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    The Importance of S-Palmitoylation in Cardiology

     

    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 cardiology?
    S-Palmitoylation regulates the activity of various ion channels and transporters in the heart. Abnormalities in channel regulation are associated with cardiac disease. S-palmitoylation has also been shown to regulate proteins involved in normal Ca2+ cycling.

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

     

    INTRODUCTION

    Considered one of the most common post-translational modifications of proteins with fatty acid moieties, palmitoylation modulates the function of a range of proteins, including ion channels and transporters. Channel activity can be modulated by changes in the environment or protein conformation, due to modification of cysteine residues with palmitate.

    The aetiology of heart disease is still an area of active research, important because its a leading cause of mortality and morbidity worldwide. It is, however, widely accepted that ion channels are critical in the propagation of action potentials, which occur as a result of an electrical stimulus, initiating the contraction of the heart. Many of the proteins involved in the coordinated opening and closing of ion channels are S-palmitoylated, and this modification is critical to their function. Defects in these channels and transporters can lead to cardiac arrhythmias and heart failure2, and alterations via S-palmitoylation can increase or decrease these risks.

    In addition to its functional significance, S-palmitoylation has also demonstrated a structural role in the development of the heart. Ablphilin 2 (Aph2) is a protein recently discovered to caltalyse S-palmitoylation with a critical role in embryonic heart development and cardiomyopathy3. This article will discuss the role of S-palmitoylation as a regulator of both heart function and structure.

    Function

    1. S-Palmitoylation of the Na+/K+ Pump

    The Na+/K+ pump utilises the hydrolysis of ATP to pump sodium out and potassium into the cell, against their respective concentration gradients, with a stoichiometry of 3Na+:2K+. The Na+/K+ pump is a multi-subunit enzyme composed of at least two subunits, α and β. The α subunit spans the membrane 10 times and contains binding sites for Na+, K+ and ATP, forming the catalytic core of the transporter. The β subunit spans the membrane once and plays an important role in trafficking to the plasma membrane4,5. In cardiac myocytes, the ion transport regulator phospholemman (FXYD1) associates with the Na+/K+ pump, and provides a link between the pump and cardiac kinases.6 Phospholemman (PLM) is abundantly found in the sarcolemma and is the primary phosphorylation site for protein kinase A (PKA) and protein kinase C (PKC).

    PLM can be S-palmitoylated at two intracellular cysteine residues, Cys­40 and Cys42, which are conserved among species7. S-palmitoylation of PLM regulates the Na+ pump through changes in phospholipid interactions, by increasing PLM half-life and inhibiting Na+ pump activity, in contrast to its phosphorylation which activates the Na+ pump.8 It has been discovered that PLM is a substrate for DHHC5, a palmitoyl acyl-transferase (PAT), which catalyses the formation of the thioester bond in S-palmitoylation. Not only can DHHC5 inhibit the Na+ pump via the palmitoylation of PLM it can also do so via the MEND pathway.9

    Although S-palmitoylation has no effect on the cell surface expression of the pump, DHHC5 can regulate pump density at the cell surface via massive endocytosis (MEND).9 MEND describes the rapid internalisation of the cell surface membrane in response to stressors such as Ca2+ overload and the clustering of S-palmitoylated proteins in lipid rafts. MEND also occurs during reperfusion of ischaemic heart muscle, implicating DHHC5 in reperfusion injuries.3,10 The Na+/K+ pump is the primary mechanism of Na+ extrusion in the heart driving the activity of other exchangers, such as NCX, and thus has an indirect control over contractility; therefore, inhibition of the Na+ pump in response to the S-palmitoylation of PLM has functional significance.

    2. S-Palmitoylation of the Na+/Ca2+ Exchanger

    The Na+/Ca2+ exchanger (NCX) plays an important role in Ca2+ homeostasis and contractility. Ca2+ has a pivotal role in excitation-contraction coupling in the heart, and the removal of Ca2+ from ventricular myocytes allows relaxation and refilling during diastole. The NCX1 splice variant is predominantly found in cardiac muscle, 60% of which is described to be palmitoylated.10

    Figure 1. Structure of the NCX, showing the two core binding domains (CBDs) and the region of the XIP.

    It was initially thought to be formed of nine trans-membrane domains within two α repeats, however recent evidence suggests there could be 10 (Figure 1). A large intracellular loop in between TM5 and TM6 contains two core-binding domains (CBDs) which mediate inactivation via Ca2+ binding. The exchanger inhibitory peptide (XIP) is a cationic site also found on this intracellular loop, which too is involved in the activation of NCX. Anionic phospholipids, such as PIP2 can bind to XIP and modulate inactivation.11

    Evidence suggests that S-palmitoylation permits the complete inactivation of NCX1 by increasing XIP function, which opposes activation mediated by PIP2.12 S-palmitoylation is known to take place at a single cysteine (C739) in the large intracellular loop. Mutated forms of NCX1 that lacked C739 did not inactivate in response to PIP2 depletion, re-emphasising the importance of S-palmitoylation for NCX inactivation. S-palmitoylation at the surface membrane promotes MEND during mitochondrial stress by forming lipid-protein domains, which was significantly reduced in NCX1 mutant model.3

    3. S-Palmitoylation of Nav1.5

    Nav1.5 is a voltage gated Na+ channel responsible for the fast depolarisation in ventricular myocytes, and is an important player in regulating cardiac rhythm. Previous studies have shown that sodium channels in the brain are subject to S-palmitoylation, however it has only recently been determined whether the same applies to contractile cells. Palmitoylation of Nav1.5 induces biophysical changes in Na+ current, increasing cell excitability, and is thought to be implicated in cardiac channelopathies.13 Prevention of S-palmitoylation through treatment with the palmitoyltransferase inhibitor 2-Br-palmitate, prevents the spontaneous activity of action potentials. Therefore, it can be concluded that S-palmitoylation of Nav1.5 is vital for the influx of sodium through Nav1.5, and therefore cyclic S-palmitoylation and depalmitoylation of Nav1.5 can regulate cell excitability.

    Research detail
    Pei et al1 incubated cardiac myocytes from rats for 24 hours with 2-Br-plamitate, a palmitoyltransferase inhibitor, resulting in protein depalmitoylation. In the control group 70% of myocytes exhibiting inward sodium current in voltage-clamp recordings showed spontaneous generation of action potentials. Treatment with 2-Br-palmitate resulted in a loss of spontaneous activity in these cells. Cells incubated with excess palmitic acid, exhibited increased spontaneous activity with an increased action potential duration and decreased firing frequency. These results demonstrate the ability of S-palmitoylation of Nav1.5 to act as a regulator of cell excitability. When studying Nav1.5, the authors observed no change in the rate of sodium channel fast inactivation when treated with excess palmitate, however, the steady state inactivation was shifted to more depolarised voltages, enabling a faster recovery from inactivation, therefore increased excitability.

    Abnormalities of Heart Development

    4. Palmitoyl Acyltransferase Aph2

    As aforementioned, S-palmitoylation is catalysed by PAT enzymes. Research conducted by Zhou et al.3 provides evidence that Aph2, initially identified as an interacting protein of nonreceptor tyrosine kinase, is in fact a PAT which utilises phospholamban (PLB) as a substrate. In mammals, 70% of Ca2+ in the cytoplasm is loaded into the sarcoplasmic reticulum (SR) following a contraction via the SR Ca2+ ATPase (SERCA), while the remaining 30% is extruded into the extracellular space via NCX. The balance between SERCA and NCX needs to be carefully regulated in order to prevent contractile abnormalities such as arrhythmogenic delayed after depolarisations (DADs) .11

    SERCA is regulated by PLB, and upon β-adrenergic stimulation, Ser16 of PLB is phosphorylated by PKA, resulting in the loss of its ability to inhibit SERCA. S-palmitoylation of PLB at Cys36 alters its interaction with PKA, regulating its phosphorylation at Ser16. An additional discovery independent of PLB revealed that Aph2-/- mice displayed an array of defects, including enlarged ventricular walls, abnormal nuclear morphology and cardiomyocyte disorganisation, all of which are characteristic features of cardiomyopathy.4

     

    In conclusion, ionic activity is paramount in regulating contractility, and maintaining a healthy heart. Modulation of ion channels and exchangers via S-palmitoylation can lead to changes in conduction and inactivation, resulting in subsequent pathological changes. The identification and modulation of S-palmitoylation has the potential as a therapeutic target for channels implicated in contractile abnormalities. S-palmitoylation mediated by Aph2 is critical in embryonic heart structure and function, and is implicated in the development of cardiomyopathy.

    Key points

    Author: Naomi Abecassis

    References

    1. Pei, Z., Xiao, Y., Meng, J., Hudmon, A., Cummins, T.R., 2016, ‘Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation’, Nature Communications, [Accessed online: 28.01.2017] Available from: http://www.nature.com/articles/ncomms12035
    2. Reilly, L., Howie J., Wypijewski, K., Ashford, M.L.J., Hilgemann, D.W., Fuller, W., 2015, ‘Palmitoylation of the Na+/Ca2+ exchanger cytoplasmic loop controls its inactivation and internalisation during stress signalling’. The FASEB Journal. 29(11), pp.4532-4543, [Accessed online: 28.01.2017]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608915/
    3. Zhou, T., Li, J., Zhao, P., Liu, H., Jia, D., Jia, H., He, L., Cang, Y., Boast, S., Chen, Y.-H., Thibault, H., Scherrer-Crosbie, M., Goff, S.P., Li, B. 2015. ‘Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy’. Proceedings of the National Academy of Sciences. pp.15666-15671. [Acessed online: 02.02.2017]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697436/
    4. Howie, J., Tulloch, L., Shattock, M., Fuller, W. 2013. ‘Regulation of the cardiac Na+ pump by palmitoylation of its catalytic and regulatory subunits’. Biochemical Society transactions. 41(1), pp.95–100, [Accessed online: 28.01.2017]. Available from: http://www.biochemsoctrans.org/content/41/1/95
    5. Purves, D., Augustine, G.J., Fitzpatrick, D., Hall, W.C., LaMantia, A.-S., McNamara, J.O. and White, L.E. 2007. Neuroscience 4e. 4th ed. Sunderland, MA: Sinauer Associates Inc.,U.S, pp 79-82
    6. Fuller, W. and Shattock, M.J. 2006. ‘Phospholemman and the cardiac sodium pump’. Editorials. 99(12), pp.1290–1292. , [Accessed online: 02.02.2017]. Available from: http://circres.ahajournals.org/content/99/12/1290
    7. Pavlovic, D., Fuller, W. and Shattock, M.J. 2013. ‘Novel regulation of cardiac Na+ pump via phospholemman’. Journal of Molecular and Cellular Cardiology. 61, pp.83–93. [Accessed online: 02.02.2017]. Available from: http://www.sciencedirect.com/science/article/pii/S0022282813001703
    8. Tulloch, LB, Shattock, MJ & Fuller, W 2010, 'Phospholemman Palmitoylation: a Novel Means of Sodium Pump Regulation’ Circulation Research, vol 122, no. 21. , [Accessed online: 02.02.2017]. Available from: http://circ.ahajournals.org/content/122/Suppl_21/A21298
    9. Howie, J., Reilly, L., Fraser, N.J., Walker, J.V.M., Wypijewski, K.J., Ashford, M.L.J., Calaghan, S.C., McClafferty, H., Tian, L., Shipston, M.J., Boguslavskyi, A., Shattock, M.J., Fuller, W. 2014. ‘Substrate recognition by the cell surface palmitoyl transferase DHHC5’. Proceedings of the National Academy of Sciences. 111(49), pp.17534–17539, [Accessed online: 10.02.2017]. Available from: http://www.pnas.org/content/111/49/17534.long
    10. Reilly, L., Howie, J., Wypijewski, K., Ashford, M., Hilgemann, D.,Fuller, W. 2015a. ‘Palmitoylation of the Na+/Ca2+ exchanger cytoplasmic loop controls its inactivation and internalization during stress signalling’. FASEB journal . 29(11), pp.4532–43, [Accessed online: 28.01.2017] Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608915/
    11. Liao, J., Li, H., Zeng, W., Sauer, D.B., Belmares, R., Jiang, Y. 2012. ‘Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger’. Science. 335(6069), pp.686–690, [Accessed online: 28.01.2017]. Available from: http://science.sciencemag.org/content/335/6069/686.long
    12. Fuller W, Reilly L, Hilgemann DW. S-palmitoylation and the regulation of NCX1. Channels. 2016;10(2):75-77, [Accessed online: 28.01.2017]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960986/
    13. Pei, Z., Hudmon, A. and Cummins, T.R. 2014. Abstract 20627: Modulation of cardiac sodium channel by Palmitoylation and its association with cardiac disease mutations. Core 4. Heart Rhythm Disorders and Resuscitation Science. 130(Suppl 2), p.20627, . [Accessed online: 02.02.2017]. Available from: http://circ.ahajournals.org/content/130/Suppl_2/A20627

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