Intracellular distribution of Cu(I): De-regulation & exploitation in pathogen-control.
Lead Research Organisation:
Durham University
Department Name: Biological and Biomedical Sciences
Abstract
Copper is widely used in the agrochemical industry as a fungicide and Syngenta are investigating new copper based formulations with greater efficacy and/or requiring lower environmental copper input. Fortuitously, one of the Cu(I)-chelator compounds produced by synthetic chemists at Syngenta appears to de-regulate copper homeostasis in S. cereviasiae, as well as inhibiting growth of several pathogenic fungi. Via such de-regulation, this Cu(I)-chelator has the potential to provide insight into the pathways that deliver this metal to its destinations inside living cells. Specifically, preliminary data suggest that the sources of copper for each copper delivery pathway are not identical. This is a rare opportunity to explore fundamental questions at the heart of the cell biology of metals, while simultaneously tackling issues of direct relevance to an agrochemical company. Syngenta would like to understand the biochemical basis via which the Cu(I)-chelator acts if it is to be pursued commercially, and the 'Metals in Cells Group' at Newcastle University are eager to use the Cu(I)-chelator to explore how copper is correctly targeted inside cells. Copper is essential for enzymes such as cytochrome oxidase, superoxide dismutase 1, and (in plants) plastocyanin. Some metals, especially copper, have a tendency to form much tighter complexes with proteins than do others. Cells must maintain exceptionally low buffered cytosolic concentrations of copper in order to minimise the mis-population of proteins that require the less competitive metals. Copper must also be tightly controlled due to its propensity to engage in redox chemistry such as the Fenton reaction which generates deadly hydroxyl radicals. To avoid copper-release in the cytosol it is supplied to copper requiring proteins under kinetic control, meaning that copper is delivered to its correct destinations by specific 'copper metallochaperones'. The metal is passed from the copper metallochaperones to their partners by sequences of ligand-exchange reactions. In most eukaryotic cells, including fungi, these include copper metallochaperones for cytochrome oxidase in mitochondria, one for superoxide dismutase 1 in the cytosol and finally one for the trans-Golgi network. However, it is unclear where the copper metallochaperones themselves obtain copper and it is also unclear how the routing of copper to these different cellular destinations is prioritised, especially when copper is in short supply. These are fundamental unknowns in regard to copper homeostasis in all organisms; plants, fungi, bacteria and animals including humans. An intriguing hypothesis is that the copper chaperones for cytochrome oxidase have access to copper released at cuproprotein turnover, while those for SOD1 and for the trans-Golgi network predominantly have access to newly imported copper. This would ensure that as copper levels decline the metal ions become predominantly routed to a most vital intracellular destination, namely cytochrome oxidase. Fungal cells treated with the Cu(I)-chelator generated by Syngenta chemists appear to detect high intracellular copper concentrations by switching on expression of (metallothionein Cup1-1 and Cup1-2) genes whose products mop up surplus copper. However, the treated cells concurrently exhibit phenotypes consistent with insufficient copper reaching cytochrome oxidase. A goal of this programme is to measure the respective cupro-enzyme activities and quantify the amounts of copper reaching the different destinations. This will establish if there are distinct sources of copper for the different copper delivery pathways.
Technical Summary
Firstly, reports in the literature suggest that plasma membrane copper importers Ctr1, Ctr3 supply copper to the copper metallochaperones CCS and Atx1 while vacuole export via Ctr2 contributes only modestly. These published data imply that the pool of copper for SOD1 and the trans-Golgi network mostly originates from cell surface importers rather than from exporters of vacuole copper. Secondly, the immediate source of copper for Cox17, hence Sco1 and Sco2, and hence the copper A and B sites of cytochrome oxidase, appears to be a low molecular weight copper complex which is present in the mitochondrial matrix, from yeast to humans. The source of copper for the low molecular weight mitochondrial copper complex remains to be defined. Finally, the sources of copper for the two nuclear copper detectors, Ace1 and Mac1, are unclear although the detection of copper by Mac1 is now known to depend upon the catalytic activity of a nuclear pool of SOD1. Treatment of S. cerevisiae with a copper-chelating fungicide generated by chemists at Syngenta prevents growth on a non-fermentable carbon source, lactate, but growth is restored by the addition of 2% glucose, indicative of a loss of activity of cytochrome oxidase. This is suggestive of a loss of copper supply via the Cox17 route. In seeming conflict with these data, beta-galactosidase activity driven by a cup1 promoter-lacZ fusion was concurrently greatly enhanced and S1 nuclease protection assays confirm greatly enhanced accumulation of cup1 transcripts. This is suggestive of enhanced copper supply to Ace1. The effects of the chelator on the intracellular distribution of copper will be determined. This is an opportunity to understand how copper is targeted to its different intracellular destinations.
Planned Impact
This programme is an IPA partly funded by Syngenta. Thus, Syngenta are the most immediate industrial beneficiaries (other forms of impact are described in the full impact plan). Syngenta have crop treatments on the market which use copper, or metal-chelators, as the active ingredients. Following an initial approach from Syngenta in late 2008, a joint consultancy agreement was negotiated which has already generated some income supporting research in the Robinson lab. The consultancy agreement is ongoing and will be extended coincident with the award of this grant. A pre-proposal version of this application was approved by an internal board within Syngenta early in 2009, giving authorisation for this IPA application to BBSRC. Syngenta will crucially provide the copper-chelator compounds required for this work, under a strict collaborative agreement. Throughout the grant there will be regular meetings either at Syngenta or in Newcastle (in preparation for this application Andy Corran has flown to Newcastle twice already in 2009 and Robinson has travelled to Jealotts Hill). Such regular meetings will ensure that discoveries with industrial impact are exploited in the swiftest possible manner.
Publications
Robinson NJ
(2011)
Structural biology: a platform for copper pumps.
in Nature
Foster AW
(2011)
Promiscuity and preferences of metallothioneins: the cell rules.
in BMC biology
Foster AW
(2014)
Metal specificity of cyanobacterial nickel-responsive repressor InrS: cells maintain zinc and copper below the detection threshold for InrS.
in Molecular microbiology
Foster AW
(2014)
A chemical potentiator of copper-accumulation used to investigate the iron-regulons of Saccharomyces cerevisiae.
in Molecular microbiology
Description | The mode of action of a candidate agrochemical was discovered informing decisions about it future development/exploitation. These studies have established that 2-(6-benzyl-2-pyridyl)quinazoline (BPQ) can be used as chemical biology reagent to explore the basis of copper toxicity in yeast and to tease out components of gene expression in response to functional iron deficiency (refer to Foster et al 2014 Molecular Microbiology, 93 317-330). Copper toxicity in yeast was shown to be mediated by damage to iron sulfur clusters. A new candidate low-iron regulated Aft1-target gene was discovered (and named Lso1) along with a paralogous non-target Lso2. The switch between these two genes is attributed to iron sparing. |
Exploitation Route | Refer to narrative impact for relevance to the industrial partner. |
Sectors | Agriculture Food and Drink Chemicals |
URL | http://onlinelibrary.wiley.com/doi/10.1111/mmi.12661/abstract;jsessionid=06083213B0B706E01879EAA7C559E5E2.f03t02 |
Description | This program involved collaboration with Syngenta. The findings informed company choices relating to the development (or otherwise) of candidate anti fungal agrochemicals. This programme also underpinned the BBSRC Metals in Biology NIBB: An employee of Syngenta served on the BBSRC NIBB management board. |
First Year Of Impact | 2015 |
Sector | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology |
Impact Types | Economic |
Title | 18 RNAseq transcriptome experiments |
Description | 18 RNAseq transcriptome experiments deposited with the NCBI Gene Expression Omnibus (Accession: GSE54045). |
Type Of Material | Database/Collection of data |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | Transcriptome data informed on the mode of action of BPQ which influenced product development by partner agrochemical. |
Title | Coordinates for the crystal structure of Cu(I)-BPQ. |
Description | Coordinates for the crystal structure of Cu(I)-BPQ deposited with the Cambridge Crystallographic Data Centre (Accession: CCDC 982656) |
Type Of Material | Database/Collection of data |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | Cu(I)-BPQ mode of action partly informed by the structure and provided to a partner agrochemical company. |
Description | Interaction with industrial sponsor |
Organisation | Procter & Gamble |
Country | United States |
Sector | Private |
PI Contribution | Regular teleconference meetings (in excess of 50 over 24 months including all forms of interaction) with industrial collaborator Reciprocal exchange of materials and biologics with industrial collaborator Reciprocal visits with industrial collaborator (associated PhD students and academic staff etc) Analytical services provided for industrial partner and others, and vice versa |
Collaborator Contribution | See above |
Impact | Ongoing and confidential |
Start Year | 2012 |
Description | Interview on Merging metals into proteomics for C&EN |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Interview for chemical and engineering news Interview about metalloproteomics methods DOI 10.1021 Greater awareness of the significance of this research. |
Year(s) Of Engagement Activity | 2011 |
Description | Invited lecture, Copper in Biology, Naples |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | ca 100 individuals attended the lecture with discussion afterwards An invitation to talk in Umea, Sweden |
Year(s) Of Engagement Activity | 2014 |
Description | Metal-Related Antimicrobials Workshop |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | We organised a workshop to bring together academics and industry to discuss opportunities in collaborative research Led to new collaborations and a volume on "Microbiology of Metals Ions" 2016 volume 70: https://www.elsevier.com/books/microbiology-of-metal-ions/author/978-0-12-812386-7 |
Year(s) Of Engagement Activity | 2015 |
URL | https://www.elsevier.com/books/microbiology-of-metal-ions/author/978-0-12-812386-7 |
Description | Metals in Biology Community Event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | We organised a workshop involving academic and industry to show case funded projects as exemplars to instigate future collaborations. |
Year(s) Of Engagement Activity | 2016 |
Description | Syngenta project review event |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Diseminated knowledge to an agrochemical company and vice versa. Joint publication with Syngenta |
Year(s) Of Engagement Activity | 2013 |