A novel regulator of human apoptosis

细胞凋亡（程序性细胞死亡）是细胞死亡的一个关键过程。细胞凋亡对于多细胞真核生物（如人类和其他哺乳动物）的正常生长、分化和发育至关重要。控制细胞凋亡通路的缺陷具有毁灭性的后果。细胞凋亡是在20世纪70年代被发现的，国际上正在努力了解其机制。与所有重要的生物现象一样，存在着复杂的调节机制，以促进对生物体发育的精细控制。几种蛋白质参与各种细胞凋亡过程，这些过程的控制和执行机制是复杂的，目前仅部分了解。重要的是，生物学研究的重点是对细胞凋亡的理解，因为显然有机会治疗源于细胞凋亡途径缺陷的疾病和发育障碍。这个项目的重点是了解一种新的人类凋亡诱导蛋白AIFM2（或凋亡诱导因子-线粒体2）的结构、机制和细胞特性，最近被描述为一种含有黄素辅助因子（FAD，或黄素腺嘌呤二核苷酸）的蛋白质，它是一种有效的细胞死亡诱导剂。事实上，它的效力优于原始的AIF蛋白（也含有FAD）。AIF位于被称为线粒体的细胞器中，线粒体是最著名的能量产生场所。然而，在响应指示细胞死亡的信号时，AIF从线粒体中释放出来，易位到细胞核，结合DNA并促进其降解-充当细胞死亡的“刽子手”。在应用前的工作中，我们通过与相对AIF的比较，定义了AIFM2的非常不同的性质。AIFM2的FAD辅因子在AIFM2本身催化的过程中通过与氧的反应进行修饰，并依赖于一种称为NADPH的辅酶。这种修改使AIFM2的颜色从黄色变为绿色。AIFM2存在于细胞质中（而不是线粒体中），我们已经证明（一旦诱导凋亡）它会转移到细胞核中。我们还证明了AIFM2与DNA结合，并且AIFM2的不同构象状态取决于DNA是否结合。这一初步工作使我们在国际上处于领先地位，我们提出的研究旨在解开AIFM2的生化机制和细胞功能。我们将努力了解其FAD氧化修饰的机制，过程动力学和反应的生化后果。我们将创建原生AIFM2，蛋白质的单个结构域和突变形式，以了解AIFM2不同部分在DNA和辅因子结合中的作用，并获得蛋白质晶体，使我们能够确定其结构。我们将解决AIFM2的细胞位置和驱动其核易位的机制。我们还将研究一个假设，涉及DNA和NADPH与AIFM2的竞争性结合，并关注AIFM2对细胞质中DNA的存在作出反应（例如在病毒感染中）以发出细胞凋亡信号的可能性。我们还将研究AIFM2在DNA结合时发生的不寻常的构象转变，并将其与功能特性联系起来。在进一步的细胞研究中，我们将确定AIFM2的结合伴侣蛋白，以进一步表征其作用模式，并推进我们对复杂的相互作用网络的认识，这些相互作用网络能够对人类细胞凋亡进行精细调节。总的来说，这些数据将为理解一个重要的生物学过程做出重大贡献，并提供一种具有迷人特性的新型人类细胞凋亡诱导蛋白的详细描述。这项工作横跨细胞生物学、生物化学和结构生物学学科，将为我们的细胞凋亡知识数据库做出重要贡献。

Apoptosis (programmed cell death) is a critical process by which cells are killed off. Apoptosis is vital for normal growth, differentiation and development of multicellular eukaryotes (e.g humans and other mammals). Defects in pathways controlling apoptosis have devastating consequences. Apoptosis was discovered in the 1970's, and international efforts are ongoing to understand its mechanisms. As with all important biological phenomena, there is intricate regulation to facilitate fine control over development of the organism. Several proteins are involved in various apoptosis processes, and mechanisms by which these are controlled and executed are complex and remain only partially understood. It is important that biological research is focused on understanding of apoptosis, since there are clearly opportunities for treatment of diseases and developmental disorders that originate from defects in apoptotic pathways. This project focuses on understanding structure, mechanism and cellular properties of a novel human apoptosis inducing protein known as AIFM2 (or apoptosis inducing factor - mitochondrion 2), recently described as a protein that contains a flavin cofactor (FAD, or flavin adenine dinucleotide) and which is a potent inducer of cell death. Indeed, its potency is superior to that of the original AIF protein (which also contains FAD). AIF is located in a cellular organelle known as the mitochondrion, which is most famous as a site of energy generation. However, in response to signals indicating cell death, AIF is released from the mitochondrion, translocates to the nucleus, binds DNA and facilitates its degradation - acting as an 'executioner' of cell death. In work leading up the application, we have defined very different properties of AIFM2 by comparison with its relative AIF. AIFM2's FAD cofactor is modified by reaction with oxygen in a process catalysed by AIFM2 itself and dependent on a coenzyme called NADPH. This modification changes AIFM2's colour from yellow to green. AIFM2 resides in the cell cytoplasm (not the mitochondrion), and we have shown that (once apoptosis is induced) it translocates to the cell nucleus. We have also shown that it binds DNA, and that there are different conformational states of AIFM2 dependent on whether DNA is bound or not. This preliminary work has given us an international lead, and the proposed study is aimed at deconvoluting the biochemical mechanism and cellular functions of AIFM2. We will do work to understand the mechanism underlying the oxidative modification of its FAD, kinetics of the process and the biochemical consequences of the reaction. We will create native AIFM2, individual domains of the protein and mutant forms to understand roles of different parts of AIFM2 in DNA and cofactor binding, and to obtain protein crystals to enable us to determine its structure. We will resolve the cellular location of AIFM2 and mechanisms that drive its nuclear translocation. We will also investigate a hypothesis that involves the competitive binding of DNA and NADPH to AIFM2, and concerns the likelihood that AIFM2 responds to presence of DNA in the cytoplasm (as e.g. in viral infections) to signal cell apoptosis. We will also investigate the unusual conformational transitions of AIFM2 that occur on DNA binding, and relate these to functional properties. In further cellular studies we will identify binding partner proteins for AIFM2 to further characterize its mode of action and to advance our knowledge of the complex web of interactions that enable fine regulation over apoptosis in human cells. Collectively, these data will make major contributions to understanding of an important biological process, and provide a detailed account of a novel human apoptosis inducing protein with fascinating properties. The work straddles cell biology, biochemistry and structural biology disciplines and will make critical contributions to our database of knowledge on apoptosis.