MRC Transition Support CDA Amandine MARECHAL

为了生存，我们需要永久的能源供应。这是通过一系列反应提供给我们的细胞的，这些反应将我们吃的食物分解成一种通用的燃料：ATP。这个过程主要发生在称为线粒体的细胞器中，称为细胞呼吸。线粒体用来产生ATP的主要机制是呼吸链。它由五个嵌入线粒体内膜的复合物组成，这些复合物共同作用，形成一种称为质子动力的电化学梯度，并驱动ATP的合成。这种梯度大部分是以质子的形式存在的，这些质子被呼吸链复合物泵送穿过线粒体内膜。越来越多的人类疾病与呼吸链组成部分的缺陷有关。在许多情况下，这是因为故障直接影响了它们通过质子梯度形成的能量产生的主要作用，或者因为它导致破坏性自由基的产生增加。细胞色素c氧化酶（CcO）是我们呼吸链的末端酶。它将我们呼吸的氧气转化为水，并极大地促进了质子梯度的产生。其结构的改变（或突变）与多种病理有关，如肌病、治疗抵抗性癫痫、神经系统疾病和前列腺癌。尽管线粒体CcO的整体化学性质已经被很好地理解，但要确定它是如何产生必要的质子梯度的，已经证明要困难得多。基于酶的可用结构，人们提出了各种假设，但在较小的细菌同源物上进行的诱变工作却提出了挑战。目前看来，在理解线粒体CcO机制，特别是人类疾病相关突变的影响方面，主要的缺陷是缺乏一种系统来产生大量含有定义点突变的纯化蛋白。值得注意的是，存在于贝克酵母线粒体中的CcO几乎与人类线粒体中的相同。编码CcO的核和线粒体dna都可以突变，因此可以对CcO结构的任何部分进行改变以研究其功能。因此，我们设计了一个酵母系统，允许大规模生产突变体，并将用它来解决与人类线粒体CcOs相关的基本问题。过去四年，我们取得了重大进展。我们已经确定了质子穿过蛋白质结构的途径，并发现了一个理想的非偶联突变，可以用来研究重要的泵送机制。我们还以最高分辨率确定了酵母CcO的三维结构，证实了它与人类的相似性，并揭示了它与其他呼吸蛋白相互作用的重要细节。后者可能是导致健康和疾病背景下CcO活动变化的原因，我们已经开发了进一步调查这一点的工具。这些成就将指导我们的努力，逐步将人类基因纳入我们的酵母系统，为人类疾病的研究和新疗法的开发和测试创造更好的模型。如果有更多的时间和持续的资金，我们将完成我们的研究计划，并在线粒体和代谢领域做出一些重大贡献。这将使我建立一个坚实的跟踪记录，建立国际声誉，并充分发挥我的潜力，以支持未来申请高级奖学金或项目资助，在竞争激烈的科学环境中发展我的实验室。我已经尽了最大的努力达到这个阶段，但也经历了一些非常困难的时期，遭受了严重的延误，我仍然要充分利用我的CDA。Transition支持将使我能够准确地实现这一点。

To live we need a permanent supply of energy. This is provided to our cells by a cascade of reactions that breaks down the food we eat into a universal fuel: ATP. This process mainly occurs in organelles called mitochondria and is known as cellular respiration. The main machinery that mitochondria use to produce ATP is the respiratory chain. It is composed of five complexes, embedded in the mitochondrial inner membrane, that work together to build up an electrochemical gradient called the proton motive force and which drives ATP synthesis. Most of this gradient is in the form of protons which are pumped across the inner mitochondrial membrane by the respiratory chain complexes.An increasing number of human pathologies are associated with defects in components of the respiratory chain. In many instances, this is because the malfunction has a direct impact on their primary role in energy production via the proton gradient that they form, or because it leads to an increased production of damaging free radicals. Cytochrome c oxidase (CcO) is the terminal enzyme of our respiratory chain. It transforms the oxygen we breathe into water and greatly contributes to the generation of the proton gradient. Alterations (or mutations) in its structure have been linked with diverse pathologies such as myopathy, therapy-resistant epilepsy, neurological diseases and prostate cancer.Although the overall chemistry of mitochondrial CcO is fairly well understood, it has proven much more difficult to determine how this produces the essential proton gradient. Various hypotheses have been formulated based on the available structures of the enzyme but were challenged by mutagenesis work performed on smaller bacterial homologues. Today it appears that the major drawback in understanding the mechanism of mitochondrial CcO, and the effects of human disease-related mutations in particular, is the lack of a system to generate large amounts of purified protein containing defined point mutations.Remarkably, the CcO that is present in Baker's yeast mitochondria is almost identical to that in human mitochondria. The nuclear and mitochondrial DNAs which encode CcO are both amenable to mutagenesis so alterations can be made in any part of the CcO structure to investigate its function. We have thus engineered a yeast system to allow large-scale production of mutants and will use it to address fundamental questions relative to human mitochondrial CcOs.We have made significant progress over the past four years. We have identified the route taken by the protons to cross the protein structure and have found an ideally uncoupled mutation with which to study the important pumping mechanism. We have also determined the 3D structure of the yeast CcO at the highest resolution which has confirmed its similarity to human and revealed important details of its interaction with other respiratory proteins. The latter might be responsible for changes in CcO activity in the context of health and disease and we have developed the tools to investigate this further. These achievements will guide our efforts to progressively incorporate the human genes in our yeast system to create an even better model for the study of human diseases and the development and testing of new therapies.Given more time and funding continuity, we will complete our research programme and make several significant contributions to the field of mitochondria and metabolism. This will allow me to build a solid track record, establish an international reputation and reach my full potential in support of future applications for a senior fellowship or programme grant to grow my laboratory within the competitive scientific environment. I have given my best to reach this stage but have also suffered significant delays while going through some very difficult times and I still have to make the most of my CDA. The Transition support would enable me to achieve exactly this.