Structural analysis of Netrin 1 signal initiation and transduction

During the development of multicellular organisms (e.g. humans), cell-to-cell communication is vital. Trillions of growing cells need to end up in the right place in order to fulfil their destiny. Even in a full grown body, our cells and tissues still need to communicate, and new cells grow and migrate to repair and renew our tissues. To achieve this crucial job, the body uses signalling molecules and receptors to direct and guide cells, and to allow communication between different cells in the body. These molecules signal like traffic lights, stopping or allowing growth and cell movement.

The netrins (in particular NET-1) are a family of signalling molecules with multiple functions in human biology. They have been initially identified as crucial signals responsible for guiding axons in the developing nervous system, for which they were named after the Sanskrit word "netr", which means "one who guides". New research has also associated netrins to the development and maintenance of non-neural tissue, such as the vascular and muscle systems as well as mammary gland and the heart.

The importance of netrins signalling in human disease has become increasingly clear as mutations in netrins and their receptors have been linked to Parkinson's disease and amyotrophic lateral sclerosis (ALS), suggesting that netrin-mediated changes in synapse function may influence human neurodegenerative diseases. Moreover, it has been shown that netrins are activated and serving as pro-cancer factors in various human cancers, such as pancreatic adenocarcinomas, metastatic breast cancer, non-small-cell lung cancer and neuroblastoma. Specific disruption of netrin binding to its receptors could therefore represent an efficient anti-cancer strategy.

Genetic studies have provided a broad brush-stroke understanding of these mechanisms, however much still remains to be found out about impaired netrin signalling leading to human disease. Crucially, the details (fine brush-stroke) of netrin recognition at the surface of cells and how information is transmitted into the cell are only poorly understood. This level of detail can greatly aid drug design. We aim to unravel these fundamental mechanisms by providing molecular snapshots of netrin-receptor complexes using the methods of X-ray crystallography combined with functional, biophysical and cell-based experiments. Our results will be placed in a context of neuronal systems and whole organisms via collaborations with neurobiologists to get an integrated picture and a deeper understanding. This will potentially allow the structure-guided design of new therapeutic and/or diagnostic approaches.

We will study the molecular mechanisms of NET-1 signalling, a crucial pathway in cell and axon migration and cell-to-cell contacts. NET-1 signalling is important for human health with implications spanning from neurodegenerative diseases to cancer. We will study the molecular mechanisms of NET-1 signal initiation by NET-1 interactions with its receptors and signal transduction at the plasma membrane. We will use a combination of X-ray crystallography to obtain high-resolution molecular snapshots of extracellular NET-1 receptor complexes, biophysical methods to characterise interfaces and complex stoichiometries and live-cell fluorescence microscopy, using full-length NET-1 receptors, to relate our molecular analysis to the cellular and physiological context. Interrelated aims are:

- To determine crystal structures of NET-1 complexes with the ectodomain constructs of NEO1, DCC and UNC5 receptors to obtain detailed molecular interface information, interaction stoichiometries as well as insights into the mechanisms of NET-1 signal modulation.

- To biophysically characterise the NET-1-NEO1/DCC-UNC5 extracellular stoichiometry and interface properties using SPR, ITC, MALS and SAXS. Our structural data will allow us to design site-directed mutants that selectively impair the ability of NET-1 to interact with its receptors.

- To validate the observed NET-1-NEO1/DCC binding mode within a cellular context, using full-length transmembrane NEO1/DCC receptors. We will employ FRET/FLIM in live COS7 cells to unravel signal transduction via the NET-1 receptors DCC, NEO1 and UNC5 and test the impact of structure-guided mutations in the observed interfaces.

- To test the importance of the competition between NET-1 and two other protein families that bind NEO1 and DCC respectively, RGMs and CBLN4. We will employ competition binding assays to test the effect of NET-1 on RGM-NEO1 and CBLN-DCC binding and on NEO-1/DCC clustering in FRET/FLIM experiments in live COS7 cells.

A detailed, molecular level characterization of the signalling mechanisms that drive cell motility and connectivity is an essential step towards understanding the complex process of tissue morphogenesis. NET1 and its receptors are crucial players in this process, as disregulation of this pathway leads to developmental defects primarily in the nervous system. Such impairments are often incompatible with survival, but in in milder forms they can lead to neurological and psychiatric disorders or, later in life, to a broad panel of cancer forms.

Although this is essentially a basic science proposal, without doubt the long-term beneficiary of this work is the general public. According to the World Health Organization (http://www.who.int/mental_health/policy/services/integratingmhintoprimarycare/en/index.html), disorders linked to synaptic dysfunction currently affect hundreds of millions of people worldwide. This poses a huge burden to the world's economic output. Without the concerted efforts of academia and industry, this situation is likely to exacerbate considering the predicted increase of life expectancy worldwide. For example, within the UK, research commissioned by the Alzheimer's Society projects that the 2011 number of 750,000 people suffering from dementia may increase to 1 million by 2021 and 1.7 million by 2051.

Cancer is a major cause of death worldwide, accounting for 7.6 million deaths (around 13% of all deaths) in 2008 with a projection to continue rising, with an estimated 13.1 million deaths in 2030 (http://www.who.int/mediacentre/factsheets/fs297/en/). There are more than 200 different types of cancer identified in 60 different organs in the body. The knowledge about the causes of cancer, and strategies to prevent and manage the disease is extensive. However, looking at the statistics it is eminent that a lot of work has to be done. Targeted therapies provide a type of anti-cancer medication that significantly changed the treatment of cancer over the past 10 years. The idea is to block the growth of cancer cells by interfering with specific targeted molecules needed for tumor growth, rather than by simply inhibit all rapidly dividing cells (such as chemotherapy or radiotherapy). This may provide a more effective strategy for curing cancer than current treatments, and less harmful to normal cells.

The crystal structures we will solve, and novel biological insights, can provide a solid grounding needed to develop novel therapeutics (including antibodies), that may trigger fewer side effects compared to those designed using the traditional "silver bullet" approach. While our own laboratories are not directly focused on drug design, both the PI and the Co-I have actively followed the translation aspects of structural work in the past, together with Cancer Research Technology (PI Siebold, on developing small molecule inhibitors that block Hh morphogen binding to its receptors) and the MRC-Technology (Co-I Aricescu, on developing structure-guided modulators of ionotropic glutamate receptors), respectively. Furthermore, the Co-I has recently been awarded an MRC Confidence in Concept award aimed at developing, together with clinical researchers and a company focused on microarray design, novel diagnostic strategies for neurological autoimmune disorders linked to cell surface receptors involved in synapse formation and function. We will continue to pursue such avenues and thus contribute to translating the knowledge generated by the work proposed in this grant application into tangible benefits for the society.