Molecular dissection of DHHC protein targeting and its importance for post-synaptic palmitoylation dynamics
Lead Research Organisation:
University of Strathclyde
Department Name: Inst of Pharmacy and Biomedical Sci
Abstract
Genes present within the DNA of living organisms encode for the production of specific proteins. The thousands of proteins that are produced within a single cell interact to drive a multitude of pathways, such as cell growth and division. Protein modifications can enhance protein diversity beyond that encoded at the DNA level. For example, many proteins are modified by the attachment of the fatty acid palmitate, a process termed palmitoylation.
Communication between neurons, specialised cells in the brain, underlies every movement, thought and sensation; this neuronal communication occurs at specialised sites termed synapses. Palmitoylation of several proteins that are essential for neuronal communication mediates their targeting to synapses; modulating the extent of this targeting affects synaptic communication. It is well established that changes in synaptic communication are important for events such as learning and memory. Despite the importance of palmitoylation for normal synaptic function, there is very little known about how the enzymes that mediate palmitoylation reactions are regulated in neurons. Recent work identified a family of 24 'DHHC' proteins that are responsible for essentially all cellular palmitoylation activity. The importance of DHHC proteins for normal brain function is highlighted by work linking genetic mutations in these proteins with schizophrenia and mental retardation.
This research project will focus on DHHC2, which is one of the most highly expressed DHHC proteins in brain. Furthermore, DHHC2 is targeted to synaptic regions, where it has been shown to palmitoylate a protein called PSD95; this protein plays an important role in stabilising neurotransmitter receptors and is therefore essential for synaptic communication. Palmitoylation of PSD95 by DHHC2 leads to an increase in synaptic targeting of PSD95, which in turn affects synaptic dynamics of neurotransmitter receptors. In this project, we will investigate the mechanisms that regulate DHHC2 movement to synapses where it palmitoylates PSD95. Furthermore, we will examine how interfering with the mobility of DHHC2 at synapses impacts neuronal communication. This work will play a major role in delineating how palmitoylation dynamics are regulated at synapses and the downstream effects of this regulation on neurotransmitter receptor dynamics.
There is currently much interest in DHHC proteins as potential drug targets for the treatment of diverse human disorders, thus delineating the mechanisms whereby specific DHHC proteins regulate cellular dynamics is of major importance.
Communication between neurons, specialised cells in the brain, underlies every movement, thought and sensation; this neuronal communication occurs at specialised sites termed synapses. Palmitoylation of several proteins that are essential for neuronal communication mediates their targeting to synapses; modulating the extent of this targeting affects synaptic communication. It is well established that changes in synaptic communication are important for events such as learning and memory. Despite the importance of palmitoylation for normal synaptic function, there is very little known about how the enzymes that mediate palmitoylation reactions are regulated in neurons. Recent work identified a family of 24 'DHHC' proteins that are responsible for essentially all cellular palmitoylation activity. The importance of DHHC proteins for normal brain function is highlighted by work linking genetic mutations in these proteins with schizophrenia and mental retardation.
This research project will focus on DHHC2, which is one of the most highly expressed DHHC proteins in brain. Furthermore, DHHC2 is targeted to synaptic regions, where it has been shown to palmitoylate a protein called PSD95; this protein plays an important role in stabilising neurotransmitter receptors and is therefore essential for synaptic communication. Palmitoylation of PSD95 by DHHC2 leads to an increase in synaptic targeting of PSD95, which in turn affects synaptic dynamics of neurotransmitter receptors. In this project, we will investigate the mechanisms that regulate DHHC2 movement to synapses where it palmitoylates PSD95. Furthermore, we will examine how interfering with the mobility of DHHC2 at synapses impacts neuronal communication. This work will play a major role in delineating how palmitoylation dynamics are regulated at synapses and the downstream effects of this regulation on neurotransmitter receptor dynamics.
There is currently much interest in DHHC proteins as potential drug targets for the treatment of diverse human disorders, thus delineating the mechanisms whereby specific DHHC proteins regulate cellular dynamics is of major importance.
Technical Summary
Palmitoylation, a reversible protein modification, is catalysed in mammalian cells by a family of 24 'DHHC' proteins. Hundreds of neuronal proteins are regulated by palmitoylation, for example, clustering and trafficking of proteins at the post-synaptic density (PSD) can be regulated by activity-dependent changes in their palmitoylation status. Recent work reported that DHHC2 is one of the major DHHC proteins expressed in neurons and neuroendocrince cells. DHHC2 was also identified as the key DHHC protein responsible for activity-dependent palmitoylation of PSD95, and hence is central to the regulation of synaptic targeting/dynamics of both PSD95 and AMPA receptors.
Our recent published work has shown that DHHC2 is targeted to a dynamic cycling pathway operating between the plasma membrane and recycling endosomes in neuroendocrine cells; targeting of DHHC2 into this cycling pathway is dependent upon its cytoplasmic C-terminal tail. The over-arching hypothesis that the current project will test is that cycling of DHHC2 through this pathway is a fundamental mechanism to regulate PSD95 palmitoylation, and subsequent regulation of AMPA receptor dynamics. The described study will build upon our recently published work and pinpoint the precise signals and cellular factors that mediate post-Golgi sorting and dynamic cycling of DHHC2. Furthermore, we will define the importance of DHHC2 cycling in regulating palmitoylation of PSD95, and examine how this is affected by synaptic activity. The downstream effects of DHHC2 cycling on AMPA receptor dynamics will also be investigated. Overall, this work will provide a detailed description of the signals and mechanisms underlying post-Golgi sorting and membrane cycling of DHHC2, and will determine the importance of this cycling pathway for activity-dependent regulation of PSD95 palmitoylation and AMPA receptor dynamics.
Our recent published work has shown that DHHC2 is targeted to a dynamic cycling pathway operating between the plasma membrane and recycling endosomes in neuroendocrine cells; targeting of DHHC2 into this cycling pathway is dependent upon its cytoplasmic C-terminal tail. The over-arching hypothesis that the current project will test is that cycling of DHHC2 through this pathway is a fundamental mechanism to regulate PSD95 palmitoylation, and subsequent regulation of AMPA receptor dynamics. The described study will build upon our recently published work and pinpoint the precise signals and cellular factors that mediate post-Golgi sorting and dynamic cycling of DHHC2. Furthermore, we will define the importance of DHHC2 cycling in regulating palmitoylation of PSD95, and examine how this is affected by synaptic activity. The downstream effects of DHHC2 cycling on AMPA receptor dynamics will also be investigated. Overall, this work will provide a detailed description of the signals and mechanisms underlying post-Golgi sorting and membrane cycling of DHHC2, and will determine the importance of this cycling pathway for activity-dependent regulation of PSD95 palmitoylation and AMPA receptor dynamics.
Planned Impact
This research will contribute in the longer term (next ten years) to improving the health and well-being of the wider community. The impact of this work to health and well-being will be through the delineation of novel fundamental cellular pathways that regulate neuronal function. To develop the most specific and effective treatments for medical conditions and to ensure life-long health requires a detailed understanding of cell and tissue physiology in health and disease. Palmitoylation and DHHC proteins are relevant to many cellular pathways in every cell type, and thus the results will have wide and general relevance to our understanding of normal physiological processes. Indeed changes in palmitoylation have been linked with many important clinical conditions: mutations in DHHC proteins are associated with disorders, including schizophrenia, mental retardation and cancer. Thus, progress made in understanding DHHC protein regulation is likely to have a direct impact upon future strategies and treatments for a range of important clinical conditions. Indeed, there is significant interest in DHHC proteins as drug targets, and thus excellent basic research that unravels the (as yet poorly defined) functions of these proteins is essential.
The researchers employed on this project will also benefit from developing expertise in a wide-range of advanced scientific techniques, including fluorescence recovery after photo-bleaching and photoactivation techniques. Developing this expertise is important to ensure that we have the skills base to maintain the rapid progress and standing of UK science. Furthermore, the quantitative and problem-solving skills developed by employees will be widely applicable to a range of different professions outside of laboratory-based research.
The researchers employed on this project will also benefit from developing expertise in a wide-range of advanced scientific techniques, including fluorescence recovery after photo-bleaching and photoactivation techniques. Developing this expertise is important to ensure that we have the skills base to maintain the rapid progress and standing of UK science. Furthermore, the quantitative and problem-solving skills developed by employees will be widely applicable to a range of different professions outside of laboratory-based research.
Publications
Lemonidis K
(2017)
Peptide array-based screening reveals a large number of proteins interacting with the ankyrin-repeat domain of the zDHHC17 S-acyltransferase.
in The Journal of biological chemistry
Salaun C
(2017)
The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.
in Molecular and cellular neurosciences
Description | Brain activities are mediated via interactions between neuronal cells, which use neurotransmitter chemicals to communicate with each other at contact sites called synapses. Changes in the level of neurotransmitter receptors at synapses affects the strength of connections between different neurons and modulates synaptic "plasticity", which is associated with processes such as learning and memory. Recent work identified the enzyme zDHHC2 as a potential novel regulator of synaptic plasticity. In particular, dynamic changes in the localisation of this enzyme are linked to the modulation of neurotransmitter receptor expression at synapses. In this project, we have identified novel signals within zDHHC2 that regulate enzyme localisation and also identified a new mechanism potentially controlling dynamic changes in zDHHC2 localisation in response to different states of neuronal activity. We are currently using zDHHC2 mutants as tools to dissect how zDHHC2 localisation affects neurotransmitter receptor expression and synaptic function. These studies will offer new insight into mechanisms regulating synaptic plasticity and associated physiological processes in the brain. |
Exploitation Route | The work undertaken in this project provides novel information about pathways involved in synaptic plasticity. In particular, the research gives new insight into how neuronal activity and protein trafficking at the synapse might be linked. It also highlights the potential for dynamic changes in the localisation of protein palmitoylation enzymes to impact synaptic function. There is significant interest from other investigators in molecular mechanisms regulating synaptic function and plasticity and we will publish the work in peer-reviewed scientific journals and present our findings at conferences to ensure that these new discoveries reach these potential academic beneficiaries. In addition, changes in palmitoylation are linked to a number of neurological disorders such as intellectual disability, neuronal ceroid lipofuscinosis, and Huntington's disease. Novel information on the mechanisms regulating localisation and function of palmitoylation enzymes can be utilised to identify new therapeutic targets and sites for intervention. We will ensure that our research in this area reaches potential beneficiaries in the healthcare and pharmaceutical sector by engaging with representatives at organised events and by highlighting our findings in flyers and brochures targeted at these groups. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | The findings from this grant contributed to investment by a major pharmaceutical company (Ono Pharma Ltd). The company supported a 2.5 year grant that developed high-throughput screens and novel inhibitors of S-acylation. These inhibitors can now be further developed and have potential to be used as novel therapeutics. |
First Year Of Impact | 2018 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
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 | Funding from pharmaceutical company |
Amount | £345,000 (GBP) |
Organisation | Ono Pharmaceutical |
Sector | Private |
Country | Japan |
Start | 06/2015 |
End | 12/2018 |
Description | Porject grant |
Amount | £62,000 (GBP) |
Organisation | Ono Pharmaceutical |
Sector | Private |
Country | Japan |
Start | 09/2016 |
End | 03/2017 |
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 |
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 | 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 |
Description | ZDHHC Knockout Mice |
Organisation | University of Strathclyde |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provide expertise in DHHC enzyme biology and protein palmitoylation. |
Collaborator Contribution | Expertise in behavioural testing |
Impact | Project grant application under consideration at MRC. Multidisciplinary project using Biochemistry, Cell Biology, and in vivo biology. |
Start Year | 2013 |
Description | Art-Science collaboration- Huntington's Disease |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Several hundred people visited to see the outputs that were generated by 10 artists who participated with us on a collaboration focused on Huntington's disease. Members of the public reported an increased understanding of current research focused on Huntington' disease. |
Year(s) Of Engagement Activity | 2014 |
URL | http://chamberlainlab.co.uk |
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 |