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    The Importance of S-Palmitoylation in Neurodevelopment

     

    Summary

    What is S-palmitoylation?
    S-palmitoylation is a reversible post-translational protein modification process, whereby palmitic acid forms a thioester bond with the sulphur atom of the cysteine residue of a protein.

    How is this relevant in neurodevelopment?
    The S-palmitoylation of the postsynaptic scaffolding protein PSD-95 influences synaptic strength and plasticity, due to its involvement in the regulation of postsynaptic glutamate expression. Dysregulation of PSD-95 S-palmitoylation is implicated in Huntington’s Disease, whereby altered glutamate expression contributes to disease pathology.

    What if this applies to my research?
    The CAPTUREomeTM S-Palmitoylated Protein Kit can be used to determine whether this fatty acid modification is occurring in your experimental systems.

    Click below to find out more!

     

    Figure 1

    A simple animation showing palmitic acid thioester bonded to the sulphur atom in the side chain of a cysteine residue

     

    INTRODUCTION

    S-palmitoylation is the post-translational process by which a thioester bond reversibly attaches a fatty acid to a target protein. S-palmitoylation is considered to be especially vital in the central nervous system (CNS) – in fact, over 250 proteins expressed in neurones have been identified as S-palmitoylated proteins, and palmitic acid is the fatty acid in the highest concentration in the brain1,2.

    Examples of S-palmitoylated proteins or protein subunits in the CNS include α subunits of Na+ and β2a subunits of Ca2+ channels , serotonin 1B and 4A receptors, D1 and D2L dopamine receptors, α7 subunit of nicotinic and γ2 subunits of GABAA receptors, as well as various scaffolding proteins , GTPases, chaperones and myelin- associated proteins1,3.

    In cells, S-palmitoylation often alters how the protein interacts with the phospholipid bilayer – as the attached fatty acid often acts as a hydrophobic membrane anchor – regulating protein-protein interactions, stability and expression, and localisation4.This suggests that, in neuronal cells, the roles of S-palmitoylation may include regulating neurotransmitter receptor expression5 , synaptic strength6 and action potential propagation7. One notable palmitoylated protein is the postsynaptic scaffolding protein postsynaptic density-95 (PSD-95)8.

     
    1. Location, structure and function of PSD-95

    In the 1990s, metabolic labelling of brain slices and cultured cells identified PSD-95 as one of the major palmitoylated proteins in the CNS8. PSD-95 is a scaffolding protein which anchors various proteins, including glutamate receptors, to the postsynaptic membrane at excitatory synapses8. Importantly, S-palmitoylation has been identified to regulate PSD-95, and therefore the S-palmitoylation of this protein is key in influencing both glutamate receptor expression and synaptic plasticity.

    S-palmitoylation regulates PSD-95 activity, which influences both glutamate receptor expression and synaptic plasticity.

     
     

    PSD-95, a member of the membrane associated guanylate kinase family (MAGUK), is the most abundant postsynaptic scaffolding protein9,10 and is predominately expressed in the postsynaptic density – a protein dense specialisation of the postsynaptic cell’s membrane, involved in the modulation of synaptic adhesion, receptor sensitivity and receptor localisation11 as shown in figure 2.

    This specialisation contains various different proteins, including scaffolding proteins, glutamate receptors and cytoplasmic signalling molecules12. Specifically, postsynaptic densities usually contain between 200 and 300 copies of PSD-959.

    As with other members of the PSD-95 related MAGUK family, PSD-95 expresses three conserved PDZ domains and one Src homology 3-guanylate kinase (SH3-GK module)9. The PDZ domains are 80-90 residues in length, and consist of six β-strands and two α-helices13. The β -strands form a partially accessible barrel, and the sides of these are capped by each α-helix13. PDZ domains recognize 5-7 residues at the C-terminal end of their target proteins (e.g. glutamate receptors), which bind to the extended groove shaped by the β-strands and α-helices13. Similarly, SH3-GK modules play a role in protein-protein interaction, and are formed by linker regions connecting SH3 and GK14.

    It is vitally important to understand PSD-95 S-palmitoylation as, within the postsynaptic density, S-palmitoylation regulates the PSD-95 function of anchoring target proteins to the postsynaptic membrane15. PSD-95 is thought to be responsible for targeting and stabilising the expression of clusters of neuroligin, NMDA receptors (NMDAR), AMPA receptors (AMPAR), potassium channels and other scaffolding proteins in the postsynaptic density, therefore contributing to neurotransmission  and synaptic strength9, 16. These functional effects contribute to synaptogenesis and synaptic plasticity – hence the need for a total understanding of S-palmitoylation regulation.

    PSD-95 functional effects contribute to synaptogenesis and synaptic plasticity – hence the need for a total understanding of S-palmitoylation regulation.

    2. S-palmitoylation and depalmitoylation in normal neurodevelopment

    A plethora of experiments have identified PSD-95 as a palmitoylated protein. As mentioned, the postsynaptic clustering of PSD-95 is crucial to its function of regulating the expression and clustering of target proteins in the postsynaptic membrane, and research using green fluorescent protein- (GFP-) labelled PSD-95, expressed in cultured hippocampal neurones, showed that this clustering relied on S-palmitoylation of the protein on cysteine residue(s) in the N-terminal domain, the first two PDZ domains and a C-terminal targeting motif17.

    When PSD-95 anchors target proteins to the membrane, it is in its palmitoylated state. Protein palmitoyltransferases (PATs) catalyse the addition of fatty acids to target proteins, and several specific PSD-95 PATs (P-PATs) have been identified, including DHHC2, DHHC3, DHHC7, DHHC15, DHHC1718, 19, 20. These P-PATs have been found to attach palmitic acid to cysteines 3 and 5 of PDS-95 - this is induced by the consensus hydrophobic sequence of five consecutive NH2-terminal residues of PSD-9515. Importantly, S-palmitoylation is reversible. Protein thioesterases (PTTs) catalyse depalmitoylation by hydrolysing the thioester bond between the fatty acid and target protein – these enzymes are thought to belong to the metabolic serine hydrolase superfamily10. The exact enzymes responsible for PSD-95 depalmitoylation have not been identified. However, hexadecylfluorophosphonates (HDFP) are inhibitors of serine hydrolases which mostly have an α/β-hydrolase catalytic domain (ABHD), and HDFPs have been found to block the depalmitoylation of the palmitoylated proteins Gα, Ras GTPases, and MAGUK proteins10. This suggests that ABHD-containing proteins may function as depalmitoylating enzymes for PSD-9510 – specifically, S-palmitoylation of PSD-95 was selectively reduced in the presence of ABHD17, suggesting that ABHD17 depalmitoylates PSD-9510. The palmitate cycle of PSD-95 (the process of S-palmitoylation and depalmitoylation) is regulated by glutamate receptor activation15. Activation of glutamate receptors, such as AMPAR or NMDARs, results in depalmitoylation of PSD-9515. The loss of palmitic acid from PSD-95 results in PSD-95 dissociation from the postsynaptic density, destabilising the expression of glutamate receptors and promoting receptor endocytosis and internalisation, as shown in figure 3, down-regulating synaptic strength and G protein signalling5. Evidence for the above was also shown by Yokoi et al. (2016), who found that ABHD17 reduced synaptic clustering of AMPARs.

     

    As a result, the glutamate-induced palmitoylation cycle of PSD-95 is vitally important in synaptic strength and plasticity.

    3. PSD-95 S-palmitoylation in Huntington’s Disease

    Huntington’s Disease (HD) is a neurodegenerative disorder caused by an increase in CAG repeats (the codon for glutamine) in the HTT gene (located at position 4p16.3), which encodes the large protein (>350 kDa) huntingtin (HTT)21. Individuals with wild type (WT) HTT have between 9 and 36 CAG repeats in exon 1, whilst individuals with HD have 37 copies or more, resulting in increased glutamine near the N terminus of the encoded protein22, 21. The exact physiological function of HTT is unknown, but it is thought to be involved in neuronal signalling, protein transportation and expression and protecting cells from apoptosis21. Mutant HTT (mHTT) results in a toxic function of the huntingtin protein, glutamate-mediated excitotoxicity, and altered cellular and synaptic function – most prominent in the GABAergic spiny projection neurons (SNPs) in the striatum and in the hippocampus – which results in apoptosis and overt motor symptoms23, 24.

    Excess PSD-95 S-palmitoylation may cause NMDAR excitotoxicity
    In the hippocampus of healthy individuals, HTT in the cytoplasm binds to PSD-95 via its SH3 domain, trapping it and preventing it being localised to the postsynaptic membrane23. Research suggests that the glutamine expansion in mHTT reduces binding to PSD-95, increasing the levels of ‘free’ PSD-95 localised to the postsynaptic density. As a result, more PSD-95 is available for S-palmitoylation at the postsynaptic membrane, and these increased levels of S-palmitoylated PSD-95 promotes the anchoring of excessive levels of NMDARs to the membrane, resulting in excitotoxicity, which is associated with HD pathology23.

    Glutamine expansion in mHTT reduces binding to PSD-95, increasing free PSD-95, promoting the anchoring of excessive levels of NMDARs to the postsynaptic membrane.

    However, PSD-95-induced overexpression of NMDARs is not the only pathological pathway proposed to contribute to disease development.

    Reduced PSD-95 S-palmitoylation results in AMPA internalisation
    Both HTT and PSD-95 are S-palmitoylated by DHHC17, which is also known as HIP14, a neuronal PAT localised to the Golgi apparatus and cytoplasmic vesicles20. HIP14 palmitoylates HTT at Cys21421 and is regulated by normal HTT expression – the presence of HTT promotes the S-palmitoylation activity of HIP14, whilst the presence of mHTT reduces it20.

    Consequently, it’s said that the interaction between HIP14 and HTT is inversely correlated with the number of glutamine repeats in HTT21.

    Therefore, the presence of mHTT in HD results in reduced HIP14 PAT activity in the striatum, and reduced S-palmitoylation of PSD-9520. As S-palmitoylation of PSD-95 promotes AMPA expression on the postsynaptic membrane, it is concluded that reduced S-palmitoylation of PSD-95 promotes the internalisation of AMPA receptors, which may contribute to the behavioural and motor symptoms associated with HD25. This theory is supported by further research which found that AMPA expression was reduced in the putamen of the striatum of individuals with HD25.

    Research detail
    HIP14, which interacts with HTT via it’s ankyrin repeat domain, S-palmitoylates HTT at Cys21421 and is regulated by WT HTT expression. In HD, the expression of mHTT results in the reduced activity of HIP14 – potentially increasing levels of depalmitoylated HTT and PSD-95. However, research has found that HTT S-palmitoylation is not reduced in HIP14-/- knockout mice vs. WT mice20. Most likely, the paralog HIP14L PAT is able to compensate for the loss of HIP14, and S-palmitoylates HTT in the place of HIP1420, suggesting that reduced S-palmitoylation of HTT by HIP14 does not contribute to HD.

    However, PSD-95 S-palmitoylation was significantly reduced in HIP14-/- 20. As the HIP14-/- phenotype shows both reduced PSD-95 S-palmitoylation and also several hallmarks of HD – e.g. decreased striatal volume and striatal medium spiny neurons (MSN) – it is thought that mHTT results in reduced S-palmitoylation of PSD-95, and this may be part of the pathogenesis of HD20. Moreover, further research observed that the level of AMPARs expressed on postsynaptic membranes is reduced in the putamen of the striatum in individuals with HD – consequently, it is predicted that reduced S-palmitoylation of PSD-95 in the presence of mHTT results in the internalisation of AMPAs, which may contribute to the behavioural and motor symptoms associated with HD25.

    Key points

    • Among various roles, PSD-95 is key in regulating normal glutamate receptor expression at the postsynaptic membrane, via its palmitoylation cycle
    • Dysregulation of PSD-95 S-palmitoylation results in defective neurotransmission and alterations in synaptic strength
    • In Huntington’s Disease, mutant huntingtin reduces the activity of the palmitoyltransferase HIP14, resulting in decreased S-palmitoylation of PSD-95 and reduced AMPA expression in the striatum, which may contribute to the behavioural and motor defects associated with HD.

    Author: Hilary Robinson

    References

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