Litaf, a novel driver of membrane protrusion pathways

The behaviour of cells within a tissue is controlled by their response to the environment. Receptor molecules at the cell surface receive a large number of chemical and physical stimuli that transmit signals to the interior of the cell and control important processes such cell migration, metabolism, cell proliferation and differentiation. Anomalies in the transmission of such signals result in pathological states that derive in diseases like cancer, diabetes, muscular dystrophy and neurological degeneration.

Amongst the most important signals that cells receive are from circulating small proteins called growth factors. These bind to specialised receptors, that in response to growth factor engagement alter their pattern of interactions with many molecules inside the cell to generate a so-called 'mitogenic response'. This response encompasses many changes to the cell's behaviour (such as growth, division and migration). One of the best-studied examples of a growth factor receptor is the epidermal growth factor receptor (EGFR), which controls many aspects of cell growth. Increased expression of EGFR is linked to several types of cancer. In order to prevent overstimulation of the cell response to growth factors, there are mechanisms of regulation to ensure that these responses are not sustained endlessly, which would lead to uncontrolled cell division and proliferation. These mechanisms encompass the internalisation of the receptor upon stimulation, leading it from the cell surface to specialised membrane-enclosed compartments within the cell (called endosomes). From endosomes, receptors pass to another membrane-enclosed compartment called the lysosome, where they are ultimately destroyed. The pathway to the endosome is termed the endocytic pathway.

We discovered key elements of the cellular pathway that controls the uptake of EGFR and its movement through the endocytic pathway. These elements recognise the stimulated receptor and re-shape the local membrane around it so that the receptor can move from the cell surface to endosomes and then to lysosomes. The current application concerns a new protein element that we have discovered, called LITAF. Our preliminary data suggest that LITAF can alter the shape of membranes and thus aid the transport of EGFR through the endocytic pathway.

The endocytic pathway is also critical in protecting against viral and bacterial infections and to eliminate protein aggregates that otherwise accumulate inside cells and result in the neurodegeneration observed in Alzheimer's, Parkinson's and Huntington's diseases. In fact, LITAF (lipopolysaccharide-induced TNF factor) was first identified as a protein that the cell produces in high quantity as a response to exposure to bacterial toxins. It is likely that the function of LITAF is linked in some way to the function of the endocytic pathway in helping to generate immune responses, though we do not yet know what the link is. In addition, mutations within LITAF that occur in the population lead to a debilitating paralysis termed Charcot Marie Tooth disease, caused by loss of myelin sheaths from the peripheral nervous system. We hope that information we gain from this project will help us understand how the health of the myelin sheath is normally maintained

In summary, the knowledge gained by our investigations will bring new insights into the molecular basis of many diseases caused by mutations in proteins that control the endocytic pathway, and in the long-term will guide further efforts for pharmacological intervention.

Routing of cell surface-derived receptors through the endocytic pathway is fundamental to how cells interact with their environment. Defects in endocytic receptor sorting are linked to many diseases, including cancer and neurodegeneration and, hence, a comprehensive understanding of the endocytic pathway is of crucial importance. This proposal focuses on the function of LITAF (lipopolysaccharide [LPS]-induced TNF factor), a ubiquitiously expressed small integral membrane protein that is involved in sorting mitogenic receptors to the multivesicular body (MVB), but whose molecular function is uncharacterised. This project will build on preliminary data showing that LITAF is required at a late stage of MVB formation, and thus may drive deformation of the endosomal membrane in order to generate intralumenal vesicles within the MVB. We will then test our central hypothesis that LITAF is a key element in driving membrane protrusion away from the cytoplasm. This hypothesis will be tested from a number of directions.

We will identify the mechanism by which LITAF is integrated into the membrane of the endoplasmic reticulum, since we believe the membrane topology of LITAF may be key to explaining its biological function. For these experiments we will in vitro translate LITAF in the presence of canine pancreatic microsomes. We will also examine the interaction of recombinant LITAF with artificial lipid membranes. We will then test key structural determinants of LITAF, namely a putative Zn-finger domain, to test whether this plays a role in LITAF function.

Having identified the mechanisms by which LITAF integrates with lipid bilayers, we will examine whether this mode of insertion plays a crucial role for LITAF function at the MVB. We will also test more broadly whether LITAF plays a role in membrane protrusion processes, and to what extent these processes utilise the same pathways as MVB formation.

Although the precise cellular function of LITAF is poorly characterised, it has emerged as a protein of great potential importance for both the biomedical and agricultural sectors. Hence, defining the cellular functions of LITAF will provide the essential groundwork for future studies into the underlying mechanisms of diseases as widespread as demyelination (Charcot Marie Tooth disease), cardiac disease, cancer and inflammatory disease (Crohn's disease). It is quite feasible that LITAF might offer new targets for novel therapeutic strategies.

The exposure of commercially important fisheries and livestock species to a range of pathogenic bacteria and viruses results in a many-fold induction of the LITAF gene. Hence, it has the potential to act as a biomarker for bacterial infection in commercial stocks, particularly when its physiological functions are clearly defined. This area of potential exploitation meets the BBSRC strategic priority of healthy and safe food.

The immediate academic impact of this project will be with research scientists working in related areas of the life sciences, including signalling receptor trafficking and cell migration. Our work will provide new knowledge, insight and scientific advances that will be relevant to all those that are active in these areas, irrespective of whether their research is being carried out from a basic or a medical perspective. Clearly, however, our work will direct relevance to clinical scientists. Another major stakeholder with a fundamental interest in this field is the pharmaceutical industry, with LITAF offering a potentially new clinical target.

The applicants are both strongly committed to engaging with the general public about the importance of Science and the specific goals of their research, actively participating in a variety of outreach activities. These range from Faculty and Institute open days to giving scientific presentations at English Literature conferences and informal spoken word events designed for a wide audience. We strongly encourage our staff and students to take part in raising the profile and understanding of science in the wider society, and Ling Zhang the PDRA will participate in outreach activities throughout the course of the project. In addition, the project offers further scope for Ling to develop his range of advanced EM skills, making him well placed to pursue a long-term career in science, thereby contributing to the UK knowledge base and economy. Since Ling is a Chinese national, there is also an important international context to his academic development.