A novel regulator of human apoptosis

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.

Apoptosis is a key process in eukaryote development. A plethora of proteins are involved in induction and execution of apoptosis, with several cell death effectors contained in the mitochondrion. Cell death signalling leads to mitochondrial disruption and release of effector molecules (e.g. cytochrome c and the FAD-containing apoptosis inducing factor (AIF)). AIF translocates to the nucleus and binds DNA, leading to its destruction and to progression of apoptosis. We have characterized a novel human AIF-like protein (AIFM2), which is a more potent apoptosis inducing protein than AIF. We have expressed/purified AIFM2 and shown it binds a modified flavin (6-hydroxy FAD), which it auto-hydroxylates in a NADPH-dependent manner. Among other intriguing findings, we have shown AIFM2 binds DNA and undergoes conformational changes. In this study we will resolve several key features of AIFM2 structure, mechanism and cell biology. Priorities are (i) establishing AIFM2 expression in human cell lines, resolution of its cellular location and proof that apoptosis induction leads to its nuclear translocation; (ii) in vitro studies of interactions with DNA/coenzyme, to determine Kd values, binding kinetics/thermodynamics and conformational transitions; (iii) analysis of effects of cytoplasmic nucleic acids on AIFM2 behaviour, to probe a model relating antagonistic binding of NAD(P)H/DNA to ROS production and survival signalling; (iv) cellular expression of wild-type, mutant and domain constructs of AIFM2 to explore cellular behaviour, capacity to induce apoptosis and response to oxidative and DNA damage stress; (v) analysis of the FAD auto-oxidation reaction and the source of oxygen required; (vi) structural analysis of AIFM2 and/or domains, and characterization of its DNA binding site by labelling and mass spectrometry. Collectively, data will provide the first integrated account of cellular and biochemical properties of a key human apoptosis inducing protein.