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

线粒体转位蛋白（mTSPO）在细胞稳态中的作用：信号传导和自我保护机制中的分子途径

• 批准号: BB/I013695/1
• 负责人: Michelangelo Campanella
• 金额: $ 52.8万
• 依托单位: Royal Veterinary College
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI013695%2F1
• 关键词: Role; mitochondrial; Translocator; Protein; mTSPO

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.

为了寻找可能设定细胞代谢速度并定义其健康稳态的分子，我们现在对阐明mTSPO的细胞生物学作用感兴趣。尽管与许多病理生理条件相关，但其在保护细胞完整性（如自噬和凋亡）过程中的作用尚不清楚，其在线粒体性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学调节如何定义Ca2+信号，ROS动力学，ATP定量和氧化还原状态将具有指导意义。细胞Ca2+的生理、ATP平衡、程序性凋亡细胞死亡以及自噬都依赖于线粒体对Ca2+的有效处理。然而，尽管有一些小的尝试，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它，认为这是mTSPO对线粒体偶联贡献的枢纽，ATP合成、ROS生成和网络重构通过靶向自噬产生。然而，ROS信号的修饰也会影响下游效应物：促进VDAC非生理性磷酸化的激酶。因此，这种间接途径以及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：基于实时成像、发光和荧光的方法将用于评估mTSPO在细胞自噬和信号传导中的作用。a)通过传统的分子生物学技术，对mTSPO蛋白进行短暂的操作，以增加（+）或减少（-）其表达；这种操作的结果将确定b)自噬，c) Ca2+信号传导，d) ROS生成和e)缓冲Ca2+和ROS条件下的自噬。2. 将使用共聚焦显微镜、荧光和基于生化的方法来确定mTSPO在线粒体偶联效率和重塑中的作用。在mTSPO的瞬时调节或药物结合后，将解决以下问题：a)线粒体氧化还原状态，b) ATP生成，c)线粒体膜电位将被评估；d)计算线粒体网络的体积；e)与细胞器发生相关的基因表达将被分析，f)线粒体触发的自噬活性将被研究。3. 活细胞成像和标准分析将测试激酶的活性，由此产生的VDAC磷酸化，以及线粒体调节的凋亡死亡的结果。mTSPO的分子和药理调节：a)将评估PKA和PKCepsilon的易位效率和活性；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。该对象还将看到PKCepsilon KO小鼠MEF细胞系的利用。4. 将采用免疫组织化学、生化和基于酵母的方法来鉴定mTSPO和VDAC之间的分子相互作用：a)将监测mTSPO和VDAC亚型之间的细胞共定位；b)通过双杂交策略和共免疫沉淀研究相互作用及其类型；c)评价了VDAC的同聚物。所有这些都将揭示一个重要但迄今为止被忽视的调节途径的分子和细胞生理学。我们的研究结果将为未来的研究提供一个概念和实验框架，旨在确定mTSPO中一种新的生物标志物和靶点，用于与代谢和增殖重塑相关的疾病的药物干预。

项目成果

Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index.

• DOI: 10.1371/journal.pone.0169454
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Beffagna G;Sammarco A;Bedin C;Romualdi C;Mainenti M;Mollo A;Cavicchioli L;Ferro S;Trez D;De Maria R;Nitti D;Saccani A;Campanella M;Agostini M;Zappulli V
• 通讯作者: Zappulli V

Peptide targeting of mitochondria elicits testosterone formation.

线粒体肽靶向引发睾酮形成。

• DOI: 10.1038/mt.2014.171
• 发表时间: 2014
• 期刊: the journal of the American Society of Gene Therapy
• 作者: Campanella M

Mitochondrial pharmacology: A need in modern biomedicine.

线粒体药理学：现代生物医学的需要。

• DOI: 10.1016/j.phrs.2015.10.011
• 发表时间: 2016
• 期刊: Pharmacological research
• 影响因子: 9.3
• 作者: Campanella M

The shrimp mitochondrial FoF1-ATPase inhibitory factor 1 (IF1).

虾线粒体 FoF1-ATP 酶抑制因子 1 (IF1)。

• DOI: 10.1007/s10863-015-9621-0
• 发表时间: 2015
• 期刊: Journal of bioenergetics and biomembranes
• 影响因子: 3
• 作者: Chimeo C

TSPO the unrested: challenged opinions of a resourceful mitochondrial protein

不安分的 TSPO：对足智多谋的线粒体蛋白的观点提出质疑

• DOI: 10.1016/j.tem.2015.05.003
• 发表时间: 2015
• 期刊: Trends in Endocrinology & Metabolism
• 影响因子: 10.9
• 作者: Campanella M