A novel regulator of human apoptosis

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Publications

10 25 50
publication icon
Girvan HM (2013) Heme sensor proteins. in The Journal of biological chemistry

 
Description The purpose of this study was to characterize an unusual human flavin cofactor-binding enzyme named AIFM2 (apoptosis inducing factor mitochondrion-associated 2). AIFM2 shows some structural and sequence relationships to AIF (apoptosis inducing factor), a protein that plays a key role in the progression of the process of apoptosis (or programmed cell death) in higher organisms. AIF moves from the mitochondrion (the energy generating organelle in cells) to the nucleus as apoptosis begins, and participates in the process of DNA degradation that leads to the death of the cell. Apoptosis is an essential process in development, but the role of the AIFM2 protein is uncertain, despite its similarity to AIF. We expressed and purified the AIFM2 protein and showed key differences between this protein and AIF. The AIF protein binds the flavin cofactor FAD, and interacts with the electron donating NADH cofactor, possibly to enable a role in respiration that is distinct from its role in apoptosis. AIF in its resting (oxidized) form has a yellow colour derived from the FAD cofactor. However, unlike AIF, the AIFM2 protein interacts more effectively with the phosphorylated cofactor NADPH, and in its oxidized form is green in colour, which we showed to originate from hydroxylation of its FAD cofactor to a 6-hydroxy-FAD derivative. Work in this project then moved on to analysis of the cellular localization of AIFM2. By expressing the AIFM2 protein in a number of human cell lines, we were able to probe its cellular localization and examine whether this localization was changed when the cells were exposed to stresses, including the induction of apoptosis. Through a series of studies we were able to conclude that the major location of AIFM2 is in the Golgi apparatus - a cellular organelle involved in protein processing and secretion. A model developed was that AIFM2 may be an apoptosis sensing protein specific to the Golgi. In other studies AIFM2 was shown to bind to nucleic acids, and the process of 6-hydroxylation of the FAD in AIFM2 was shown to be autocatalytic and to occur through redox cycling (i.e. reduction of the AIFM2 FAD cofactor by NADPH and its non-productive reoxidation through subsequent enzymatic reduction of oxygen). The auto-hydroxylation process is not very efficient, but occurs sufficiently quickly that AIFM2 protein isolated from bacterial cells expressing the protein is completely in the 6-hydroxy FAD (green) form. The catalytic properties of AIFM2 were investigated in detail, and the enzyme was shown to exist in a mixture of monomeric and dimeric states that can be affected by binding to NADPH or DNA. Several novel insights into the biochemical properties of AIFM2 were made in this project, and while its ability to induce apoptosis appears unlikely (unless it is expressed to very high levels within cells), its primary cellular localization and biochemical properties are now much more clearly understood.
Exploitation Route The work done defined more clearly aspects of the biochemical and catalytic properties of the human AIFM2 protein. Further work should now be done to establish firmly the major cellular functions of this enzyme, including how its positioning in the Golgi may relate to its role in human biochemistry. Recent studies suggest putative roles for AIFM2 in areas such as cellular respiration and gene regulation, and interest continues to accumulate in this important protein.
Sectors Energy,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://gtr.rcuk.ac.uk/projects?ref=BB%2FG008558%2F1
 
Description The findings made in this project have led to further studies to define biochemical and structural features of this enzyme, including the mechanism of the auto-hydroxylation process and its influence on the properties of the AIFM2, and the apparent localization of AIFM2 in the Golgi. We have demonstrated that AIFM2 undergoes a conversion from a typical yellow flavin state to form a green-coloured enzyme as a consequence of auto-hydroxylation of the flavin during turnover driven by NADPH. AIFM2 was also demonstrated to be a DNA-binding protein. Our studies have helped stimulate other work in the area, and increasing numbers of publications are now appearing that report novel roles on AIFM2 in e.g. gene regulation and cellular respiration, likely linked to a primary role in apoptosis.
First Year Of Impact 2005
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Manchester Institute of Biotechnology Open Day - annual event from 2012 onwards 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Scientific demonstrations to senior secondary school students to enthuse them about a scientific career and to provide advice on career development and the courses on offer at the University of Manchester.

Annual event - such that lessons are learned from one year's activity and are carried forward to the following year's presentations.
Year(s) Of Engagement Activity 2012,2013,2014,2015
 
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011