Materials: We applied co-imuno-precipitations (co-IP) and subjected to liquid chromatography -mass spectrometry/mass spectrometry (LC-MS/MS) to identify MGCRABGAP interactors. The molecule-biological skills (e.g., Immunofluorescence staining, Cloning, Transfection, and Co-immunoprecipitation assay) used in this study.
Results:. 1) Several MgcRabGAP interacted-proteins have been identified through co-IP and LC-MS/MS e.g., RAB10, RAB5C, RAP1. 2) We also verified the binding/activating ability between RAB10 and MGCRABGAP through transfection and co-immuno-precipiateion. 3) Further, MGCRABGAP-RAB10 complexes are co-expressed at the developmental stages of the sperm-head and tail formation.
Conclusion: MGCRABGAP-RAB10 complexes are involved in sperm-head and tail formation at post- meiotic stages. This is first study suggesting the functional roles of MGCRABGAP-RAB10 complexes during mouse and human spermatogenesis.