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Degradation of Mitochondrial Inner Membrane Protein Disrupts the Structural Interaction between Mitofilin and Cyclophilin D and Determines the Extent of Ischemia/reperfusion injury

线粒体内膜蛋白的降解破坏 Mitofilin 和亲环蛋白 D 之间的结构相互作用并确定缺血/再灌注损伤的程度

基本信息

批准号：
10200128
负责人：
Jean Chrisostome Bopassa
金额：
$ 37.76万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-15 至 2023-06-30
项目状态：
已结题

项目摘要

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, and peripheral vascular disease, continue to be among the most frequent causes of devastating disease and main causes of death in the US. Reperfusion of ischemic tissue triggers many pro-death signaling pathways which converge on to the mitochondria. Indeed, reoxygenation of cardiomyocytes leads to mitochondrial Ca2+ overload and an increase in reactive oxygen species (ROS) generation that triggers the opening of the mitochondrial permeability transition pore (mPTP). Although several proteins have been proposed as contributing to mPTP formation and function, its exact molecular identity and mechanism still need to be elucidated. Therefore, the current proposal seeks to establish the impact of mitofilin, which controls mitochondrial cristae morphology, regulation in mPTP formation that is responsible for triggering mitochondrial permeability transition is of fundamental importance for advancing our basic understanding of the mechanisms of I/R injury and represents a particularly exciting approach that will open new possibilities for therapeutic interventions against various diseases including I/R injury. Using 2D-DIGE and mass spectrometry, we identified mitofilin as a protein whose expression is significantly reduced after I/R versus sham. We found that versus WT, mitofilin-/- mice subjected to I/R exhibit an increase in myocardial infarct size, a reduction in cardiac functional recovery and Ca2+ retention capacity required to induce the mPTP opening, as well as an increase in mitochondrial Parkin expression and mitofilin ubiquitination. We further found that knockdown of mitofilin in H9c2 myoblasts with siRNA led to an increase in apoptosis via the AIF-PARP1 pathway that is associated with S phase arrest of the cell cycle, an increase in mitochondrial cristae disorganization, ROS production and Calpain activity, as well as a decrease in intracellular ATP production and mitochondrial membrane potential versus scramble siRNA. Interestingly, we also revealed that mitofilin structurally binds to Cyclophilin D and this interaction is abridged after mPTP opening triggered by Ca2+ overload. Our central hypothesis is that degradation mitofilin during I/R disrupts the CypD-mitofilin interaction resulting in pore formation that triggers mitochondrial permeability transition, thus activating necrotic signaling cascades. We will: 1) Define whether protection of mitofilin from degradation induces protective effects against I/R injury and anti-inflammatory effects in vivo, as well as establish the mechanism by which mitofilin down-regulation promotes apoptosis in transfected H9c2 myoblasts; 2. Define the impact of the MEK/ERK/GSK-3 pathway in I/R-induced mitofilin degradation, and reveal the mechanisms by which I/R stress induces mitofilin loss by increasing mitofilin ubiquitination, promoting excessive mitophagy, and increasing Calpain activity; 3. Determine the impact of the mitofilin-Cyclophilin D interaction in mPTP formation. In this proposal, we will test a panel of novel therapeutic approaches that could be ultimately used to improve the survival and outcomes of I/R injury.

以缺血/再灌注 (I/R) 为特征的疾病，例如心肌梗塞、中风和外周血管疾病仍然是破坏性疾病的最常见原因之一，也是导致美国的死亡原因。缺血组织的再灌注会触发许多促死亡信号通路，汇聚到线粒体上。事实上，心肌细胞的再氧合会导致线粒体 Ca2+ 过载和活性氧 (ROS) 生成的增加触发了线粒体通透性转换孔（mPTP）。尽管有几种蛋白质被提议作为有助于 mPTP 的形成和功能，其确切的分子特性和机制仍需进一步研究阐明了。因此，当前的提案旨在确定米托菲林的影响，米托菲林控制线粒体嵴形态、mPTP 形成的调节负责触发线粒体渗透性转变对于增进我们对机制的基本理解至关重要 I/R 损伤的研究代表了一种特别令人兴奋的方法，将为治疗开辟新的可能性针对包括缺血再灌注损伤在内的各种疾病的干预措施。使用 2D-DIGE 和质谱分析，我们鉴定出 mitofilin 是一种蛋白质，与假手术相比，I/R 后其表达显着降低。我们发现与 WT 相比，经过 I/R 处理的 mitofilin-/- 小鼠表现出心肌梗塞面积增加、心脏功能减少诱导 mPTP 打开所需的功能恢复和 Ca2+ 保留能力，以及增加线粒体 Parkin 表达和 mitofilin 泛素化。我们进一步发现 mitofilin 的敲低使用 siRNA 的 H9c2 成肌细胞通过 AIF-PARP1 途径导致细胞凋亡增加，该途径与细胞周期 S 期停滞、线粒体嵴解体增加、ROS 产生和钙蛋白酶活性，以及细胞内 ATP 产生和线粒体膜电位的降低与扰乱 siRNA 相比。有趣的是，我们还揭示了 mitofilin 在结构上与亲环蛋白 D 结合，并且这 Ca2+ 过载触发 mPTP 打开后，相互作用会减少。我们的中心假设是 I/R 过程中线粒体分裂素的降解破坏了 CypD-线粒体分裂素相互作用，导致孔形成，从而触发线粒体通透性转变，从而激活坏死信号级联。我们将： 1）定义是否保护 mitofilin 免遭降解可诱导 I/R 损伤和抗炎保护作用体内影响，并建立线粒体丝分裂素下调促进细胞凋亡的机制转染H9c2成肌细胞； 2. 定义 MEK/ERK/GSK-3 通路对 I/R 诱导的 mitofilin 的影响降解，并揭示 I/R 应激通过增加 mitofilin 诱导 mitofilin 损失的机制泛素化，促进过度线粒体自噬，并增加钙蛋白酶活性； 3. 确定影响 mPTP 形成中丝裂蛋白-亲环蛋白 D 的相互作用。在本提案中，我们将测试一组新型治疗方法最终可用于改善 I/R 损伤的生存率和结果的方法。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inner mitochondrial membrane protein MPV17 mutant mice display increased myocardial injury after ischemia/reperfusion.

线粒体内膜蛋白 MPV17 突变小鼠在缺血/再灌注后表现出心肌损伤增加。

DOI：
发表时间：
2020
期刊：
American journal of translational research
影响因子：
2.2
作者：
Madungwe,NgonidzasheB;Feng,Yansheng;ImamAliagan,Abdulhafiz;Tombo,Nathalie;Kaya,Ferdinand;Bopassa,JeanC
通讯作者：
Bopassa,JeanC

Estrogen rescues heart failure through estrogen receptor Beta activation.

DOI：
10.1186/s13293-018-0206-6
发表时间：
2018-10-30
期刊：
Biology of sex differences
影响因子：
7.9
作者：
Iorga A;Umar S;Ruffenach G;Aryan L;Li J;Sharma S;Motayagheni N;Nadadur RD;Bopassa JC;Eghbali M
通讯作者：
Eghbali M

Mitochondrial inner membrane protein, Mic60/mitofilin in mammalian organ protection.

DOI：
10.1002/jcp.27314
发表时间：
2019-04
期刊：
Journal of cellular physiology
影响因子：
5.6
作者：
Feng Y;Madungwe NB;Bopassa JC
通讯作者：
Bopassa JC

TrpA1 activation in peripheral sensory neurons underlies the ionic basis of pain hypersensitivity in response to vinca alkaloids.

DOI：
10.1371/journal.pone.0186888
发表时间：
2017
期刊：
PloS one
影响因子：
3.7
作者：
Boiko N;Medrano G;Montano E;Jiang N;Williams CR;Madungwe NB;Bopassa JC;Kim CC;Parrish JZ;Hargreaves KM;Stockand JD;Eaton BA
通讯作者：
Eaton BA

Activation of G protein-coupled oestrogen receptor 1 at the onset of reperfusion protects the myocardium against ischemia/reperfusion injury by reducing mitochondrial dysfunction and mitophagy.