Elucidating the pathways and machinery of constitutive secretion

The human body is made up from billions of cells and each of these cells is further organised into a series of compartments. Cells in some ways resemble miniature cities and these compartments are just like factories that make specific products, for example the endoplasmic reticulum is involved in making proteins and lipids. Once a protein or lipid has been made, its function is often required somewhere else within the cell. To transport these proteins and lipids to the site where they are needed, small packages (vesicles) break off from one compartment and fuse with another, thereby delivering their contents. These vesicles are just like lorries taking goods to different destinations within the city. In some cases the newly made proteins are required outside the cell so the vesicles carrying these proteins fuse with the membrane that covers the cell surface (plasma membrane). This process is called constitutive secretion and is required for many important processes such as inflammation and immune system function. Changes in secretion can cause disease and there is an increasing number of rare genetic disorders cause by mutations in proteins required for secretion.

My laboratory is interested in identifying and characterising proteins required for generating, transporting and fusing secretory vesicles with the plasma membrane. To identify this machinery we have developed new ways of measuring secretion and used them with technologies that allow the function of individual genes to be disrupted. Using this approach we have identified two proteins, STX19 and SNAP29, that are required for secretion. This proposal aims to characterise the function of these proteins, in particular we are interested in understanding how SNAP29 and STX19 are regulated and how these proteins are targeted to the correct place within the cell. We are also interested in determining whether SNAP29 and STX19 are involved in the secretion of all types of proteins from cells or just a specific subset.

This work will benefit society because it will increase our understanding of a fundamental cellular process and in the future may help in the development of new diagnostics or therapeutics for the treatment of diseases caused by defective secretion. In particular, our work should help in understanding the pathologies which occur in the rare fatal disease CEDNIK (CErebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma) syndrome which is caused by the loss of SNAP29.

Constitutive secretion is a fundamental cellular process required for the delivery of newly synthesised proteins and lipids to the plasma membrane as well as the exocytosis of extracellular factors such as cytokines and antibodies. Perturbations in secretion can cause disease and there are now several examples of rare genetic disorders caused by mutations in the secretory machinery. Very little is known about the post-Golgi pathways and machinery required for secretion in metazoans. To address this my lab has taken a functional genomics approach and performed a targeted siRNA screen against SNAREs. Our screen identified the post-Golgi SNAREs, SNAP29 and STX19, as having a role in constitutive secretion.

This proposal aims to elucidate the roles of STX19 and SNAP29 in post-Golgi trafficking and secretion. In particular, we are interested in addressing whether STX19 and SNAP29 have an endocytic function, if they are involved in the endocytic and/or biosynthetic transport of GPI linked cargo proteins and the identification of factors required for targeting STX19 and SNAP29 on to endocytic and/or biosynthetic membranes.

To achieve our aims, we will use immuno-EM to localise SNAP29 and STX19 in relation to endocytic and biosynthetic markers. We will determine how SNAP29 and STX19 are recruited on to post-Golgi membranes by mutating key residues in SNAP29 and STX19 and by identifying novel interacting partners for these SNAREs using yeast two hybrid screens and mass-spectrometry. The role of SNAP29 and STX19 in endocytic and biosynthetic transport of GPI linked protein will be assessed using the "knocked sideways" approach in combination with novel GPI linked protein transport assays.

If we are to effectively treat human disease we need to have a detailed understanding of the basic cellular process that occur within the body. However, our knowledge of many key biological processes remains poor. For example, we do not understand the cellular machinery and pathways required for the post-Golgi trafficking and secretion of antibodies, cytokines and extracellular matrix components, even though these are biologically important molecules. This proposal aims to address this by elucidating the role of SNAREs (SNAP29 and STX19) in secretion.

This research has the potential to improve lifelong health and well-being of our society because it will provide the fundamental knowledge required for the development of novel diagnostics and therapeutics for the treatment of diseases associated with secretion. There are three main groups in society who will benefit from our research:

Health care professionals
Our research should significantly increase our understanding of intracellular transport pathways and allow for the function of novel proteins to be assigned. This in turn will aid those investigating the links between genetic mutations and human disorders. Our research may also assist in the development of novel biomarkers for the diagnosis of disease.

Industry
There is significant interest in developing drugs that target SNAREs for the treatment of diseases such as chronic pain and acromegaly. However, the role of SNAREs in post-Golgi trafficking and secretion is poorly defined so it is unclear what pathways these drugs may target. Our work will provide critical information regarding the specificity and safety of these new drugs.

Patients
The development of novel diagnostics and therapeutics should allow for treatment and possible cure of many secretory-based diseases for which no treatments are currently available.

As well as benefiting these communities, the postdoctoral researcher employed on this grant will directly benefit from being trained in a variety of cutting edge molecular cell biological techniques and gain expertise in data analysis. The transferable skill gained in the laboratory will prepare this person for a career in either academia or industry.