Investigating the role of cardiolipin metabolism in mitochondrial DNA replication and mitochondrial division

线粒体提供人体细胞中的主要能量来源，并控制许多代谢途径。能量产生机制的13个亚基由线粒体中存在的DNA（线粒体DNA，mtDNA）编码;而绝大多数线粒体蛋白质组（> 1，500种预测蛋白质）由核基因组编码，并从细胞质主动输入到细胞器中。线粒体疾病是由核和mtDNA编码的线粒体基因突变引起的遗传性疾病，这些突变损害了线粒体功能，是最常见的遗传性神经系统疾病之一，每4,300人中就有1人受到影响。它们往往造成严重的疾病，导致儿童和成人严重残疾和寿命缩短。不幸的是，目前还没有有效的治疗方法来阻止或逆转疾病的进展。一种新兴的，但特征不佳的线粒体疾病类别与磷脂代谢受损有关。心磷脂（CL）是一种磷脂（PL），仅存在于线粒体膜中，具有许多重要的线粒体功能。CL生物合成是一个复杂的过程，涉及内质网（ER），细胞质内的膜小管网络，与核膜和线粒体相连。然而，它最终由线粒体内膜（IMM）内的磷脂酸（PA）合成。PA从ER转移到IMM的关键是TRIAP 1-PRELID 1复合物。ER和线粒体之间的内在联系的进一步证据最近出现的证据表明，线粒体DNA复制发生在ER-线粒体接触位点，从而耦合线粒体DNA合成和线粒体分裂。然而，线粒体DNA合成与线粒体分裂之间的联系机制，以及内质网-线粒体接触位点的干扰对线粒体DNA复制的影响，仍然知之甚少。我已经确定了一个病人，在NHS英格兰高度专业化的线粒体疾病服务中，我在国立神经病学和神经外科医院帮助运行，在人TRIAP 1基因中具有第一致病突变（导致蛋白质的移码），其蛋白质产物对CL生物合成至关重要。我已经进行了详细的调查，揭示受损的CL代谢和异常的mtDNA复制在患者来源的成纤维细胞和肌肉组织。这一新发现支持了线粒体DNA维持中磷脂稳态的重要性，并为推进对线粒体生物学基本方面的理解提供了一个重要的机会。我的奖学金的总体研究目标是更深入地了解CL代谢如何影响线粒体DNA合成和线粒体分裂。主要目标是：1）阐明CL代谢受干扰如何影响mtDNA复制和线粒体分裂; 2）定义TRIAP 1相互作用组并鉴定CL生物合成和PL运输所需的新蛋白; 3）研究肌肉特异性CL生物合成和PL运输途径。这些目标将通过研究以下细胞模型中的CL代谢和PL运输途径来实现：突变和敲除（KO）TRIAP 1成纤维细胞;突变TRIAP 1人诱导多能干细胞（hiPSC）衍生的成肌细胞，使用细胞生物学和蛋白质组学方法。这项研究将在世界领先的神经科学中心伦敦大学学院神经病学研究所进行，与剑桥大学MRC线粒体生物学单位合作（Massimo Zeviani教授），一个国际公认的线粒体生物学和医学研究卓越中心;以及科隆大学衰老相关疾病细胞应激反应卓越研究组（托马斯兰格教授），欧洲领先的线粒体生物学和衰老研究机构。

Mitochondria provide the major source of energy in human cells and control numerous metabolic pathways. Thirteen subunits of the energy producing machinery are encoded by DNA present in the mitochondria (mitochondrial DNA, mtDNA); while the vast majority of the mitochondrial proteome (>1,500 predicted proteins) are encoded by the nuclear genome and are actively imported into the organelles from the cytosol. Mitochondrial diseases, inherited conditions caused by mutations in nuclear- and mtDNA-encoded mitochondrial genes which impair mitochondrial function, are among the most common genetic neurological disorders, affecting 1 in 4,300 individuals. They often cause devastating illness associated with severe disability and shortened lifespan in children and adults. Unfortunately, there are currently no effective treatments that halt or reverse progression of the disease.One emerging, but poorly-characterised, category of mitochondrial diseases relates to impaired phospholipid metabolism. Cardiolipin (CL) is a phospholipid (PL) found only in mitochondrial membranes with numerous essential mitochondrial functions. CL biosynthesis is a complex process, involving the endoplasmic reticulum (ER), a network of membranous tubules within the cytoplasm of the cell, continuous with the nuclear membrane, and the mitochondria. However, it is ultimately synthesised from phosphatidic acid (PA) within the inner mitochondrial membrane (IMM). Crucial for the transfer of PA from the ER to the IMM is the TRIAP1-PRELID1 complex. Further evidence for the intrinsic connection between the ER and mitochondria has recently emerged with evidence that mtDNA replication occurs at ER-mitochondria contact sites, thus coupling mtDNA synthesis and mitochondrial division. However, the mechanism that links mtDNA synthesis to mitochondrial division, and the impact of perturbed ER-mitochondria contact sites on mtDNA replication, remains poorly-understood.I have identified a patient, in the NHS England Highly Specialised Services for Mitochondrial Disorders that I help run at the National Hospital for Neurology and Neurosurgery, with the first pathogenic mutations (resulting in frameshift of the protein) in the human TRIAP1 gene, the protein product of which is crucial to CL biosynthesis. I have undertaken detailed investigations that reveal impaired CL metabolism and aberrant mtDNA replication in both patient-derived fibroblasts and muscle tissue. This new discovery supports the importance of phospholipid homeostasis in mtDNA maintenance and presents a significant opportunity to advance understanding of fundamental aspects of mitochondrial biology.The overarching research aim of my fellowship is to gain a deeper understanding of how CL metabolism influences mtDNA synthesis and mitochondrial division. The key objectives are to: 1) characterise how perturbed CL metabolism influences mtDNA replication and mitochondrial division; 2) define the TRIAP1 interactome and identifying novel proteins required for CL biosynthesis and PL trafficking; and 3) investigate muscle-specific CL biosynthesis and PL trafficking pathways. These objectives will be achieved by studying CL metabolism and PL trafficking pathways in the following cell models: mutant and knockout (KO) TRIAP1 fibroblasts; mutant TRIAP1 human induced pluripotent stem cell (hiPSC)-derived myoblasts, using a combined cell biology and proteomics approach. The research will be undertaken at the UCL Institute of Neurology, a world-leading neuroscience centre, in collaboration with: the MRC-Mitochondrial Biology Unit, University of Cambridge (Professor Massimo Zeviani), an internationally-recognised centre of excellence for the study of mitochondrial biology and medicine; and the Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases, University of Cologne (Professor Thomas Langer), a leading European institution for mitochondrial biology and aging.