Palmitoylation of Exocytic Proteins: Role in Membrane Compartmentalization, Intracellular Trafficking, and Function

Lead Research Organisation: University of Strathclyde
Department Name: Inst of Pharmacy and Biomedical Sci

Abstract

Certain cells contain small sacs or ‘vesicles‘ filled with important cargo, such as hormones or neurotransmitters. Appropriate stimuli trigger the fusion of these vesicles with the cell membrane, resulting in cargo secretion from the cell. A large number of proteins are required for membrane fusion, and it is essential to determine how these various proteins are regulated. Interestingly, we have shown that the attachment of palmitate (a fatty acid) to certain proteins modifies their localisation in the cell membrane and regulates their ability to support membrane fusion. This proposal will precisely determine how palmitate addition regulates the activity of proteins that mediate membrane fusion, and identify whether dynamic addition/removal of palmitate is important for fusion activity. Furthermore, we will determine how palmitate regulates the sorting of proteins to specific locations in the cell. These analyses will provide valuable and novel information on the regulation of membrane fusion by palmitate, and will also serve as an important paradigm to understand how palmitate regulates the sorting and membrane distribution of proteins in general. The results generated from this study will hopefully contribute to the design of treatments for conditions such as epilepsy and other brain disorders, and metabolic disorders such as diabetes.

Technical Summary

The fusion of intracellular secretory vesicles with the plasma membrane (exocytosis) mediates cellular secretion of essential molecules such as neurotransmitters and hormones. Exocytosis in neuronal cells is dependent upon the interaction of the plasma membrane SNAREs, SNAP25 and syntaxin 1A, with the vesicle SNARE, VAMP2. Indeed, the formation of this ‘SNARE complex‘ may directly catalyze membrane fusion. A large number of other proteins function in exocytosis, including the chaperone cysteine-string protein (CSP). Despite a good understanding of the proteins and protein-protein interactions that mediate exocytosis, far less is known about how lipid modifications, such as protein palmitoylation, regulate this process.
We identified a relationship between the number of potential palmitoylation sites in SNAP25, or its ubiquitous homologue SNAP23, and the extent of exocytosis. These effects of palmitoylation on exocytosis may be mediated by compartmentalisation of SNAP25/23 into specific micro-domains of the plasma membrane. To extend upon these novel observations, we will utilise mass spectrometry to analyze the extent of SNAP25/23 palmitoylation, and whether differentially palmitoylated pools of these proteins are present in cells. Immunogold labelling will then be used to precisely map the micro-localisation of SNAP25/23 at the plasma membrane, and determine how this is modulated by palmitoylation. Furthermore, we will examine the activity-dependent regulation of SNAP25/23 palmitoylation, and of other cellular proteins, and determine the importance of this for exocytosis. Finally, total internal reflection microscopy and amperometry will be used to pinpoint the aspect(s) of exocytosis that is modulated by specific SNAP25/23 cysteine mutants or palmitoylating enzymes.
In addition to regulating micro-localization and function of SNAP25/23, we propose that palmitoylation also regulates the intracellular sorting of SNAP25 and CSP. Indeed, recent work has highlighted an important role for palmitoylation in the intracellular trafficking of certain proteins. However, little is known about how palmitoylated proteins that lack identifiable primary membrane targeting determinants, such as SNAP25 and CSP, are trafficked within the cell. To address this, we will use a combination of confocal imaging, fractionation and inhibitor studies to identify the intracellular trafficking pathways of SNAP25 and CSP, and determine how trafficking and correct sorting is regulated by palmitoylation. In addition, we will determine how palmitoylation code regulates correct intracellular sorting to specific pre/post-synaptic sites in neurons. Finally, the role of palmitoylation in regulating the exocytic function of CSP will be tested.
Overall, these analyses will provide novel data on the role of palmitoylation in regulating protein trafficking, micro-localization and function.

Publications

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Bournazos S (2009) Association of FcgammaRIIa (CD32a) with lipid rafts regulates ligand binding activity. in Journal of immunology (Baltimore, Md. : 1950)

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Chamberlain LH (2013) Palmitoylation and the trafficking of peripheral membrane proteins. in Biochemical Society transactions

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Greaves J (2011) DHHC palmitoyl transferases: substrate interactions and (patho)physiology. in Trends in biochemical sciences

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Greaves J (2010) Regulation of SNAP-25 trafficking and function by palmitoylation. in Biochemical Society transactions

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Greaves J (2007) Palmitoylation-dependent protein sorting. in The Journal of cell biology

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Greaves J (2010) S-acylation by the DHHC protein family. in Biochemical Society transactions

 
Description Analysis of the substrate network and neurodevelopmental functions of the intellectual disability enzyme, zDHHC9
Amount £472,115 (GBP)
Funding ID MR/S011080/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 04/2022
 
Description British Heart Foundation Project Grant
Amount £249,000 (GBP)
Funding ID 29782. CRM:0001933 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2013 
End 12/2015
 
Description Diabetes UK studentship
Amount £82,700 (GBP)
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description MRC project grant
Amount £543,378 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2011 
End 07/2014
 
Description Responsive mode
Amount £460,841 (GBP)
Funding ID BB/L022087/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 09/2017
 
Description Responsive mode research grants
Amount £405,771 (GBP)
Funding ID MR/R011842/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 05/2021
 
Description Wellcome Trust project grant
Amount £264,655 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 06/2015
 
Description responsive mode
Amount £396,454 (GBP)
Funding ID BB/J006432/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2012 
End 11/2015
 
Title Azide and alkyne derivatives of fatty acids for analysis of S-acylation by click chemistry 
Description Azide/alkyne derivatives of different fatty acids have been developed to assay the fatty acid selectivity of enzymes involves in S-acylation. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact manuscripts in preparation 
 
Title CSP Isoform Antibodies 
Description Cysteine-string protein (CSP) plays an essential neuroprotective function in the brain. Recently, two novel isoforms (CSPb and CSPg) were identified. We have successfully generated isoform-specific antibodies against these proteins. 
Type Of Material Antibody 
Year Produced 2012 
Provided To Others? Yes  
Impact manuscript published: 20499929 
 
Title DHHC2 
Description Novel antibodies against DHHC2 have been generated in rabbits 
Type Of Material Antibody 
Year Produced 2012 
Provided To Others? Yes  
Impact manuscript published: 21471008 
 
Title SNAP25a and SNAP25b antibodies 
Description SNAP25a and SNAP25b play essential but non-overlapping functions in brain physiology. The proteins are highly similar at the amino acid level and several research groups had failed to develop specific antibodies. We have now generated and characterised antibodies that specifically recognise SNAP25a and SNAP25b. These tools will allow a careful assessment of the gross localisation and microlocalisation of these proteins in the brain. 
Type Of Material Antibody 
Year Produced 2012 
Provided To Others? Yes  
Impact manuscript published: 21526988 
 
Description BDSRA application 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Prepared a grant application for submission based on previous work from the group on DNAJC5
Collaborator Contribution Provided expertise to support application
Impact biochemistry, cell biology and in vivo biology
Start Year 2013
 
Description DHHC substrate specificity 
Organisation National Institute for Physiological Sciences
Country Japan 
Sector Academic/University 
PI Contribution Designed assays to map the substrate specificities of this important enzyme family.
Collaborator Contribution Supply of a cloned DNA encoding a family of 23 DHHC palmitoyl transferases. These have been essential for 2 recent publications.
Impact PMID: 18596047 PMID: 19158383
Start Year 2006
 
Description GM1 Antibodies 
Organisation University of Glasgow
Department Institute of Biomedical and Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in the analysis of lipid rafts.
Collaborator Contribution Shared publication of a mansucript.
Impact PMID: 19221437
Start Year 2007
 
Description Neuronal Ceroid Lipofuscinosis 
Organisation Washington University in St Louis
Department Department of Psychiatry
Country United States 
Sector Academic/University 
PI Contribution My research team was the major partner in this research collaboration. We identified a novel interplay between genetic mutations and post-translational modifications in the induction of protein aggregation linked with neurodegeneration. We initiated interaction with the partner to obtain human brain samples to further this investigation.
Collaborator Contribution Supply of human brain tissue from patients with neuronal ceroid lipofuscinosis.
Impact Greaves J., Lemonidis K., Gorleku O.A., Cruchaga C., Grefen C., and Chamberlain L.H. (2012). Palmitoylation-induced aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis. Journal of Biological Chemistry 2012 Aug 19.
Start Year 2011
 
Description Raft Association of CD32a 
Organisation University of Edinburgh
Department MRC Centre for Inflammation Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in the analysis of protein palmitoylation and targeting of proteins to lipid rafts. Design of experiments and drafting of mansucript.
Collaborator Contribution Joint publication of a manuscript on palmitoylation and raft targeting of CD32a.
Impact PMID: 19494328
Start Year 2008
 
Description Regulation of Potassium Channels by Palmitoylation 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in the use of radiolabelling to detect palmitoylation of BK channels. Contributed to experimental design and drafting of manuscript.
Collaborator Contribution Shared publication on regulation of BK potassium channels by palmitoylation.
Impact PMID: 19098106
Start Year 2008
 
Description SNARE Protein Trafficking 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom 
Sector Academic/University 
PI Contribution Shared expertise in subject area and helped with design of experiments
Collaborator Contribution This joint study resulted in a publication describing how SNARE proteins traffic to the plasma membrane and complementing our other independent studies in this area (i.e. PMID: 19158383 ).
Impact PMID: 18057031
Start Year 2007
 
Description Tetherin 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed palmitoylation assays on tetherin proteins
Collaborator Contribution Performed assays looking at raft association and localisation of tetherin proteins
Impact PMC3828773
Start Year 2012
 
Description partnership application 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I have established a research alliance between scientists interested in protein palmitoylation. The aim of this alliance is to establish and develop techniques and reagents to advance the study of palmitoylation. A preliminary application for a partnership grant was put to the MRC, and we have been invited to submit a full application for funding.
Collaborator Contribution The research partners were involved in drafting a full application for MRC funding.
Impact none yet
Start Year 2010
 
Description partnership application 
Organisation University of Glasgow
Department Institute of Molecular Cell and Systems Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution I have established a research alliance between scientists interested in protein palmitoylation. The aim of this alliance is to establish and develop techniques and reagents to advance the study of palmitoylation. A preliminary application for a partnership grant was put to the MRC, and we have been invited to submit a full application for funding.
Collaborator Contribution The research partners were involved in drafting a full application for MRC funding.
Impact none yet
Start Year 2010
 
Description partnership in analysis of zDHHC9 function in neuronal physiology 
Organisation University of British Columbia
Country Canada 
Sector Academic/University 
PI Contribution Back-crossing and supply of ZDHHC9 knockout mice
Collaborator Contribution Analysis of neuronal function in knockout mice.
Impact In preparation
Start Year 2017
 
Description kickstart 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact A small group (5-6 students) from low-achieving schools. We discussed with the students our work and science in general, encouraging them to apply for University places.

none
Year(s) Of Engagement Activity 2009,2010
 
Description school science project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 12 school pupils (age 15-16) and their teacher visited our laboratory to learn more about our research related to neurodegeneration. There was both a theoretical and a practical component. Several pupils reported an interest in pursuing a higher degree in biology-related subjects and this was reinforced by the visit. The school has also reported that they would be interested in participating in this event again.
Year(s) Of Engagement Activity 2016