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: 8931603
• 负责人: Steven Sollott
• 金额: $ 82.23万
• 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/8931603
• 关键词: 70-kDa Ribosomal Protein S6 Kinases; Affinity; Age; Aging; Antifungal Agents; Apoptosis; Area; BCL-2 Protein; BCL2 gene; Binding; Binding Proteins; Biological Markers; 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; Muscle satellite cell; 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; 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; VDAC1 gene; VDAC2 gene; age related; burden of illness; cariporide; design; fitness; healthy aging; hexokinase; human FRAP1 protein; inhibitor/antagonist; insight; knock-down; mitochondrial K(ATP) channel; mitochondrial permeability transition pore; novel; oxidant stress; oxidation; preconditioning; prevent; research study; response; sarcopenia; 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. Experiments are ongoing examining the hypothesis that the sensitivity of mPTP to ROS can serve as a biomarker of mitochondrial fitness during aging. The mitochondrial fitness of muscle stem cells may in part underlie the development of age-related sarcopenia.

环境压力集中在线粒体上，可以触发或抑制细胞死亡。可兴奋的有丝分裂后细胞（例如心脏中的心肌细胞和大脑中的神经元）响应亚致死性有害应激，参与提供保护免受后续损伤的机制。这些保护机制涉及内源性信号传导的激活，可以对氧化剂和与缺氧/复氧（即心脏病发作或中风期间）相关的其他应激产生显着的抵抗力，从而促进细胞生存能力的增强。然而，上游信号机制仍然是一个活跃争论的领域，而末端执行器仍未得到解决。 我们发现，长时间缺氧后的复氧会降低心肌细胞中线粒体通透性转变（mPTP）的活性氧（ROS-）阈值，并且细胞存活率与去极化线粒体的比例呈显着负相关。我们证明，通过不同上游机制发挥作用的多种心脏/神经保护剂均通过限制 mPTP 诱导来促进细胞存活。我们发现，可以通过两种依赖和独立于调节性线粒体肿胀的一般方式触发保护，这些调节性线粒体肿胀通过抑制末端效应器（渗透性过渡孔复合物）上的 GSK-3b 来收敛，从而防止 mPTP。表现出记忆（即“预处理”）的细胞保护是由触发的线粒体肿胀（由于通过流入和/或保留增强 K+ 积累）引起的，导致底物氧化和 ROS 产生增强，导致 PKC 的氧化还原激活，进而抑制 GSK- 3b（通过 Ser-9 磷酸化）。例如，二氮嗪激活的线粒体 ATP 依赖性 K+ 通道（mitoKATP，我们已经鉴定并正在表征）和 Ca2+ 激活的 K+ 通道都可以充当线粒体 K+ 流入机制（可以介导线粒体调节肿胀依赖性）保护等）。 δ-阿片肽 DADLE 和 NHE 抑制剂 HOE 642（卡立波利）和 HOE 694 各自产生独立于 mitoKATP 的线粒体调节性肿胀依赖性保护。或者，受体酪氨酸激酶或某些 G 蛋白偶联受体激活可通过 PKB/Akt 和 mTOR/p70s6k、PKC 或 PKA 途径抑制 GSK-3b，从而引发细胞保护（无线粒体肿胀或持久记忆）。后一类的例子包括胰岛素（通过 Akt 和 mTOR/p70s6k）和直接 GSK-3 抑制剂 Li+。 我们发现，GSK-3b（而不是 GSK-3a）的 siRNA 敲低会诱导保护状态，并且转基因小鼠表达心脏限制性、组成性活性、非抑制形式的 GSK-3b (GSK-3b S9A)对一系列上游信号具有抵抗力，这些信号可有效诱导 WT 小鼠的保护状态。 我们得出的结论是，GSK-3b（特别是其失活）是作用于负责心脏保护的末端效应器（线粒体通透性过渡孔）的多个上游信号的主要必需整合点。 当细胞保护信号通路被激活时，我们发现 Bcl-2 家族成员将来自 GSK-3b 的信号传递到孔处或附近的靶标上。 因此，通过抑制 GSK-3b（通过 Bcl-2 蛋白传递），这些信号通路的汇聚对末端效应器（通透性转换孔复合物）的影响，以限制 mPTP 诱导，是心肌细胞保护的一般机制。 我们提出，旨在抑制主开关激酶 GSK-3b 的临床治疗策略，以保护通透性转换孔复合物免受 mPTP 诱导，可通过预防患者死亡，有效减少心脏病发作或中风发作期间的梗塞范围。心肌细胞和神经元（分别）。 正在检查与年龄相关的缺血预处理能力丧失背后的信号缺陷，这可能会导致与保持健康衰老相关的可测试的临床疗法。 作为一种关键的糖酵解酶，己糖激酶 (HK) 通过控制线粒体 ATP 的葡萄糖磷酸化优先利用，在线粒体和细胞质之间提供功能性联系。在 20 世纪 70 年代末和 80 年代初，人们证明 HKI 和 II 是 VDAC 结合蛋白，从那时起，这种糖酵解酶就被认为是主要的 VDAC 调节剂。在过去的几年里，关于 HKII（线粒体结合亚型之一）在 mPTP 调节中的作用存在相当多的争论，我们试图明确回答这个问题。 葡萄糖和 ATP 既充当 HK-VDAC 结合的催化底物又充当调节剂。此外，不同的药物可能会促进 HK 从线粒体中释放，从而模仿葡萄糖-6-磷酸的自然作用。抗真菌药物如克霉唑和联苯苄唑是有效的己糖激酶释放剂。事实上，我们发现克霉唑确实降低了心肌细胞中的 mPTP ROS 阈值，尽管该药物可能具有其他非特异性作用。由于已知线粒体 HKI 和 II 的 N 端序列对 VDAC 具有高亲和力，因此对应于 HKI 和 HKII 共有的 N 端 29 个 AA 残基结构域的肽（HKII-VDB；VDAC 结合结构域） ）被构建并被发现可以有效释放线粒体结合的 HK 库。 我们发现该肽显着降低了心肌细胞中的 mPTP ROS 阈值（IC50 400 nM），而对照肽则没有这种效果（证明了活性肽的特异性）。研究还发现，该肽强制释放 HKII 会引发细胞死亡信号，从而导致 mPTP 打开。令人惊讶的是，这种肽在 VDAC1/3 -/- 成纤维细胞中也完全有效（其中还发现 VDAC2 同种型不与 HKII 结合），证明 VDAC 在诱导这些细胞中的凋亡方面完全是可有可无的。因此，HKII 从另一个位点脱离可能是通过从 OMM 到 IMM 以及 mPTP 成分/调节剂的构象变化传递启动细胞凋亡的信号，与 VDAC 的存在无关。 实验正在进行中，以验证 mPTP 对 ROS 的敏感性可以作为衰老过程中线粒体健康度的生物标志物的假设。 肌肉干细胞的线粒体适应性可能在一定程度上是与年龄相关的肌少症发生的基础。