School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR China; National Engineering Research Center of Seafood, Dalian 116034, PR China.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
Food Res Int. 2018 Jul;109:481-488. doi: 10.1016/j.foodres.2018.04.067. Epub 2018 May 1.
The objective of this study was to investigate the differential role of cathepsin B, L, D and calpain in degradation and disassembly of myofilament. Myofibrillar protein of grass carp (Ctenopharyngodon idella) was incubated with proteases monotonously, simultaneously or sequentially. Subsequently, protein degradation were detected using SDS-PAGE and myofilament disassembly induced by changes of non-covalent interactions were measured through SDS-PAGE using l-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC) as a zero length cross-linker. Additionally, content of heat shock proteins which functioned in stabilizing assembly architecture of myofibrillar protein was determined. Results showed that calpain and cathepsin B, calpain and cathepisn L could act in a stepwise and complimentary manner to synergistically dissociate and degrade myofibrillar protein. In synergistic action, cathepsin B disrupted the thick filament assembly through lowering the UNC45 and HSP90 concentration in myofibrillar protein, facilitating the degradation of dissociated MHC by calpain. Meanwhile, Cathepsin L was shown to preferentially remove the actin from thin filament via lowering the content of HSP27 and αb-crystallin, to create dissociated actin as substrate supply for calpain.
本研究旨在探究组织蛋白酶 B、L、D 和钙蛋白酶在肌丝降解和组装中的差异作用。采用蛋白酶单一、同时或顺序孵育草鱼（Ctenopharyngodon idella）肌原纤维蛋白，随后通过 SDS-PAGE 检测蛋白降解，通过 SDS-PAGE 用 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）作为零长度交联剂测量非共价相互作用变化诱导的肌丝组装。此外，还测定了热休克蛋白的含量，热休克蛋白在稳定肌原纤维蛋白组装结构中起作用。结果表明，钙蛋白酶和组织蛋白酶 B、钙蛋白酶和组织蛋白酶 L 可以以逐步和互补的方式协同作用，协同解离和降解肌原纤维蛋白。在协同作用中，组织蛋白酶 B 通过降低肌原纤维蛋白中 UNC45 和 HSP90 的浓度，破坏粗丝组装，从而促进钙蛋白酶对解离 MHC 的降解。同时，组织蛋白酶 L 优先通过降低 HSP27 和 αb-晶状体蛋白的含量，从细肌丝中去除肌动蛋白，为钙蛋白酶提供解离的肌动蛋白作为底物供应。
