谷氨酰胺通过蛋白质二硫键异构酶调控MG53蛋白修饰促进烧伤后骨骼肌膜修复的机制研究

Skeletal muscle membrane repair disorders is one of the key reasons leading to the muscle decomposition after burn injury. It is known that glutamine (Gln) can mitigate skeletal muscle catabolism postburn, but whether it can promote damaged muscle membrane repair is unclear. Our previous study has demonstrated that Gln can improve MG53, a important membrane repair protein, synthesis and modification, and accelerate skeletal muscle membrane repair in burned mice. Previous studies have found that protein disulfide isomerase (PDI) is a key enzyme in protein modification, may play a crucial role in MG53 dimer formation. Hence, we deduce that Gln maintain PDI structure and function, facilitate MG53 modification, and accelerate damaged muscle membrane restore probably through maintaining intracellular acid-base and oxidation-reduction equilibrium. In order to confirm the hypothesis, we will first verify the effect of PDI on MG53 synthesis and modification by using site directed mutagenesis and siRNA inhibit PDI expression. Then, analysis the effect of Gln on PDI structure and function using NMR, three-dimensional electron microscopy and enzyme kinetics analysis. Finally, will observe the effect of Gln on PDI structure and function, MG53 synthesis and modification, as well as muscle membrane repair capacity through a variety of means to regulate Gln metabolism. This study is expected to partly clarify the molecular mechanisms of Gln promote damaged muscle membrane restoration, and it has important theoretical significance and potential application value.

肌膜损伤后修复障碍是导致烧伤后骨骼肌分解的重要原因，现已知谷氨酰胺(Gln)能减轻烧伤后骨骼肌分解代谢，但是否能促进受损肌膜修复，不甚清楚。课题组前期发现，Gln能促进烧伤小鼠骨骼肌修复蛋白MG53形成二聚体，加快受损肌膜修复。蛋白质二硫键异构酶(PDI)是蛋白修饰的关键酶，可能在MG53二聚体的形成中挥了关键作用。据此提出Gln可能通过调节细胞内酸碱和氧化还原平衡，维护PDI的结构与功能，促进MG53蛋白修饰，加速受损肌膜修复的假说。为此，本项目拟采用定点突变、siRNA等手段抑制PDI合成，观察对MG53修饰的影响，进而采用NMR、三维电镜、酶动力学等手段，观察Gln对PDI结构与功能的影响，最后通过多种手段调控Gln代谢，观察Gln对PDI结构与功能，MG53合成与修饰，以及膜损伤后修复的影响。本研究有望初步阐明Gln促进受损肌膜修复的分子机制，具有重要的理论意义和潜在的应用价值。

本课题通过四年的研究，初步阐明了谷氨酰胺通过蛋白质二硫键异构酶促进MG53合成与修饰，加快烧伤后骨骼肌膜修复的分子机制。本研究采用了烧伤、心肌毒素CTX以及运动损伤等多种致伤模型，观察了伤后骨骼肌膜损伤修复情况。发现上述致伤因素可导致骨骼肌膜受损，修复延迟。进一步研究发现，各种损伤因素均可导致MG53合成减少，特别是具有生理功能的二聚体明显减少。这与内质网中负责MG53修饰的蛋白质二硫键异构酶（PDI）活性降低有关。深入研究发现，该家族蛋白中的PDIA3是调控MG53合成与修饰的关键酶，其活性改变直接影响骨骼肌损伤后修复。进一步研究发现，多种病理状态下的内质网应激可导致PDIA3微环境中氧化还原和酸碱平衡丧失，PDIA3酶活性受到抑制，从而导致MG53合成与修饰障碍，抑制骨骼肌膜修复。本课题研究证实，谷氨酰胺是合成 GSH 的前体物质，是机体潜在的氧化还原平衡物质。在氧化应激的情况下，谷氨酰胺能提高谷胱甘肽还原酶活性，加快 GSSG 向 GSH 的转化，维持其动态平衡，从而减轻烧伤后PDI亚硝基化，促进MG53二聚体的形成加速骨骼肌膜修复。综上，谷氨酰胺能减轻烧伤后PDI亚硝基化调控MG53的修饰与合成促进骨骼肌膜修复。通过本课题研究，为后期研究谷氨酰胺代谢与PDI和MG53空间结构的关系奠定了基础。项目资助发表核心论文1篇，SCI论文3篇，国家发明专利1项，培养研究生2名，获得硕士学位1名，博士学位1名。