Screening Of Porcine Calsarcin-1 Interacting Proteins By Yeast Two-hybrid

Calsarcins are calmodulin dependent,calcium-activated protein phosphatase that plays an important role in transducing calcium-dependent signals in a variety of cell types. They comprise a novel family of muscle-specific calcineurin-interacting proteins and play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells.The expression of calsarcin-1 is restricted to slow-twitch skeletal muscle fibres,whereas that of both calsarcin-2 and calsarcin-3 is enriched in fast-twitch fibres. Calsarcin-1 knockout mice show enhanced calcineurin activity and an excess of skeletal muscle fibres,indicating that calsarcin-1 negatively modulates the activation of calcineurin.The calsarcins may also play an important role in myofibrillar differentiation and development via their ability to regulate other related gene express,as well as a possible involvement in calcium ion signal transduction pathways.The colocalization with several key Z-disc proteins such asα-actinin,γ-filamin and telethonin suggests that calsarcins serve to cross-link these proteins,thereby likely contributing to the formation and maintanance of the Z-disc.To study the regulation of calcium ion by calsarcin-1 gene and its interacted proteins in myocyte will provide new insights on molecule mechanisms of muscle fiber determination and differentiated.We performed yeast two-hybrid sceen to identify calsarcin-1-interacting proteins and preliminary analysis on the biological functions of the interaction,for investigate the function of calsarcin-1.The main results are as follows:1.The coding region of calsarcin-1 was amplified by PCR using pEGFP-N1-CS1 fusion plasmids as a template,to produce the pGBKT7-CS1 bait expression vector which expressed successful and nontoxic.2.Isolated and purified ds cDNA from pig muscle tissue to construct the prey library.3.We have identified 29 interaction proteins through co-transformation with bait vector,ds cDNA and prey library.4.After PCR,enzyme digestion and sequencing,we found three interested proteins CASQ1,TNNT3 and ACTN3.CASQ1 and TNNT3 participated in calcium ion regulation and ACTN3 was important in maintaining the structure of Z-disc.