Role of the mitochondrial Translocator Protein (mTSPO) in cell homeostasis: a molecular pathway in signalling and self-conservation mechanisms

为了寻找可能决定细胞代谢速度和定义其健康稳态的分子，我们现在有兴趣阐明mTSPO的细胞生物学作用。虽然与许多病理生理条件有关，但它对保护细胞完整性的过程，如自噬和凋亡的作用尚不清楚，它在线粒体功能中的作用也不明确。为了阐明这两个基本过程，确定mTSPO与VDAC的表达比率及其药理调节如何定义钙信号传递、ROS动态、ATP配给和氧化还原状态将是有指导意义的。细胞内钙离子的生理、三磷酸腺苷的平衡、细胞程序性死亡以及自噬都依赖于线粒体对钙离子的有效处理。然而，尽管进行了一些较小的尝试，但mTSPO对这一信号通路的贡献在很大程度上被忽视了，我们现在的目标是探索和阐明mTSPO对线粒体耦合的贡献，通过靶向自噬，线粒体偶联是ATP合成、ROS产生和网络重塑的起源。然而，ROS信号的修饰也会影响下游的效应器：促进VDAC非生理性磷酸化的激酶。因此，这一间接途径以及mTSPO和VDAC之间的直接物理相互作用将被视为潜在的潜在机制。以下实验将利用小鼠胚胎成纤维细胞(MEF)细胞系阐明上述观点：1.将使用基于实时成像、发光和荧光的方法来评估mTSPO在细胞自噬和信号转导中的作用。A)mTSPO蛋白将通过传统的分子生物学技术被瞬时操纵以增加(+)或降低(-)其表达；然后，这种操纵的结果将在b)自噬、c)钙信号传递、d)ROS产生和e)在缓冲的Ca+和ROS条件下的自噬来确定。2.利用共聚焦显微镜、荧光和生化等方法研究mTSPO在线粒体偶联效率和重塑中的作用。在瞬时调节mTSPO或药物结合后，将涉及以下内容：a)线粒体的氧化还原状态，b)ATP的产生，以及c)线粒体膜电位；d)线粒体网络的体积；e)与细胞器发生相关的基因的表达情况；f)线粒体触发的自噬的活性。3.活体细胞成像和标准分析将测试这些激酶的活性，由此产生的VDAC磷酸化，以及线粒体调控的凋亡死亡的结果。根据mTSPO的分子和药理调控：a)评估PKA和PKCepsilon的转位效率和活性；b)揭示VDAC的磷酸化状态；c)评估线粒体依赖性细胞凋亡的效率。这一目标还将看到来自PKCepsilon KO小鼠的MEF细胞系的利用。4.将采用免疫组织化学、生物化学和酵母为基础的方法来鉴定mTSPO和VDAC之间的分子相互作用：a)监测mTSPO和VDAC亚型之间的细胞共定位；b)通过双杂交策略和免疫共沉淀研究相互作用及其类型；以及c)评估VDAC的同源低聚物。所有这些都将揭示一个重要但迄今被忽视的调控途径的分子和细胞生理学。我们的研究结果将形成未来研究的概念和实验框架，旨在确定mTSPO中一种新的生物标志物和药物干预与新陈代谢和增殖相关的条件的靶点。

In search of the molecules that may set the pace of cell metabolism and define its healthy homeostasis, we are now interested in elucidating the cell biological role of the mTSPO. Although associated with many pathophysiological conditions, its contribution to processes guarding cell integrity such as autophagy and apoptosis is obscure, and its role in mitochondrial performance ill-defined. In order to shed light on these two fundamental processes, it will be instructive to ascertain how mTSPO ratio of expression with that of the VDAC, and its pharmacological modulation, defines the signalling of Ca2+, dynamics of the ROS, ATP rationing and Redox State. The physiology of cellular Ca2+, the ATP balance, the programmed apoptotic cell death as well as the Autophagy all depend on the efficient handling of Ca2+ by the mitochondrion. Despite some minor attempts, however, the contribution of mTSPO to this pathway of signalling has been largely ignored, and we now aim to explore and elucidate it, considering this as the hub in the mTSPO contribution to mitochondrial coupling by which ATP synthesis, ROS generation and network remodelling, via targeted autophagy, originate. However, modifications in ROS signalling would impinge also on downstream effectors: the kinases which promote the unphysiological phosphorylation of VDAC. This indirect pathway, as well as the direct physical interaction between mTSPO and VDAC, will therefore be addressed as potential underlying mechanisms. The following experiments will clarify the above points using Mouse Embryonic Fibroblasts (MEF) cell lines: 1. Live imaging, luminescent and fluorescence based methods will be used to assess mTSPO role in cell autopagy and signalling. a) The mTSPO protein will be transiently manipulated to increase (+) or decrease (-) its expression via conventional techniques of molecular biology; the outcome of this manipulation will then be ascertained on b) Autophagy, c) Ca2+ signalling d) ROS generation and e) Autophagy in conditions of buffered Ca2+ and ROS. 2. Confocal Microscopy, fluorescence and biochemical based methods will be used to determine the role of mTSPO in mitochondrial coupling efficiency and remodelling. After transient modulation of mTSPO or pharmacological binding the following will be addressed: a) The mitochondrial redox state, b) ATP generation, and c) Mitochondrial membrane potential, will be evaluated; d) The volume of the mitochondrial network will be calculated; e) The expression of genes associated with the organelle's genesis will be profiled, and f) The activity of mitochondria-triggered autophagy will be investigated. 3. Live cell imaging and standard assays will test the kinases' activity, VDAC phosphorylation which this generates, and the outcome in mitochondrial regulated apoptotic demise. Following molecular and pharmacological modulation of mTSPO: a) The efficiency of translocation and activity of PKA and PKCepsilon will be evaluated; b) The phosphorylation state of VDAC revealed; and c) The efficiency of mitochondrial-dependent apoptosis assessed. This object will also see the utilization of MEF cell lines from PKCepsilon KO mice. 4. Immunohistochemical, biochemical and yeast based methods will be employed to identify the molecular interplay between mTSPO and VDAC: a) Cellular co-localization between mTSPO and VDAC isoforms will be monitored; b) The interaction and its type studied via two-hybrid strategy and co-immunoprecipitation; and c) The homo-oligomers of VDAC evaluated. All this will reveal the molecular and cellular physiology of an important but so far neglected regulatory pathway. The results of our studies will form a conceptual and experimental framework for future investigations aiming to identify in mTSPO a novel bio-marker and target for pharmacological intervention of conditions associated with remodelling of metabolism and proliferation.