Molecular biology

Asthma and allergies: spotlight on leukotrienes

In July 2007, European researchers resolved the structure of leukotriene C4 synthase (LTC4 synthase) with unprecedented accuracy. This enzyme is of particular interest to scientists because it plays a key role in the complex process governing certain allergies, notably asthma. This promising new discovery was the result of collaboration between two European research projects.

Frontal view of LTC4 synthase inside the cell membrane. The grey, green and purple sections represent the three protomers (sub-units) comprising this homotrimer (protein comprising three sub-units). © Daniel Martinez Molina Frontal view of LTC4 synthase inside the cell membrane. The grey, green and purple sections represent the three protomers (sub-units) comprising this homotrimer (protein comprising three sub-units). © Daniel Martinez Molina
Cytosolic view of LTC4 synthase. The active sites are represented by dots. © Daniel Martinez Molina Cytosolic view of LTC4 synthase. The active sites are represented by dots. © Daniel Martinez Molina
Representation of the surface of LTC4 synthase. Each colour symbolises a different type of molecule. © Daniel Martinez Molina Representation of the surface of LTC4 synthase. Each colour symbolises a different type of molecule. © Daniel Martinez Molina

What does leukotriene C4 synthase (or LTC4 synthase) look like? It has taken researchers working on the two European projects – Eicosanox and E-MeP – more than eight years to ascertain with optimal accuracy the three-dimensional profile of this membrane protein. Determining the protein’s structure at a resolution never before achieved, of 2 Å(1), represents a major step forward in the development of new therapies against such allergies as allergic rhinitis (hayfever) and certain forms of asthma. LTC4 synthase is an enzyme essential to the synthesis of leukotriene C4 (LTC4) – a neurotransmitter that acts as a bronchoconstrictor agent which patients with such allergies produce to excess. Controlling the production of LTC4 would make it possible to foil the complex inflammation mechanisms triggering asthma and allergic rhinitis, as this hormone is responsible for constricting the bronchial tubes and causing excess mucus production.

Attacking the disease at source

Jesper Z. Haeggström, Professor of Bio - chemistry at Karolinska Institutet (SE) and chief coordinator of Eicosanox, explains that this new image of LTC4 synthase reveals a molecule formed of three identical sub-units, each comprising five spiral-shaped structures. “This has allowed us to determine the exact position and characteristics of the enzyme’s active sites. Having located these sites to which the activatory or inhibitory molecules attach themselves, it is now possible to synthesise new proteins specifically designed to block the action of LTC4 synthase, and hence the synthesis of LTC4 which is responsible for hayfever and asthma.”

Most of the drugs currently prescribed for these pathologies target the symptoms caused by the overproduction of leukotrienes. Although anti-leukotriene agents like Montelukast have recently come onto the market, their efficacy is limited because they do not target the production mechanism with any great accuracy. Discovering more about LTC4 synthase therefore makes it possible to develop compounds tailored specifically to a disease.

“We have been working on LTC4 synthase since 1999, well before the Eicosanox and E-MeP projects were launched. We integrated the LTC4 synthase subprogramme into Eicosanox in response to the 6th Framework Programme’s new guidelines advocating the inclusion of basic research objectives with a higher failure risk. This subprogramme is one of the Eicosanox programme’s boldest aspirations, and the crowning achievement of this long-drawn-out research effort was the publi- cation of our results in the journal Nature, nearly two years before the main project ends”, said Jesper Z. Haeggström proudly.

On the molecular front

Two ambitious research projects were behind this exceptional European scientific achievement. Eicosanox aims to improve our understanding of how two types of neurotransmitter function in the human body: eicosanoids and nitric oxide (NO). These molecules play an important role in numerous physiological mechanisms such as inflammation, pain and fever regulators. They are also significantly responsible for certain cardiovascular, brain and even tumorous dysfunctions, which explains the keen interest of Eicosanox researchers and, indirectly, the European Commission, which has allocated a massive €10 million to the project.

The European Membrane Protein Consor - tium, E-MeP, is a vast research platform that seeks to overcome the current technological obstacles to resolving the structure of membrane proteins. Indeed, some 40 % of all proteins of pharmaceutical interest are membrane proteins and one-quarter of the human genome is devoted to synthesising them. These molecules, which are found in the membranes of all human cells, fulfil a variety of functions, notably in intra- and extracellular exchanges. As membrane proteins are involved in the development of many diseases, finding out more about them is likely to lead to new treatments, particularly for incurable diseases like cystic fibrosis or Alzheimer’s disease.

With a little help from our friends

None of this explains how the Eicosanox and E-MeP projects actually came to cooperate in the first place. “The answer is simple”, says Jesper Z. Haeggström. “Pär Nordlund, Professor of Biochemistry and Biophysics at the University of Stockholm (SE) and E-MeP project researcher is an old friend of mine. When we realised how much our research objectives for LTC4 synthase overlapped, we soon decided to pool our efforts.” So it seems as though there’s nothing like friendly relations to encourage interproject collaboration. Despite European Union efforts to promote a degree of cross-disciplinarity in scientific research, such collaboration is all too rare in the European Research Area. According to Jesper Z. Haeggström, despite the EU’s reputation for complex administrative procedures, no special authorisation was required for their collaboration venture, which should be very welcome news to European researchers.

Julie Van Rossom

  1. 1 Å = 1 ångström = 10-10 meter

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Producing the portrait of a protein: no easy matter

Resolving the structure of a protein can be an extremely painstaking task, as in the case of LTC4 synthase. The whole process starts with isolating the portion of DNA responsible for synthesising the molecule and then cloning this fragment to make a large number of copies. Then, a means must be found to induce the clones to produce the protein in a proper expression system. To do this, the fragment is inserted into a host cell (usually a bacterium or yeast), which allows it to be expressed in exactly the same way as under natural conditions. Once the protein has been produced and extracted, it must be stabilised using detergents to prevent it from breaking up.

The next stage is to crystallise (or solidify) the molecule that has now been encapsulated in a drop of water.  Researchers use the vapourphase crystal growth technique, where the drop  of water containing the protein crystal is placed in a vessel containing a moisture-absorbing hygroscopic substance. To complete the puzzle, an X-ray image is taken of the crystal. In the case of LTC4 synthase, E-MeP and Eicosanox researchers were able to obtain their excellent 2 Å resolution by using the European Synchrotron Radiation Facility based in Grenoble (FR), the most powerful synchrotron light source in Europe, consisting of very bright X-rays.



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To find out more

  • Eicosanox
    12 partners - 7 countries (SE, DE, IT, UK, IE, ES, CA)
    www.eicosanox.org
  • E-Mep
    18 partners - 6 countries (UK, FR, DE, SE, CH, NL)
    www.e-mep.org