Metallothionein mediated metal trafficking in the model invertebrate C. elegans: dissection of structural and functional differentiation
Lead Research Organisation:
University of Warwick
Department Name: Chemistry
Abstract
The free-living roundworm (or nematode) C.elegans is extensively used as a model organism to study processes that are pertinent to life. Its genome has been fully sequenced, and basic biological mechanisms operating in C.elegans frequently parallel those in man, and thus results obtained from C.elegans have been instrumental in providing a detailed insight into human biology and physiology. As any other living organism, C.elegans obtains essential nutrients from the environment whilst avoiding being poisoned by noxious compounds. However, some elements are essential at low concentrations, but toxic at elevated levels. The heavy metal zinc, for example, is indispensable to virtually all biological processes, and zinc deficiency has been documented to have deleterious effects on reproduction, growth, and development. Similarly, copper, although usually present in biological systems at lower amounts than zinc, is equally essential to many biological processes, such as energy production and the formation of cartilage and connective tissue. Both heavy metals underlie a stringent control as excess amounts are highly toxic. A promising player in the control of available levels of essential metals is the family of Metallothioneins (MTs), small metal-binding proteins that are present in all higher life forms (plants, fungi and animals). However, even after a five decade spanning time period since its discovery, the precise biological role(s) of mammalian MTs remain enigmatic. One persisting hurdle is the presence of over 20 different genes within the human genome, and at least four in the genome of the mouse. In consequence, it has been very difficult to pin down an isoform specific function for each MT. In contrast, the fully sequenced C.elegans genome has only two isoforms. In previous work by one of the applicants, it was shown that both genes (individually or together) decrease the susceptibility towards heavy metal poisoning. In addition, cadmium, copper and zinc trigger the expression of both mtl-1 and mtl-2 in the cells of the worm's gut. However, mtl-1 is notably expressed at all times in the pharynx suggesting a role as a metal (possibly zinc) sensor. A particular point of interest is the difference between the two genes and the corresponding proteins, namely mtl-1's 15 additional residues in its C-terminus, three of which are capable of binding metal ions. We propose to use cutting-edge molecular biology, genetic and whole animal life-cycle parameters to study the differential functions of the two proteins with particular focus on the 15 C-terminal residues. In parallel, we will exhaustively characterise the metal binding properties of both proteins, and also determine their 3-dimensional structures, as we believe that biomolecules and their function can only be understood with a detailed knowledge of their structure and the molecular mechanisms of their action. The two labs are experts in either of the two areas, and we are convinced that through our interaction we will achieve a level of knowledge and understanding that would otherwise be impossible to obtain.
Technical Summary
The two C. elegans metallothionein (MT) isoforms show intriguing differences between their primary structures and their expression pattern: Although mtl-1 and mtl-2 share 62% sequence identity, and their expression in the gut can be induced by external heavy metals (Cd, Cu, Zn), mtl-1 has a 15 amino-acid C-terminal extension with 3 additional potential metal-binding residues. Most remarkable is the finding that only mtl-1 is constitutively expressed in the lower bulb of the pharynx. Our recent investigations have shown that, besides a role in Cd detoxification, the MTs are likely to have more general roles in metal ion homeostasis. We propose to determine these functions in detail, and carry out a comprehensive in vitro characterisation of the biophysical and biochemical properties of these two MTs, to advance knowledge of the relation between structure, solution properties, and function of metallothioneins. In addition, we will pay particular attention to the significance of the constitutive expression and C-terminal extension in mtl-1. Functional characterisation will be achieved by phenotyping C. elegans strains which over-express mtl-1 and mtl-2, by studying the impact of Zn and Cu depletion on a mtl-null strain, and by studying strains expressing mtls in which the C-termini have been mutated. In addition. all strains created will be evaluated in response to their susceptibility towards paraquat induced oxidative stress. We will also explore gene regulation and the genetic interaction network of mtl-1 and mtl-2 using customised micro-arrays. Biophysical and biochemical studies will be carried out on recombinantly (E. coli) expressed and purified proteins, and will include full structure determinations by state-of-the-art solution NMR methods. Metal ion binding thermodynamics and kinetics will be determined using multinuclear (1H, 19F and 111Cd) NMR spectroscopy, high-resolution electrospray mass spectrometry, ICP-MS and absorption spectroscopies.
People |
ORCID iD |
Claudia Blindauer (Principal Investigator) |
Publications
Blindauer CA
(2010)
Metallothioneins: unparalleled diversity in structures and functions for metal ion homeostasis and more.
in Natural product reports
Kowald GR
(2016)
Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.
in International journal of molecular sciences
Leszczyszyn OI
(2011)
Tools for metal ion sorting: in vitro evidence for partitioning of zinc and cadmium in C. elegans metallothionein isoforms.
in Chemical communications (Cambridge, England)
Zeitoun-Ghandour S
(2010)
The two Caenorhabditis elegans metallothioneins (CeMT-1 and CeMT-2) discriminate between essential zinc and toxic cadmium
in The FEBS Journal
Zeitoun-Ghandour S
(2011)
C. elegans metallothioneins: response to and defence against ROS toxicity.
in Molecular bioSystems
Description | As any other living organism, C.elegans obtains essential nutrients from the environment whilst avoiding being poisoned by noxious compounds. The heavy metal zinc, for example, is indispensable to virtually all biological processes, and zinc deficiency has been documented to have deleterious effects on reproduction, growth, and development. Since excess amounts are however highly toxic, its concentration and distribution in biological systems are under stringent control. Furthermore, the soil-dwelling worms are also exposed to the toxic element cadmium, which competes with essential zinc. Our research was aimed at understanding how organisms may discriminate between toxic essential elements. 1. We demonstrated how the two metallothionein (MT) proteins discriminate, in vitro, between toxic Cd and essential Zn. We showed that biological metal "selectivity" exploits relative affinities: although both proteins bind Cd more strongly than Zn, MTL-2 binds Cd more strongly than MTL-1 by two orders of magnitude, leading to predominant sequestration of Cd by MTL-2, and leaving MTL-1 to deal with Zn. Our observations correlate directly with those made in vivo by our collaborators. 2. We demonstrated by mass spectrometry that Zn-loaded MTL-1 and MTL-2 are both capable of reacting with hydrogen peroxide; this in vitro reaction correlates with observations in vivo. Intriguingly, MTL-1 yielded a sulfinic acid derivative, unprecedented in MTs. 3. Structural studies found that MTL-1 contains an isolated ZnHis3Cys site in the C-terminal extension, unprecedented in MTs. The site displays an absolute preference for Zn over Cd, and is the origin of the sulfinic acid derivative. |
Exploitation Route | Understanding metal metabolism is fundamentally important for any healthy organism. Our work contributes to this understanding, in particulatr in the area of discrimination between toxic and essential elements. |
Sectors | Agriculture, Food and Drink,Chemicals,Environment |
Description | Ongoing collaborations with SR Sturzenbaum |
Organisation | King's College London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Biophysical Studies of diverse metallothioneins. |
Collaborator Contribution | Partner designed and produced expression constructs. |
Impact | PhD Thesis Gregory Kowald 2012. (publication(s) pending) |
Start Year | 2008 |
Description | Cinderella complexes: The good ones go into the crop, the bad ones go to pot: novel metallothioneins to discriminate between essential zinc and toxic cadmium |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Lecture to research groups at the University of Edinburgh, resulting in scientific discussions. Raised the national profile of my research group. Increased awareness of the research presented and wider implications. |
Year(s) Of Engagement Activity | 2011 |
Description | Discrimination between essential zinc and toxic cadmium by soil-dwelling organisms: metallothioneins behaving in unexpected manners |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Keynote lecture at the 11th International Symposium on Applied Bioinorganic Chemistry (ISABC) (Barcelona, Spain, 2-5th December 2011). Abstract and Lecture Increase in international recognition of the research team's work. Increased awareness of the work presented and the wider implications. |
Year(s) Of Engagement Activity | 2011 |
URL | http://www.qi.ub.es/isabc11/News_.htm |
Description | Emergence of non-classical metallothionein metal binding sites: influence on structure, metal dynamics and function |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | poster presentation |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Poster presentation at the 2nd Georgian Bay Biological Inorganic Chemistry Conference (Parry Sound, Ontario, Canada, May 26-29 2009). Abstract and Poster Lively discussions with other attendees. |
Year(s) Of Engagement Activity | 2009 |
URL | http://www.canbic.ca/ |
Description | Glimpses of How Biological Systems Exploit Coordination Chemistry to Distinguish Essential from Toxic Metal Ions |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Lecture to research groups at the University of Campinas, Brazil Lecture Questions and discussions. |
Year(s) Of Engagement Activity | 2010 |
Description | How two metallothioneins CAN work together to discriminate between essential Zn and toxic Cd in C. elegans |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | A lecture on the 2012 Cadmium Symposium, Sassari, Sardinia, explaining how the thermodynamics of zinc and cadmium binding to the two C. elegans metallothioneins allows for predominant binding of cadmium to MTL-2, leaving MTL-1 to deal with zinc. Several interesting discussions with other attendees after the talk. Increased my research group's international profile amongst a new research community. Increase awareness of the implications of the work presented. |
Year(s) Of Engagement Activity | 2012 |
URL | http://www.cadmiumsymposium2012.uniss.it/ |
Description | Metallothioneins as intracellular metal filters that discriminate between zinc and cadmium |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited Lecture at the 3rd Georgian Bay Biological Inorganic Chemistry Conference (CanBIC 3) (Parry Sound, Ontario, Canada, May 31st-June 4th 2011) Abstract and Lecture Increased the research group's international profile. Increase awareness of he research presented and its implications. |
Year(s) Of Engagement Activity | 2011 |
URL | http://www.canbic.ca/ |
Description | Multiple Schools Outreach activities |
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 | Oksana Leszczyszyn (as well as several other Blindauer group members) played a significant part in the Department's schools outreach activities (we interact with ca. 4,000 pupils per year), and regularly participated in events bringing Biological Chemistry to school pupils. These events include visits to schools (e.g Whitley Abbey and Burton Green School, Coventry), as well as hosting pupils for a day in Warwick. In particular, Dr Leszczyszyn worked with pupils with learning and behavioural difficulties from the Pupil Referral Unit in Coventry, engaging these children in laboratory experiments. Enthused school children for Science. Changing pupils' with difficulties outlook. |
Year(s) Of Engagement Activity | 2008,2009,2010 |
URL | http://www2.warwick.ac.uk/fac/sci/chemistry/about/schools/ |
Description | New insights into Nature's tricks to distinguish between toxic cadmium and essential zinc |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Lecture at the IBDG Symposium on Metal Ion Homeostasis, University of Newcastle, Sept. 10/11th 2010, was received with interest and led to pertinent questions. Increased awareness of attendees of the work presented. |
Year(s) Of Engagement Activity | 2010,2015 |
URL | http://www.rsc.org/events/detail/4534?CFID=77794738&CFTOKEN=ad66dd299798c28-910ABABE-D24F-E094-A910D... |
Description | Outreach and public engagement - mutiple activities - IGGY |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Dr Oksana Leszczyszyn took a very active part in various outreach activities - these are now separately listed 1. She was academic staff member on three two-week courses for IGGY (International Gateway for Gifted Youth; http://www2.warwick.ac.uk/study/iggy/events/) during 2008-2010 (held in Singapore, Warwick, and Botswana). These programmes aim to reach out to the brightest young people around the globe. Inspired young people with an interest in Science. |
Year(s) Of Engagement Activity | 2008,2009,2010 |
URL | http://www2.warwick.ac.uk/study/iggy/events/ |
Description | Outreach to University of Botswana |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Undergraduate students |
Results and Impact | Dr. Leszcyszyn introduced Chemistry students at the University of Botswana to interdisciplinary science at the interface with the Life Sciences, giving two talks. see below |
Year(s) Of Engagement Activity | 2010 |
Description | Proteins as metal filters: novel metallothioneins for the discrimination between toxic cadmium and essential zinc |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Lecture to research groups and students at the University of Galway, Ireland (NUI). Lively discussion with students ensued. Increased awareness of attendees of the work presented. |
Year(s) Of Engagement Activity | 2013 |
URL | https://sites.google.com/site/ancumannceimice/photos/socs_day_1_2010/guest-speakers |
Description | Public Lecture at OneWorldWeek - Future of food |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Gave a presentation on bio-fortification of vegetable crops with zinc, to raise awareness of importance of zinc in nutrition, and to stimulate debate in the audience. This event was organised by students. |
Year(s) Of Engagement Activity | 2015 |
URL | https://www.warwicksu.com/societies/oneworldweek/ |
Description | Towards understanding metal specificity of selected class II metallothioneins |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | paper presentation |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Lecture at the 10th EuroBIC (Thessaloniki, Greece, June 22-26 2010). Abstract and Lecture Increased international recognition of the group's research. Increased audience's awareness of the wrok presented as well its wider implications. |
Year(s) Of Engagement Activity | 2010 |
URL | http://www.medimond.com/proceedings/moreinfo/20100622.htm |
Description | Work experience for pupils |
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 | Dr Leszcyszyn introduced two work experience students to her research. Giving pupils insight into a scientist's life and work. |
Year(s) Of Engagement Activity | 2009,2010 |