Mechanisms of mitochondrial Drp1 inducing excessive opening of mPTP channel through LRRK2-HK2 after hypoxia

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Mechanisms of mitochondrial Drp1 inducing excessive opening of mPTP channel through LRRK2-HK2 after hypoxia

Basic research | 更新时间：2025-10-21

- Mechanisms of mitochondrial Drp1 inducing excessive opening of mPTP channel through LRRK2-HK2 after hypoxia
- Journal of Chongqing Medical University Vol. 48, Issue 2, Pages: 117-123(2023)
- 作者机构：
  
  重庆医科大学第二附属医院麻醉科，重庆 400010
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.003176
  CLC： R364
- Received：20 August 2021，
  
  Revised：2022-02-24，
  
  Accepted：16 March 2022，
  
  Published：28 February 2023
- 稿件说明：
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段晨阳.缺氧后线粒体Drp1通过LRRK2-HK2诱导mPTP过度开放的机制研究[J].重庆医科大学学报,2023,48(2):117-123.

Duan Chenyang.Mechanisms of mitochondrial Drp1 inducing excessive opening of mPTP channel through LRRK2-HK2 after hypoxia[J].Journal of Chongqing Medical University,2023,48(2):117-123.
段晨阳.缺氧后线粒体Drp1通过LRRK2-HK2诱导mPTP过度开放的机制研究[J].重庆医科大学学报,2023,48(2):117-123. DOI： 10.13406/j.cnki.cyxb.003176.

Duan Chenyang.Mechanisms of mitochondrial Drp1 inducing excessive opening of mPTP channel through LRRK2-HK2 after hypoxia[J].Journal of Chongqing Medical University,2023,48(2):117-123. DOI： 10.13406/j.cnki.cyxb.003176.

摘要

目的

2

探究缺氧后线粒体动力相关蛋白1（dynamin-related protein 1，Drp1）对线粒体膜通透性转换通道（mitochondrial permeability transition pore，mPTP）开放的调控机制。

方法

2

在血管平滑肌细胞中通过免疫荧光方法观察缺氧后mPTP开放情况；通过超速离心法分离线粒体和细胞质成分蛋白；使用Co-IP和Western blot检测缺氧或者干预Drp1、干预己糖激酶-2（hexokinase-2，HK2）后Drp1的表达分布情况、HK2与电压依赖性阴离子通道蛋白（voltage dependent anion channel，VDAC）结合情况、Drp1与富含亮氨酸重复激酶2（leucine-rich repeat kinase 2，LRRK2）结合情况；使用蛋白分子对接和蛋白芯片方法筛选Drp1的潜在结合蛋白及结合位点；使用Drp1抑制剂和点突变法用于相应机制探究。

结果

2

缺氧后Drp1发生线粒体转位促使mPTP过度开放（

P

<

0.05）。使用Mdivi-1减少线粒体Drp1表达后可抑制mPTP开放，减少细胞色素C（cytochrome C，CytC）释放（

P

<

0.05）。缺氧后HK2-Thr473磷酸化水平减低引起的HK2线粒体分离会导致mPTP结构破坏，通过HK2 T473D点突变恢复HK2活性后，HK2与线粒体结合情况及mPTP开放情况明显改善（

P

<

0.05）。Drp1蛋白芯片结果发现缺氧后Drp1可以与LRRK2结合并封闭其活性位点，通过Drp1 T595A点突变破坏Drp1-LRRK2结合后，HK2活性及HK2线粒体分离情况明显改善（

P

<

0.05），mPTP开放情况明显减少（

P

<

0.05）。

结论

2

缺氧后线粒体Drp1通过封闭激酶LRRK2活性位点导致HK2 Thr473磷酸化水平减低及其线粒体分离，最终诱导mPTP过度开放。

Abstract

Objective

2

To explore the regulatory mechanisms of mitochondrial dynamin-related protein 1（Drp1） on excessive opening of mitochondrial permeability transition pore（mPTP） after hypoxia.

Methods

2

The opening of mPTP after hypoxia was observed by immunofluorescence in vascular smooth muscle cells（VSMCs）. Mitochondrial and cytoplasmic fractions were separated by ultracentrifugation. Co-IP and Western blot methods were used to detect the expression and distribution of Drp1，the binding of hexokinase-2（HK2） to voltage dependent anion channel（VDAC），and the binding of Drp1 to leucine-rich repeat kinase 2（LRRK2）. The potential binding proteins and binding sites of Drp1 were screened by protein molecular docking and protein chip. Mdivi-1，a Drp1 inhibitor，and mutation methods were used to explore the corresponding mechanisms.

Results

2

Mitochondrial translocation of Drp1 caused mPTP excessive opening in VSMCs after hypoxia（

P

<

0.05）. Reducing mitochondrial Drp1 expression by Mdivi-1 could inhibit mPTP opening and reduce the release

of cytochrome C（CytC）（

P

<

0.05）. The mitochondrial separation of HK2 caused by the decrease of HK2-Thr473 phosphorylation after hypoxia leaded to the destruction of mPTP structure. After restoring HK2 activity by HK2 T473D mutation，the binding of HK2 to mitochondria and the opening of mPTP after hypoxia were significantly improved（

P

<

0.05）. The results of Drp1 protein chip showed that Drp1 could bind to kinase LRRK2 and block its active site after hypoxia. After the combination of Drp1-LRRK2 was broken by the site mutation of Drp1 T595A，the activity of HK2 and the mitochondrial separation of HK2 were obviously improved（

P

<

0.05），and the opening of mPTP channel was obviously reduced（

P

<

0.05）.

Conclusion

2

Mitochondrial Drp1 may reduce HK2-Thr473 phosphorylation and cause HK2 mitochondrial separation by binding and blocking the active site of kinase LRRK2，which finally induces the excessive opening of mPTP after hypoxia.

关键词

Keywords

references

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