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Screening the proteins that interact with calpain in a human heart cDNA library using a yeast two-hybrid system
Li-Qun Jiang1, Shao-Jun Wen, Hong-Yue Wang
1Division of Biochemistry, Cardiovascular Institute and Fu Wai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, PR China.
Abstract:
Calpain, a cytosolic cysteine protease, requires calcium ions for activity. It has been reported that calpain is involved in the degradation of myofibrillar and neurofilament proteins, and the activation of phosphorylase b kinase and protein kinase C. More recently, calpain was shown to participate in apoptosis. In order to understand the calpain-related signal transduction pathway and its changes during hypertrophy, and especially in hypertension, we screened a human heart cDNA library to find proteins that interact with calpain. 1) Using PCR we amplified the full-length, domain II, domain III and domain IV cDNA of calpain (calcium-activated neutral protease, CANP) I large subunit respectively. 2) Then the fragments were cloned into pGBKT7 vector, resulting in 4 bait expression constructs (pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III, and pG BKT7-CANP IV). 3) After 4 bait vectors were transformed into AH109 by the lithium acetate-mediated method, AH109/pGBKT7-CANP, AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III, and AH109/pGBKT7-CANP IV were obtained, respectively. 4) After the human heart cDNA library was sequentially transformed into AH109/ pGBKT7-CANP, 1000-1200 positive clones were grown on SD/Trp-Leu-Ade-His-. Only 150 positive clones were obtained through a colony-lift filter assay to detect beta-galactosidase activity. 5) Total 105 clones among above 150 positive clones were eliminated through that the duplicate, pseudopositive and autoactive detection, respectively. 6) Finally, sequencing eliminated clones with a wrong open reading frame (ORF). Eight clones were cancelled with wrong ORF. The remaining 37 positive clones were analyzed using BLAST software available on the Internet and classified as follows: 1. enzymes or proteins related to signal transduction in the cell; 2. contraction proteins 3. matrix proteins 4. unknown proteins. 7) In order to determine which domain of the calpain I large subunit was involved in the interaction with these real clones, the 37 clones were transformed into AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III or AH109/pGBKT7-CANP IV. Among these 37 clones, 29 clones could interact with domain II, 5 clones could interact with domain III and 6 clones could interact with domain IV. Thus, we successfully constructed 4 bait expression vectors, pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III and pGBKT7-CANP IV, and obtained 37 real positive clones that interacted with the calpain I large subunit by screening a human heart cDNA library using pGBKT7-CANP as bait. Among them, 29 clones could interact with domain II of the calpain I large subunit, where the active site of calpain is located. Additional studies will be needed to clarify the calpain-related signal transduction pathway in greater detail.
Insights
Researchers screened a human heart cDNA library to identify proteins interacting with calpain (calcium-activated neutral protease). They found 37 interacting proteins, with 29 binding to calpain
Area of Science:
- Molecular biology
- Biochemistry
- Cardiology
Background:
- Calpain, a calcium-dependent cysteine protease, plays roles in protein degradation and cellular signaling.
- Calpain involvement in apoptosis and cardiac conditions like hypertrophy and hypertension is increasingly recognized.
- Understanding calpain-mediated signal transduction is crucial for cardiovascular research.
Purpose of the Study:
- To identify proteins interacting with the calpain I large subunit.
- To investigate the specific domains of calpain involved in these interactions.
- To elucidate calpain-related signaling pathways in the context of cardiac hypertrophy and hypertension.
Main Methods:
- Construction of four bait expression vectors encoding full-length and truncated calpain I large subunit domains.
- Yeast two-hybrid screening of a human heart cDNA library using calpain as bait.
- Confirmation of positive clones via beta-galactosidase assays, duplicate/pseudopositive/autoactive detection, and sequencing.
- Interaction mapping of positive clones with specific calpain domains (II, III, IV).
Main Results:
- Successfully constructed four bait expression vectors for calpain I large subunit.
- Identified 37 unique human heart proteins that interact with the calpain I large subunit.
- Determined that 29 of these interacting proteins bind to domain II of calpain, which contains the active site.
- Five clones interacted with domain III and six with domain IV.
Conclusions:
- The study successfully identified novel protein interactors of the calpain I large subunit.
- Domain II of calpain is a primary interaction site for identified cardiac proteins.
- These findings provide a foundation for further research into calpain's role in cardiac signaling and disease.