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，这些反应将我们所吃的食物分解成一种通用的燃料：三磷酸腺苷。这一过程主要发生在称为线粒体的细胞器中，被称为细胞呼吸。线粒体用来产生三磷酸腺苷的主要机械是呼吸链。它由嵌入线粒体内膜的五个复合体组成，它们共同作用建立一个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体泵过线粒体内膜。越来越多的人类疾病与呼吸链成分的缺陷有关。在许多情况下，这是因为故障通过它们形成的质子梯度直接影响它们在能量生产中的主要作用，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、难治性癫痫、神经系统疾病和前列腺癌。尽管线粒体CcO的整体化学已被很好地了解，但事实证明，要确定这是如何产生必要的质子梯度要困难得多。基于酶的现有结构，已经提出了各种假说，但在较小的细菌同源物上进行的诱变工作挑战了这些假说。今天，在理解线粒体CcO的机制，特别是人类疾病相关突变的影响方面，主要的缺陷似乎是缺乏一个系统来产生大量含有明确点突变的纯化蛋白。值得注意的是，存在于面包师酵母线粒体中的CcO与人类线粒体中的CcO几乎完全相同。编码CcO的核和线粒体DNA都容易发生突变，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，并将使用它来解决与人类线粒体CCOS相关的基本问题。在过去的四年里，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路线，并发现了一个理想的非偶联突变，用来研究重要的泵浦机制。我们还在最高分辨率下确定了酵母CcO的3D结构，这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研究这一点的工具。这些成就将指导我们努力逐步将人类基因整合到我们的酵母系统中，为人类疾病的研究以及新疗法的开发和测试创造一个更好的模型。如果有更多的时间和资金连续性，我们将完成我们的研究计划，并在线粒体和新陈代谢领域做出几项重大贡献。这将使我能够建立一个坚实的记录，建立国际声誉，并充分发挥我的潜力，以支持未来申请高级研究员或计划补助金，以在竞争激烈的科学环境中发展我的实验室。我已经尽了最大努力来达到这个阶段，但在经历了一些非常困难的时期也遭遇了重大延误，我仍然必须充分利用我的CDA。过渡支持将使我能够实现这一点。

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.