Stress resistance and the essential iron-sulphur cluster protein Rli1

An inherent feature of life is that organisms must cope with various forms of environmental stress. For aerobic organisms which live in an oxygenated environment, e.g. humans, oxygen itself imposes a stress as it is the source of reactive oxygen species (ROS). ROS are highly damaging to the major components of cells. Among the most ROS-sensitive molecules of cells are components of certain proteins, termed iron-sulphur (FeS) clusters. Because of their oxygen sensitivity, these FeS clusters are considered a major limitation for successful aerobic existence. Nevertheless FeS clusters are essential, and that essentiality appears to rest on the requirement organisms have for the FeS protein Rli1p. Rli1p is essential to organisms because it is required for the process of protein synthesis. It may be no coincidence that, through evolution, Rli1p has been one of the most highly conserved proteins in biology. This conservation is helpful because it means that Rli1p function can be explored in relatively simple model organisms like yeast, which is very easy to grow in the lab and to manipulate for experiments. Conveniently, yeast has also proven an ideal model for studies of stress responses in cells, having yielded a range of key insights over recent years relevant to disease processes in humans and to the biotechnological applications of microorganisms. With the yeast model, we recently discovered a novel cellular function that helps to preserve the integrity of FeS clusters during stress. That work gave new insight to how organisms cope with an aerobic lifestyle. Moreover, in related experiments, we obtained the first experimental evidence that Rli1p is a key factor determining the stress resistance of cells. This research proposal is centered on that exciting discovery, supported by our preliminary data. Specifically, we will test the hypothesis that its FeS clusters make Rli1p a target of a number of major stressors, and that the essentiality of Rli1p means that such targeting is a cause of cell death during stress. The work programme will involve both screening approaches and specific manipulations to test the hypothesis. We will examine Rli1p function over a wide range of stress conditions. We will also exploit the power of yeast genetics to identify cellular factors that impact Rli1p function during stress. In addition, we will elucidate the mechanism(s) of Rli1p targeting. These studies are important as Rli1p has not been examined previously in this context, yet the evidence we discuss indicate that Rli1p is likely to be a major player in determining the abilities of organisms to cope with certain stresses. Elucidation of that role is vital if this research may be exploited for improving biotechnological processes or health in the future.

Our preliminary data have uncovered a new role for the essential protein Rli1p, as a determinant of cellular stress resistance. This finding is particularly exciting because Rli1 is a conserved FeS cluster protein which, alone, is thought to account for the essentiality of FeS cluster biosynthesis in organisms. The function of FeS clusters is exquisitely sensitive to oxidation. The hypothesis is that the FeS clusters of Rli1p can become disabled during stress, leading to loss of protein function, and so loss of viability. By testing that hypothesis, this proposal should give a major new insight to how certain stressors work, which should have broad ramifications across the very wide range of organisms that express Rli1p or a homologue. The work will focus on the yeast Saccharomyces cerevisiae, an ideal model for the planned studies. Our preliminary data indicate that Rli1p action in stress resistance is strongly dependent on the nature of the stressor and on yeast genetic background. We will capitalise on that here to gain insight to the mechanisms involved, by determining Rli1p function under a diverse range of stress conditions, and by carrying out novel genome-wide screens to identify gene functions that modulate the role of Rli1p in stress resistance. In conjunction with tests of FeS cluster integrity in Rli1p, those screening data will set the stage for an elucidation of the mechanism of Rli1p action. To that end, the major hypotheses to be tested are that stressors either indirectly (via ROS generation) or directly (by binding) interfere with FeS cluster integrity in Rli1p, so causing loss of this essential protein's function.

'Living with environmental change' is a strategic priority of the BBSRC, and this research falls firmly within that priority. New insight into how organisms manage and respond to environmental stress can have wide-ranging impacts. For example, results from research such as this have the potential ultimately to influence environmental policy, e.g., by provoking revisions to recommended safe limits for specific environmental pollutants. The new determinant of stress resistance on which this research focuses (Rli1p) is also an essential protein. Therefore, the results are relevant to cell health on more than one front. Findings with yeast (as used here) are also commonly extrapolated to humans, and this is a major reason why yeast is such a popular model organism. This means that the findings could impact our understanding of disease processes linked to reactive oxygen species, FeS cluster deficiency and environmental stress, including amyotrophic lateral sclerosis, Alzheimer's disease, Friedreich's ataxia and cancer. Any new insight into these diseases has the potential to impact the way in which they are managed. Therefore, the proposed research is important as the mechanistic understanding that it will provide could ultimately improve our capacity to combat these disease states in people. The work will also further the case for yeast as an alternative experimental system to animal-based research, and will help to raise awareness of this issue, a policy priority of the BBSRC. Yeasts used in fermentation procedures by the biotechnology industry are commonly exposed to stresses during processing or storage between runs. This can have a serious economic impact on operational efficiency. The insight that this project offers on Rli1p-dependent stress resistance could be exploited to improve the resilience of these strains, for example by overexpressing the RLI1 gene. If successful, such manipulations could improve the efficiency of operation and so help to promote wealth generation. In summary, the professional activities that this research is most likely to impact are scientific research, healthcare, environmental policy, and the biotechnology industry. It follows that the large proportion of the general public who benefit from at least one of these activities also stand to benefit from the proposed research. For exploitation of the research, we will build on our existing industrial links and seek to establish new ones as appropriate, in consultation with colleagues and with the Research and Innovation Services (RIS) unit at the University of Nottingham. The RIS unit has extensive experience in identifying opportunities for exploitation and commercialisation of research, and in negotiating intellectual property rights and patent protection issues. The research will be disseminated principally through publication in scientific journals and presentation at national and international conferences, as well as through external research seminars. We will also actively promote dissemination of our findings to the broader general public. This will be achieved partly by capitalizing on initiatives facilitated through the University of Nottingham. For example the University of Nottingham Media Office is very well supported, and has recently issued press releases on other aspects of our research (1) which were subsequently picked up the popular press, including the New Scientist. The University of Nottingham also has a regular programme of outreach activities to encourage public engagement. We actively publicise our research at University Open Days as well as through visits by members of our School to local schools, and through literature that is distributed to schools across the UK as part of our undergraduate and postgraduate recruitment. In addition, a full time Community Scientist is employed by the School of Biology to help broaden the impact of research by its academic staff.