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 percent 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.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/H011110/1 01/07/2010 31/12/2010 £363,401
BB/H011110/2 Transfer BB/H011110/1 30/06/2011 29/12/2013 £310,806
 
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
URL http://sites.duke.edu/copper2014/
 
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 reseach: http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2015/11/Final-programme.pdf

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 http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2017/01/Metals-in-Biology_Elements-of-the-bioeco...
 
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
URL http://prospect.rsc.org/MiB_NIBB/metals-in-biology-network-community-meeting/
 
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