A novel regulator of human apoptosis

细胞凋亡（程序性细胞死亡）是细胞被杀死的关键过程。细胞凋亡对于多细胞真核生物（例如人类和其他哺乳动物）的正常生长、分化和发育至关重要。控制细胞凋亡的途径中的缺陷具有破坏性后果。细胞凋亡在20世纪70年代被发现，国际上正在努力了解其机制。与所有重要的生物现象一样，有复杂的调节来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，并且控制和执行这些过程的机制是复杂的，并且仅部分了解。重要的是，生物学研究的重点是了解细胞凋亡，因为有明显的机会，用于治疗疾病和发育障碍，起源于细胞凋亡途径的缺陷。该项目的重点是了解一种新的人类凋亡诱导蛋白AIFM 2（或凋亡诱导因子-peptide 2）的结构，机制和细胞特性，最近被描述为含有黄素辅因子（FAD，或黄素腺嘌呤二核苷酸）的蛋白质，是细胞死亡的有效诱导剂。事实上，它的效力上级于原始AIF蛋白（也含有FAD）。AIF位于一个被称为细胞器的细胞器中，这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从细胞内释放，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”。在引导应用程序的工作中，我们通过与其相对AIF的比较，定义了AIFM 2非常不同的属性。AIFM 2的FAD辅因子通过在AIFM 2本身催化的过程中与氧气反应而被修饰，并且依赖于称为NADPH的辅酶。此修改将AIFM 2的颜色从黄色更改为绿色。AIFM 2存在于细胞质中（而不是胞核），我们已经证明（一旦诱导凋亡）它易位到细胞核。我们还表明，它结合DNA，并有不同的构象状态的AIFM 2依赖于DNA是否结合。这项初步工作为我们提供了国际领先地位，拟议的研究旨在解卷积AIFM 2的生化机制和细胞功能。我们将努力了解其FAD的氧化修饰的机制，该过程的动力学和反应的生化后果。我们将创建天然AIFM 2，蛋白质的单个结构域和突变形式，以了解AIFM 2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AIFM 2的细胞定位和驱动其核转位的机制。我们还将研究涉及DNA和NADPH与AIFM 2的竞争性结合的假设，并关注AIFM 2响应细胞质中DNA的存在（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AIFM 2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，我们将鉴定AIFM 2的结合伴侣蛋白，以进一步表征其作用模式，并提高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中的凋亡进行精细调控。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，并提供了一个新的人类凋亡诱导蛋白的详细说明，具有迷人的特性。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.