AIM:To construct the CABYR RNAi plasmid and study its relation with the nuclear factor(NF)-κB signal transduction pathway.METHODS:Human CABYR mRNA sequence was obtained from GenBank.The structure of cDNA sequence for...AIM:To construct the CABYR RNAi plasmid and study its relation with the nuclear factor(NF)-κB signal transduction pathway.METHODS:Human CABYR mRNA sequence was obtained from GenBank.The structure of cDNA sequence for the short hairpin RNA was BbsⅠ+sense+loop+ antisense+transcription terminator+KpnⅠ+Bam HⅠ.A CABYR silencing plasmid was constructed and transfected into the human embryo cell line 293T.Quantitative real-time polymerase chain reaction was used to analyze CABYR and NF-κB gene expression.RESULTS:The CABYR and NF-κB expressions were detected in 293T cells.The oligonucleotide(5'-GCT-CAGATGTTAGGTAAAG-3')efficiently silenced the expression of CABYR.The expression of NF-κB was not significantly affected by silencing CABYR(P=0.743).CONCLUSION:CABYR can be found in the human embryo cell line 293T.Cabyrmid 2 can efficiently silence its target,CABYR,indicating that CABYR is not related with the NF-κB signal transduction pathway.展开更多
Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A put...Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-dependent protein kinase A （RII） dimerisation and A kinase-anchoring protein （AKAP）-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropperin. The results showed that AKAP3 was co.immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co.immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RIl-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR＇s binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RIl-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another Rll-like domain-containing protein in the human sperm FS.展开更多
基金Supported by Grants from Natural Science Foundation of China, No.30940034
文摘AIM:To construct the CABYR RNAi plasmid and study its relation with the nuclear factor(NF)-κB signal transduction pathway.METHODS:Human CABYR mRNA sequence was obtained from GenBank.The structure of cDNA sequence for the short hairpin RNA was BbsⅠ+sense+loop+ antisense+transcription terminator+KpnⅠ+Bam HⅠ.A CABYR silencing plasmid was constructed and transfected into the human embryo cell line 293T.Quantitative real-time polymerase chain reaction was used to analyze CABYR and NF-κB gene expression.RESULTS:The CABYR and NF-κB expressions were detected in 293T cells.The oligonucleotide(5'-GCT-CAGATGTTAGGTAAAG-3')efficiently silenced the expression of CABYR.The expression of NF-κB was not significantly affected by silencing CABYR(P=0.743).CONCLUSION:CABYR can be found in the human embryo cell line 293T.Cabyrmid 2 can efficiently silence its target,CABYR,indicating that CABYR is not related with the NF-κB signal transduction pathway.
文摘Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-dependent protein kinase A （RII） dimerisation and A kinase-anchoring protein （AKAP）-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropperin. The results showed that AKAP3 was co.immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co.immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RIl-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR＇s binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RIl-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another Rll-like domain-containing protein in the human sperm FS.
