France

Every human cell is encased by a cell membrane that separates the cell contents from its surroundings. Proteins embedded in this membrane act as gates to allow molecules to enter and exit cells; they also mediate the interactions that occur between a cell and its environment. This means that membrane proteins are involved in many of the most fundamental processes in normal cell function; when these processes fail, diseases result. It is no surprise, then, that the top ten best-selling small molecule drugs of all time all target membrane proteins. There are many different membrane proteins in any given cell, grouped into over 1,500 families, each with many members. In order to study any of them in detail, it is important to understand their three-dimensional structures. Central to this is a technique called X-ray crystallography that allows scientists to obtain a detailed view of how the atoms within a protein are arranged, providing a framework for further study. Scientists use this framework to investigate how the protein functions, bringing new levels of understanding to how cells work in health and disease, and providing knowledge to develop new drugs. Tetraspanins are membrane proteins that function by interacting with a wide range of other membrane and soluble proteins, thereby affecting how cells signal, interact, change shape and move. Remarkably, tetraspanins are also involved in the process of infection for a wide range of diseases. However, because there is no known structure of any full-length tetraspanin family member, the mode of action of tetraspanins in these essential processes is not understood, leaving a major gap in our knowledge of cell biology. Obtaining the structure of any membrane protein is a major scientific challenge: It is necessary to remove the protein from the cell membrane which often results in the protein becoming so unstable that it cannot be used to make the crystals required to perform X-ray crystallography. Consequently, we know very little about many membrane protein families with important biological functions. We have now overcome this crystallization challenge for the tetraspanin, CD81. Human CD81 is one of the best understood tetraspanin family members and is the subject of our proposed research. It has well-established roles in how cells interact with each other, the immune response and fertilization. Notably CD81 is a receptor for some very important human pathogens including influenza, human immunodeficiency virus, the malarial parasite, T-cell lymphotropic virus type 1 and hepatitis C virus (HCV). It may also be a tumour promoter. Central to CD81 function (and to that of all tetraspanins) is its ability to form extensive interactions with itself and other proteins; however, we don't know what these structures look like and therefore lack the framework for further study, mentioned above. The first aim of the research outlined in our proposal is to solve the three-dimensional structure of CD81. We have made excellent progress towards this goal, having crystallized CD81 and collected X-ray diffraction data. We have also teamed up with scientists in France who can make soluble forms of the HCV protein, E2, that binds CD81. The second aim of our project is to make an HCV-E2/CD81 complex so we can characterize it and solve its structure; this will allow us to learn more about how CD81 interacts with other proteins. We believe we are the only team in the world that has all the tools to take on this challenge. Brand new developments in structural biology (e.g. high-resolution electron microscopy) have enabled us to devise a third aim, which is to look at these structures in the cell membrane (by electron tomography), linking our atomic level structural data to what is actually happening in the cell. Studying the structure of CD81 at this level of detail will allow us to begin to understand how tetraspanins work in health and disease.

Microbes are the most numerous living organism on the planet. They can cause diseases in other organisms, including humans, but they can also be helpful in everyday human activities, like digestion. Some are used to perform specific industrial or manufacturing processes such as the production of drugs or synthetic hormones, while others are used for the bioconversion of organic waste. A common trait for all these propertie is the necessity for microbes to interact with other organisms or their natural and man-made environments. For this purpose, they often use hair-like surface organelles known as pili, among which type IV pili (Tfp) are the most widespread. However, how Tfp are assembled and how they mediate the multiple functions they are linked with (adhesion to various surfaces, aggregation, motility and DNA import) remains poorly understood. We are therefore trying to answer these questions using as a model bacterium Neisseria meningitidis, which an important human pathogen responsible for 50% of the cases of bacterial meningitis worldwide. Besides helping develop more efficient therapies against human pathogens and leading to a better understanding of a fundamental biological problem, our work may lead to potential applications and benefits for other less tractable medically or commercially important species that express Tfp. In a first step, we have identified all the genes involved in Tfp assembly, which indicated that the corresponding machinery is complex since it numbers fifteen different protein components. This suggested that understanding how this machinery works would only be possibe through a reductionist approach, that is by first examining smaller pieces of it, which could explain the whole upon re-assembly. Therefore, we used bacterial genetics to determine that four different steps are necessary for the expression of functional Tfp and we could define at which one of these steps each of the above fifteen proteins is involved. The objectives of this research project are multiple and aim at understanding how the protein work together as subgroups in the above four steps to assemble functional Tfp. As a first thing, we will determine the basic biochemical properties of these fifteen proteins, such as teir abundance and localization in the cell. We will then identify the proteins that are interacting with each other in order to put the pieces of the puzzle back together. This will be done using two different and complementary techniques. Finally, we will move to a downstream stage which consists in determining the tri-dimensional structure and mode of function of the individual protein components. We will start by studying the PilW protein, which has particularly interesting properties.

Natural populations adapt to novel environments via phenotypic variation that has its origins either in contemporary mutation events or in pre-existing ancestral variation, or both. Understanding the significance of these modes of evolution in real ecological settings is central to predicting the speed of adaptation to novel challenges, and thus to informed population management intervention in the face of environmental change - but we lack suitable empirical studies. Industrial melanism in the peppered moth (Biston betularia) has emerged as a top candidate for such a study. In Britain, the black (carbonaria) form is due to a singular recent mutation, whereas in continental Europe, preliminary data suggests a surprising diversity of mutations, some of which may be adapted to a pre-industrial and pre-agricultural, forest-dominated landscape. Thus, the celebrated British case may not be generally representative of the evolutionary origins of industrial melanism across the species' range. Natural heterogeneity in resting backgrounds, associated with successional turnover and extensive mature forests, may be the unrecognised factor maintaining the striking diversity of melanic forms in this species. By revealing the identity of the mutations causing melanism in continental European populations, estimating their age, and evaluating non-industrial environmental factors maintaining melanism, this project will resolve a major puzzle in this influential evolutionary biology case study, whilst at the same time providing a novel illustration of how the interplay between genomic architecture, ecology, and geographic isolation influences mechanisms of evolution.

Society faces major challenges from viral diseases. The recent Zika and Ebola outbreaks are only two examples of the devastating impact of viral illnesses on human health, and viral pathogens infecting agriculturally important livestock and plants simultaneously reduce food production and inflict great annual financial losses worldwide. Viruses, however, also have positive impacts on health and ecology. They balance and stabilise our gut microbiome, preventing serious illnesses such as certain autoimmune diseases, and influence our climate owing to their roles in carbon cycling in the oceans. It is therefore paramount to better understand virus structure and function across the entire virosphere in order to control, and even take advantage of, viruses in medicine and biotechnology. I have demonstrated previously that mathematical approaches developed in tandem with experimentalists are drivers of discovery of functionally crucial structural viral features, revealing their novel functional roles in viral life cycles, and enabling their exploitation in therapy and biotechnology. Previously developed mathematical approaches were geared towards a specific major sub-group of the virosphere. In this research programme, I will both broaden and deepen the development of novel mathematical techniques. Working in close collaboration with leading experimental groups, at a larger scale, I will identify functionally important geometric viral features in a number of major groups of viruses. This will include: geometric strand assortment in multipartite viruses, such as the major agricultural pathogen Bluetongue virus; the assembly of retroviruses like HIV, with applications to the construction of virus-like particles from viral components as vectors for gene editing and therapy; and the structure and evolution of viruses important for the gut microbiome and marine ecology. By linking structural features with their functions, I will address open problems regarding drivers of evolution in one of the simplest yet most important groups of biological entities. This approach will unmask evolutionarily conserved functional features that can be used as novel targets in anti-viral therapy, for the development of novel safer vaccines or repurposed in bionanotechnology.