Elucidating the pathways and machinery of constitutive secretion

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The PDR position was filled in July 2014 so have made significant progress in making our key objectives.

1) Determine the subcellular localisation of SNAP29 and STX19.
We have determined the intracellular localisation of SNAP29 using a panel of monoclonal antibodies we have generated.

2) Determine how SNAP29 and STX19 are recruited to endocytic and/or biosynthetic membranes.
We have made significant progress on this aspect of the grant. Using yeast-two hybrid assays, BIO-ID and over expression experiments we have identified several key players who function on this pathway. We have identified that STX19 is palmitoylated at its C-terminus and it targets the protein to membranes. We published a manuscript at JCS as part of this project.

3) Determine if SNAP29 and STX19 are required for endocytic and/or biosynthetic transport of GPI anchored proteins
through development of a system for their rapid inactivation.
We have have generated SNAP29 knockout cells using CRISPR/CAS9 and can detect clear defects in endocytic trafficking. Using RNAi we can successfully deplete STX19 and can also observed clear trafficking defects. We are currently validating these phenotypes. We are currently writing a manuscript on this part of the project.
Exploitation Route a) Health care professionals. Our research will identify and characterise key pathways and players in constitutive secretion so uncovering the function of new genes. This will aid those investigating the links between genetic mutations and human disorders. For example our work on SNAP29 should help elucidate the molecular pathologies of CEDNIK syndrome. We currently have a collaboration with a medical geneticist in Oxford who is interested in identifying patients with CEDNIK syndrome. Our research may also aid in the development of novel tools and assays for the diagnosis of this disease.

b) Companies developing novel therapeutics. In the biotech industry there is significant interest in developing new drugs that target SNAREs. For example, Syntaxin is running clinical trials using drugs based on modified forms of Botulinum toxins. These drugs are primarily being developed for
the treatment of disorders associated with regulated secretion e.g. pain and acromegaly. However, these drugs will also target SNAREs which we believe to be involved in constitutive secretion so our work defining the role of SNAREs in post-Golgi trafficking will provide critical information regarding the specificity and safety of these new drugs. Our work will also provide information that will be invaluable for developing new applications of these drugs e.g. can these drugs be used to
treat disorder caused by dysregulated antibody secretion.

c) Patients will benefit from any new diagnostic test and/or treatments for disease resulting from this research.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.sheffield.ac.uk/bms/research/peden/index
 
Description We have developed a novel cell based assay in collaboration with Professor Davletov that allows the activity of certain bacterial toxins to measured. These toxins can currently only be assayed using mice. This assay is currently in testing/validation at National Institute of Biological Standard and in the future may aid those developing vaccines and therapeutic drugs based on these toxins. The assay has the potential to reduce costs and speed up the process of testing these toxins. We have been awarded a patent for this assay and are currently in negotiations with several botulinum and tetanus vaccine manufactures who are interested in licensing this assay. In addition, we are also in discussions with several botulinum manufacturers who are also interested in this assay.
First Year Of Impact 2018
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description A highly sensitive replacement assay for botulinum neurotoxin type B
Amount £306,073 (GBP)
Funding ID NC/S000925/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2018 
End 01/2021
 
Description PhD Studenship
Amount £70,000 (GBP)
Funding ID 1945669 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
Title Cell lines deficient for SNAP29 
Description We have generated a panel of cell lines deficient for SNAP29 using CRASPR/CAS9. These cell lines allow the function of SNAP29 to be elucidated. They will be useful models for studying the human disease CEDNIK where SNAP29 is mutated. 
Type Of Material Cell line 
Provided To Others? No  
Impact Non as yet. 
 
Title SNAP29 monoclonal antibodies 
Description We have generated a panel of monoclonal antibodies to SNAP29. 
Type Of Material Antibody 
Provided To Others? No  
Impact These antibodies will be useful for investigating the function of SNAP29 a gene mutated in CEDNIK syndrome. 
 
Description Developing novel cell lines for detecting bacterial toxins 
Organisation National Institute for Biological Standards and Control (NIBSC)
Country United Kingdom 
Sector Public 
PI Contribution We are developing novel cell lines for detecting bacterial toxins.
Collaborator Contribution They provided funds to pay for reagents and will test the novel cell lines.
Impact New knowledge. We plan to publish our findings. We have recently submitted a patent on the assay we have developed.
Start Year 2014
 
Title STABLE VAMP REPORTER ASSAY 
Description The present invention provides a polypeptide comprising an N-terminal polypeptide domain having luciferase activity and a C-terminal polypeptide domain having VAMP1, VAMP2 or VAMP3 activity where VAMP stands for vesicle-associated membrane protein. Corresponding nucleic acid molecules, expression vectors and genetically modified cells are also provided. The invention also provides methods and uses of the same. 
IP Reference WO2018150177 
Protection Patent granted
Year Protection Granted 2018
Licensed No
Impact We are currently in negotiations with several botulinum and tetanus toxin manufacturers who are interested in licensing this assay. In addition we are in discussions with several companies who manufacture botulinum toxins for cosmetic and clinal use.
 
Description Discover night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I co-organised a cell biology demonstration. The aim of the demonstration was to educate and inspire the general public about basic science.
We used microscopes and cell models.
Year(s) Of Engagement Activity 2014
 
Description Discovery night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I co-organised a cell biology demonstration. The aim of the demonstration was to educate/inspire people about basic research.
We used microscopes and cell models during the presentation.
Year(s) Of Engagement Activity 2014
 
Description Krebs Fest 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I co-organised a cell biology demonstration as part of the Krebs fest. The demonstration ran for approximately 3 hours and around 500 people attended the demonstration The aim of the pod was to inspire/educate individuals about fundamental cell biology. We had microscopes, cell models and biscuit decorating.
Year(s) Of Engagement Activity 2015
URL http://krebsfest.group.shef.ac.uk