Stress resistance and the essential iron-sulphur cluster protein Rli1

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

'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.

Publications

10 25 50
 
Description Our data have shown that the essential iron-sulphur (Fe-S)protein Rli1 is a key indirect target of reactive oxygen species (ROS). Inhibition of Rli1 function by ROS causes inhibition of yeast growth during oxidative stress. We have identified this as an important mechanism of action for pro-oxidants like copper and the antimalarial primaquine.

Our data also suggested that there was an upstream FeS-protein targeted directly by ROS, which led to the downstream defect in Rli1. We have now identified this direct target of ROS action as the mitochondrial ferredoxin Yah1. It appears that this protein's susceptibility to ROS attack is the primary cause of downstream inhibitory effects.

During the above work, we identified a novel synergistic interactions between two inhibitors of yeast growth. The combined effect was much greater than either alone, suggesting potential application as a new antifungal or fungicide. We are currently pursuing that potential with BBSRC follow-on funding.
Exploitation Route Yeasts used in in biotechnology, for brewing, baking, and as protein factories, are commonly exposed to stresses during processing or storage between runs, e.g., freeze-thaw, oxidative and osmotic stresses. This can have a serious economic impact on operational efficiency. The new insight here could be exploited to improve the resilience of these strains, for example by overexpressing the RLI1 or YAH1 genes. If successful, such manipulations could improve the efficiency of operation and so help to promote wealth generation.

The finding could also 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, this new mechanistic understanding could ultimately improve the 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.

The findings increase our knowledge of how organisms cope with stress. The knowledge should help others understand important processes such as stress resistance, cellular redox regulation, toxicology, FeS protein chemistry, and protein synthesis, as well as broader subjects like gene expression and function, protein activity, and cell health.

It is also of considerable interest to the yeast research community, especially as it complements and builds on research by a large number of labs on yeast stress responses. As these responses are well conserved - and this should be particularly true for the Rli1 and Yah1 proteins - the results will also impact communities interested in how organisms other than yeast deal with stress. This is poignant in the case of ROS stress and loss of FeS-cluster function, as both are linked to serious degenerative conditions in humans. Therefore, the finding should contribute to understanding those disease processes and potentially the development of new therapies.

Finally, the novel synergistic antifungal combination we have discovered could be exploited for antifungal or fungicidal applications. The latter are particularly important for protection of food crops and global food security, a priority of the BBSRC.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

URL http://www.molbiolcell.org/content/23/18/3582.full
 
Description Our finding of a new synergistic antifungal combination has attracted strong interest from the agrichemical industry, supported by a BBSRC follow-on pathfinder award. Based on the strength of that interest, I secured more substantial follow-on funding and have secured patent protection, followed by a CASE studentship supported by a major agrichemical company. A further follow-on proposal was recently awarded and is currently active, focused on protection of commercial materials from fungal deterioration. We will be looking to licence out this technology. If successful, the fungicide we have discovered could, among other applications, protect food crops from fungal disease in the future, so helping to preserve global food security.
Sector Agriculture, Food and Drink,Chemicals
Impact Types Societal

 
Description Pathfinder
Amount £7,658 (GBP)
Funding ID BB/M011852/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2014 
End 10/2014
 
Description Syngenta industrial partnership (CASE) studentship
Amount £30,000 (GBP)
Funding ID RIS121908 
Organisation Syngenta International AG 
Sector Public
Country Global
Start 10/2016 
End 06/2022
 
Title Novel FeS protein constructs 
Description New genetic constructs that help to investigate the impact of oxidative stress on cellular FeS proteins 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Have been used to help identify the primary protein target of oxidative stress in cells 
 
Description Individuality of yeast cells: stress resistances and virulence 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Research seminar at the University of Alicante, Department of Applied Biological Sciences

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Why yeast is good 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2012