鱼类宰后肌肉软化过程中HSP90与肌球蛋白结合规律及其对肌球蛋白纤维丝解聚的调控机制研究

Myosin filament disassembly is proved to be mainly responsible for softening of fish flesh during post mortem storage. Among factors accounting for myosin filament disassembly, HSP90 plays a major role in regulating the disassembly of myosin filament. However, it is still unclear for the action mode of HSP90 in regulating the myosin filament disassembly as well as the underlying mechanism. In the present study, with grass carp as a representative species of edible fish selected as material, firstly, the translocation of HSP90 in muscle cells will be investigated by co-immunoprecipitation and western blotting, so as to elucidate changes in association/dissociation status of HSP90 as well as its correlation with degree of myosin filament disassembly and muscle texture. Secondarily, the changes in anisotropy as well as the secondary and third structure of myosin subject to various association/dissociation status of HSP90 will be studied in order to illuminate the changes in conformational structure of myosin induced by shift of HSP90 association/dissociation status. Finally, the effect of association/dissociation status of HSP90 on self-assembly filament morphology and spatial orientation rearrangement of myosin filament will be explored by morphological observation, particle size analysis and site-directed spectroscopy of time-resolved fluorescence resonance energy transfer (TR-FRET), to better understand the allosteric regulation mode of supramolecular assembly architecture of myosin filament mediated by shift of HSP90 association/dissociation status. The allosteric regulatory mechanism of supramolecular assembly architecture of myosin filament mediated by HSP90 association/dissociation status through regulating myosin conformational structure, as well as its correlation between the myosin filament disassembly of post mortem fish will be revealed from a biochemical view and at a supramolecular level. This research is of practical significance in broadening and deepening the theory for effective control of fish texture quality during post mortem storage.

肌球蛋白纤维丝解聚是造成宰后鱼肉软化的重要原因，HSP90在调控肌球蛋白纤维丝解聚过程中起关键作用，但相关调控机制尚不清楚。本课题以大宗食用鱼种——草鱼为研究对象，首先基于免疫学技术研究宰后鱼肉中HSP90的亚细胞迁移过程，确定HSP90结合/解离状态变化规律及其与肌球蛋白聚合度和鱼肉质构间的关联；然后通过分析不同HSP90结合/解离状态下肌球蛋白的荧光各向异性及二、三级结构差异，阐明HSP90对肌球蛋白变构作用的结构域位置和构象变化类型；最后借助形态表征和荧光共振能量转移技术研究HSP90结合/解离状态对肌球蛋白纤维丝自组装聚合形态和空间取向的影响，探讨HSP90对肌球蛋白纤维丝超分子结构的变构调节模式，从生物化学角度和超分子水平阐释鱼类宰后HSP90结合/解离状态变化通过蛋白变构效应引发的肌球蛋白纤维丝超分子结构演变与其解聚间的关系。本研究将为解决鱼类宰后质构劣化问题提供理论指导。

本项目对HSP90结合状态影响宰后低温保鲜鱼类肌肉肌原纤维、肌球蛋白纤维丝解聚过程和鱼肉质构劣化的调控作用机制及相关控制技术进行了深入系统研究。初步探明了草鱼肉低温贮藏过程中HSP90在肌肉中的迁移及结合状态变化规律、肌原纤维纤维丝解聚规律及其与鱼肉质构和微观结构变化的关系；明确了HSP90从肌原纤维到肌浆的迁移引起的结合型HSP90含量下降是造成鱼肉肌原纤维尤其是肌球蛋白纤维丝解聚和保鲜鱼肉质构劣化的主导因素；宰后鱼肉中HSP90由肌原纤维迁移至肌浆中，导致结合型HSP90含量迅速降低，引起肌球蛋白轻链、肌球蛋白重链和肌动蛋白从肌原纤维中解离，造成肌球蛋白纤维丝和肌动蛋白纤维丝松散化，并伴随着肌球蛋白重链被迅速降解，肌球蛋白轻链表现为去磷酸化趋势，肌球蛋白轻链和肌动蛋白在贮藏过程中相对稳定较难降解，以胶原蛋白为代表的胞外结缔组织纤维束在贮藏期间也发生了解聚和部分降解，结合型HSP90含量下降与肌球蛋白纤维丝解聚及鱼肉质构软化密切相关；阐述了HSP90结合状态对鱼肉肌原纤维和肌球蛋白结构的调节机制，证实了随肌原纤维结合型HSP90含量增加，肌球蛋白趋向于头部折叠完整度增加而尾部疏水基团暴露并伸展的结构变化规律；发现了二羧基有机酸作为天然可食用有机酸对鱼肉结合型HSP90的稳定化作用，并通过解聚模拟体系确定了以上几种天然可食用稳定化剂作用于保鲜鱼片的有效浓度；确证了马来酸、苹果酸、酒石酸通过增加结合型HSP90含量而达到抑制肌原纤维和肌球蛋白纤维丝解聚并减缓低温保鲜草鱼肉质构劣化的效果。这些为宰后鱼肉质构品质控制提供了理论基础和应用依据。

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