GSK3b mediates convergence of protection signaling to limit mitochondrial damage

GSK3b 介导保护信号汇聚以限制线粒体损伤

• 批准号: 7964060
• 负责人: Steven Sollott
• 金额: $ 74.54万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964060
• 关键词: 70-kDa Ribosomal Protein S6 Kinases; Affinity; Age; Antifungal Agents; Apoptosis; Area; Binding; Binding Proteins; Biological Preservation; Brain; Calcium-Activated Potassium Channel; Cardiac; Cardiac Myocytes; Cell Death; Cell Death Signaling Process; Cell Survival; Cells; Cessation of life; Clinical; Clinical Treatment; Complex; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Cytosol; Defect; Development; Diazoxide; Enzymes; Exhibits; Family member; Fibroblasts; G-Protein-Coupled Receptors; Glucose; Glucose-6-Phosphate; Glycogen Synthase Kinase 3; Goals; HK2 gene; Heart; Hypoxia; Induction of Apoptosis; Infarction; Inhibitory Concentration 50; Injury; Insulin; Ischemic Preconditioning; Lead; Leucine-2-Alanine Enkephalin; Link; Mediating; Memory; Mitochondria; Mitochondrial Swelling; Mitotic; Mus; Myocardial Infarction; N-terminal; Nature; Neurons; Neuroprotective Agents; Opioid Peptide; Oxidants; Oxidation-Reduction; Pathway interactions; Peptides; Permeability; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Potassium Channel; Production; Protein Isoforms; Proteins; Reactive Oxygen Species; Receptor Activation; Receptor Protein-Tyrosine Kinases; Regulation; Resistance; Role; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Specificity; Stress; Stroke; Stroke prevention; Swelling; Transgenic Mice; VDAC2 gene; burden of illness; cariporide; design; healthy aging; hexokinase; human FRAP1 protein; inhibitor/antagonist; insight; knock-down; mitochondrial K(ATP) channel; mitochondrial permeability transition pore; novel; oxidation; preconditioning; prevent; response; treatment strategy

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, post-mitotic cells (such as cardiac myocytes in heart, and neurons in brain), in response to sub-lethal noxious stress engage mechanisms affording protection from subsequent insults. These protection mechanisms involve activation of endogenous signaling which can confer significant resistance to oxidant and other stresses associated with hypoxia/reoxygenation (i.e., during a heart attack or stroke), which promotes the enhanced capacity for cell survival. However, the upstream signaling mechanisms have remained an area of active debate, and the end effector(s) have remained unsolved. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species- (ROS-) threshold for the mitochondrial permeability transition (mPTP) in cardiac myocytes, and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. We demonstrate that a wide variety of cardio/neuroprotective agents acting via distinct upstream mechanisms all promote cell survival by limiting mPTP induction. We found that protection can be triggered in 2 general ways dependent and independent of regulatory mitochondrial swelling which converge via inhibition of GSK-3b on the end effector, the permeability transition pore complex, preventing the mPTP. Cell protection exhibiting a memory (i.e., "preconditioning") results from triggered mitochondrial swelling (due to enhanced K+ accumulation via influx and/or retention) causing enhanced substrate oxidation and ROS production, leading to redox activation of PKC which in turn inhibits GSK-3b (via phosphorylation of ser-9). Both the diazoxide-activated mitochondrial ATP dependent K+ channel (mitoKATP, which we have identified and are now chararcterizing), and the Ca2+-activated K+ channel, for example, can serve as mitochondrial K+ influx mechanisms (that can mediate mitochondrial regulatory swelling-dependent protection, etc). The delta-opioid peptide, DADLE, and the NHE-inhibitors, HOE 642 (cariporide) and HOE 694, each produce mitochondrial regulatory swelling-dependent protection independently of the mitoKATP. Alternatively, receptor tyrosine kinase or certain G-protein coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3b, via either PKB/Akt and mTOR/p70s6k, PKC, or PKA pathways. Examples of this latter class include insulin (via Akt and mTOR/p70s6k) and the direct GSK-3 inhibitor, Li+. We found that siRNA knock-down of GSK-3b, but not GSK-3a, induced the protection state, and that transgenic mice expressing a cardiac-restricted, constitutively active, non-inhibitable form of GSK-3b (GSK-3b S9A) were resistant to a whole battery of upstream signals that were effective to induce the protected state in WT mice. We concluded that GSK-3b (and specifically its inactivation) is a major, required integration point for a multitude of upstream signals acting on an end-effector responsible for cardioprotection (the mitochondrial permeability transition pore). When cell protection signaling pathways are activated, we found that the Bcl-2 family members relay the signal from GSK-3b onto a target at or in close proximity to the pore. Thus, the effect of the convergence of these signaling pathways via inhibition of GSK-3b, relayed through Bcl-2 proteins, on the end effector, the permeability transition pore complex, to limit mPTP induction, is the general mechanism of cardiomyocyte protection. We propose that clinical treatment strategies designed to inhibit the master switch kinase, GSK-3b, to protect the permeability transition pore complex from mPTP induction, would be effective to reduce the size of infarction during episodes of heart attack or stroke by preventing the death of cardiac myocytes and neurons (respectively). Signaling defects underlying the age-assocciated loss of the capacity for ischemic preconditioning are being examined which could lead to testable clinical therapies relevant to the preservation of healthy aging. As a key glycolytic enzyme, hexokinase (HK) provides a functional link between mitochondria and cytosol by governing the preferential utilization of mitochondrial ATP for glucose phosphorylation. In the late 1970s and early 1980s it was demonstrated that HKI & II are VDAC-binding proteins, and since then this glycolytic enzyme has been recognized as the main VDAC modulator. There had been considerable debate over the past several years regarding the role of HKII (one of the mitochondrial-bound isoforms) in mPTP regulation, and we sought to definitively answer the question. Glucose and ATP act both as catalytic substrates and modulators of HK-VDAC binding. In addition, different drugs may facilitate the release of HK from mitochondria, thereby mimicking the natural effect of glucose-6-phosphate. Antifungal drugs such as clotrimazol and bifonazol are effective hexokinase-releasing agents. Indeed, we found that clotrimazol does reduce the mPTP ROS-threshold in cardiomyocytes, although this drug could have other non-specific actions. Since it is known that the N-terminal sequence of the mitochondrial HKI and II has high affinity to VDAC, a peptide corresponding to the N-terminal 29 AA residue domain common for both the HKI and HKII (HKII-VDB; VDAC-binding domain) was constructed and found to effectively release the mitochondrial-bound HK pool. We found that this peptide substantially decreased the mPTP ROS-threshold in cardiomyocytes (IC50 400 nM), while a control peptide had no such effect (proving the specificity of the active peptide). It was also found that forced release of HKII by this peptide invokes a cell death signal that engages mPTP opening. Surprisingly, this peptide was also fully effective in VDAC1/3 -/- fibroblasts (where the VDAC2 isoform was also found not to be bound to HKII) proving the complete dispensability of VDACs for the induction of apoptosis in these cells. Thus, HKII detachment from another site might be the signal to initiate apoptosis by relay of a conformational change from OMM to IMM and to components/modulators of the mPTP, independently of VDACs presence.

环境应力会在线粒体上汇聚，该线粒体会触发或抑制细胞死亡。可激发的，可激发的有丝分裂后细胞（例如心脏中的心肌细胞和大脑中的神经元），以响应亚洲性有害压力的响应，从而使机制具有保护，从而可以保护免受随后的侮辱。这些保护机制涉及激活内源性信号传导，该信号传导可以赋予对氧化剂的显着抗性和与缺氧/重氧相关的其他应力（即心脏病发作或中风期间），从而促进了细胞存活的增强能力。但是，上游信号传导机制仍然是主动争论的领域，末端效应子仍未解决。 我们表明，长时间缺氧后的重氧降低了心肌细胞中线粒体通透性过渡（MPTP）的活性氧（ROS-）阈值，并且该细胞的存活与地下细胞体的脱极层的分数陡峭地呈负相关。我们证明，通过不同上游机制作用的各种心脏/神经保护剂都通过限制MPTP诱导来促进细胞存活。我们发现，可以以两种一般方式触发保护，并独立于调节线粒体肿胀，这些线粒体肿胀通过抑制GSK-3B对末端效应器的抑制（渗透性过渡孔复合物）融合，从而防止MPTP。细胞保护表现出记忆（即“预处理”）是由触发的线粒体肿胀引起的（由于涌入和/或保留的K+积累增强而导致底物氧化和ROS产生增强，从而导致PKC的氧化还原激活，从而导致PKC的氧化还原激活，从而通过Ser-3b抑制了GSK-3B（通过Ser-9的磷酸化）。二氮氧化物激活的线粒体ATP依赖性K+通道（MitokATP，我们已经确定并正在炭化），例如，Ca2+激活的K+通道（例如，可以用作线粒体K+涌入机制）（可以介导线粒体调节性肿胀依赖性依赖性依赖性依赖性依赖性依赖）。 Delta-Apeid肽，Dadle和NHE抑制剂，HOE 642（Cariporide）和HOE 694，每种都会产生线粒体调节性肿胀依赖性依赖性依赖性保护，独立于MitokATP。另外，通过抑制PKB/AKT和MTOR/P70S6K，PKC，PKC或PKA通路，通过抑制GSK-3B，受体酪氨酸激酶或某些G蛋白偶联受体激活会引起细胞保护（无线粒体肿胀或耐用记忆）。后一类的例子包括胰岛素（通过AKT和MTOR/P70S6K）和直接的GSK-3抑制剂LI+。 We found that siRNA knock-down of GSK-3b, but not GSK-3a, induced the protection state, and that transgenic mice expressing a cardiac-restricted, constitutively active, non-inhibitable form of GSK-3b (GSK-3b S9A) were resistant to a whole battery of upstream signals that were effective to induce the protected state in WT mice. 我们得出的结论是，GSK-3B（尤其是其失活的）是主要的，所需的集成点，用于作用于负责心脏保护的最终效应器（线粒体渗透性过渡孔）的众多上游信号。 当激活细胞保护信号通路时，我们发现Bcl-2家族成员将信号从GSK-3B传递到孔或近距离接近孔的目标。 因此，通过抑制GSK-3B的抑制，通过Bcl-2蛋白继电器对末端效应子（渗透性过渡孔复合物）限制MPTP诱导的融合的影响，是心肌细胞保护的一般机制。 我们建议旨在抑制主开关激酶GSK-3B的临床治疗策略，以保护渗透性过渡孔复合物免受MPTP诱导的影响，将有效地减少心脏病发作或中风期间梗塞的大小，从而通过防止心肌细胞和神经元的死亡（分别可以防止心脏肌细胞和神经元）（分别防止）。 正在检查与年龄相关的缺血性预处理损失背后的信号缺陷，这可能会导致与保存健康衰老有关的可测试临床疗法。 作为关键糖酵解酶，己糖激酶（HK）通过管理线粒体ATP的优先利用来促葡萄糖磷酸化，从而提供了线粒体和细胞质之间的功能联系。在1970年代末和1980年代初，已证明HKI和II是VDAC结合蛋白，从那时起，这种糖酵解酶就被公认为是主要的VDAC调节剂。在过去的几年中，关于HKII（线粒体结合的同工型）在MPTP调节中的作用有很多争论，我们试图确切地回答这个问题。 葡萄糖和ATP均充当HK-VDAC结合的催化底物和调节剂。此外，不同的药物可能有助于从线粒体中释放HK，从而模仿6-磷酸葡萄糖的自然作用。抗真菌药物（例如氯吡唑和双唑醇）是有效的己糖酶释放剂。的确，我们发现克替三唑确实会减少心肌细胞中MPTP ROS阈值，尽管该药物可能具有其他非特异性作用。 Since it is known that the N-terminal sequence of the mitochondrial HKI and II has high affinity to VDAC, a peptide corresponding to the N-terminal 29 AA residue domain common for both the HKI and HKII (HKII-VDB; VDAC-binding domain) was constructed and found to effectively release the mitochondrial-bound HK pool. 我们发现，该肽显着降低了心肌细胞（IC50 400 nm）中的MPTP ROS阈值，而对照肽没有这种作用（证明活性肽的特异性）。还发现，这种肽强迫释放HKII会引起与MPTP打开的细胞死亡信号。令人惊讶的是，该肽在VDAC1/3 - / - 成纤维细胞中也完全有效（在其中也发现VDAC2同工型不与HKII结合），证明VDAC对这些细胞中凋亡的诱导具有完全的可配合性。因此，HKII脱离另一个位点可能是通过从OMM到IMM到MPTP的组件/调节剂的构象变化来引发凋亡的信号，独立于VDACS的存在。