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缺氧后活化Drp1通过偶联LRRK2影响多组织细胞线粒体功能的机制研究
Activated mitochondrial dynamin-related protein 1 couples with LRRK2 to cause mitochondrial dysfunction in hypoxic cells
- 第三军医大学学报 2020年42卷第4期 页码：326-333
- 作者机构：
400042 重庆，陆军军医大学(第三军医大学)野战外科研究所战伤休克与输血研究室，创伤、烧伤与复合伤国家重点实验室
- 作者简介：
[李涛，E-mail: lt200132@163.com LI Tao, E-mail: lt200132@163.com
- 基金信息：国家自然科学基金重点项目(81730059);国家自然科学基金青年科学基金(81700429)
DOI：10.16016/j.1000-5404.201910153
中图分类号： R341; R363.21; R364.4收稿：2019-10-22，
修回：2019-12-02，
纸质出版：2020-02-28
- 稿件说明：
移动端阅览
段晨阳, 向鑫明, 匡磊, 等. 缺氧后活化Drp1通过偶联LRRK2影响多组织细胞线粒体功能的机制研究[J]. 第三军医大学学报, 2020,42(4):326-333.
Chenyang DUAN, Xinming XIANG, Lei KUANG, et al. Activated mitochondrial dynamin-related protein 1 couples with LRRK2 to cause mitochondrial dysfunction in hypoxic cells[J]. Journal of Third Military Medical University, 2020, 42(4): 326-333.
段晨阳, 向鑫明, 匡磊, 等. 缺氧后活化Drp1通过偶联LRRK2影响多组织细胞线粒体功能的机制研究[J]. 第三军医大学学报, 2020,42(4):326-333. DOI： 10.16016/j.1000-5404.201910153.
Chenyang DUAN, Xinming XIANG, Lei KUANG, et al. Activated mitochondrial dynamin-related protein 1 couples with LRRK2 to cause mitochondrial dysfunction in hypoxic cells[J]. Journal of Third Military Medical University, 2020, 42(4): 326-333. DOI： 10.16016/j.1000-5404.201910153.
摘要
目的
2
探究缺氧后活化Drp1对线粒体能量代谢和有氧呼吸的调控机制。
方法
2
在心肌细胞(H9C2)、血管平滑肌细胞系(vascular smooth muscle cells，VSMC)、肠上皮细胞系(intestinal epithelial cells，IEC)中通过免疫共沉淀、免疫荧光方法观察多种细胞类型缺氧后Drp1活性变化及与线粒体损伤的关系；利用ZDOCK方法建立Drp1与同源激酶LRRK2蛋白互作模型，验证缺氧后Drp1-LRRK2偶联情况；以VSMC细胞为代表，通过Drp1点突变T595A破坏缺氧后Drp1-LRRK2的偶联，分别检测了培养基酸化程度、细胞外乳酸含量等反映无氧呼吸的指标和线粒体膜电位、ATP生成量等反映有氧呼吸的指标。
结果
2
缺氧后Drp1的Thr磷酸化水平增高、线粒体损伤明显。进一步研究发现缺氧后Drp1蛋白与LRRK2蛋白界面残基间存在大量氢键，导致缺氧后Drp1-LRRK2偶联从而影响线粒体功能。具体表现为糖酵解过程中乳酸生成的增多和线粒体有氧呼吸过程中膜电位的减低。
结论
2
缺氧后活化Drp1可通过偶联LRRK2破坏线粒体能量代谢和有氧呼吸，加重多器官组织线粒体损伤。
Abstract
Objective
2
To explore the mechanism of activated mitochondrial dynamin-related protein 1 (Drp1) in regulating mitochondrial energy metabolism and aerobic respiration after hypoxia.
Methods
2
We observed the changes of Drp1 activity and its relationship with mitochondrial damage by coimmunoprecipitation and immunofluorescence in cardiac myocyte line H9C2
vascular smooth muscle cells (VSMCs) and intestinal epithelial cells (IECs). The protein interaction model of Drp1 and LRRK2 was established using ZDOCK and verified by coimmunoprecipitation. A point mutation (T595A) was introduced in Drp1 gene to disrupt the coupling of Drp1 and LRRK2 in VSMCs
and the acidification of the culture medium
extracellular lactate content
mitochondrial membrane potential and ATP production of the cells were examined.
Results
2
Hypoxia caused significantly increased level of Thr phosphorylation in Drp1 and obvious mitochondrial damage in the cells. After hypoxia
numerus hydrogen bonds were found between the interface residues of Drp1 and LRRK2 proteins
which resulted in close binding between them to cause mitochondrial dysfunction
manifested by an increased lactate production during glycolysis and a decreased mitochondrial membrane potential during mitochondrial aerobic respiration.
Conclusion
2
Activated Drp1 causes impairment of mitochondrial energy metabolism and aerobic respiration by coupling with LRRK2 to aggravate mitochondrial damage in multiple tissues and organs after hypoxia.
关键词
Keywords
references
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