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S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

Keith T Woodley, Mark O Collins

Preprint posted on December 06, 2018 https://www.biorxiv.org/content/early/2018/12/06/481861

Let’s stick together! Regulation of cell adhesion by biology’s next top post-translational modification, palmitoylation!

Selected by Abagael Lasseigne

Categories: biochemistry, cell biology

Background:

Palmitoylation is a reversible, post-translational modification where a lipid is attached to cysteine residues to increase the hydrophobicity of a protein. Little is know about the regulation of palmitoyl acyltransferases (PATs), which attach these lipids via their catalytic site. DHHC5 is a human PAT, with palmitoylation sites of its own, that localizes to the plasma membrane and is known to be involved in tumor growth, massive palmitoylation-dependent endocytosis in the heart, and synaptic plasticity. Up until now, it was unclear how DHHC5 was regulated and what precise molecular roles it might be playing.

Key findings:

This paper identified another protein, Golga7b, that interacts with DHHC5 and is required for its membrane localization in a palmitoylation-dependent manner. When expressed alone in cell culture, DHHC5 localizes to the cytoplasm. However, in the presence of Golga7b, it localizes to the plasma membrane but only if Golga7b can be palmitoylated. This suggests that Golga7b palmitoylation may be initiating the formation of a complex with DHHC5 at the cell membrane. The authors next wanted to understand how this complex might be functioning, so, utilizing mass spectrometry, they identified proteins that interacted strongly with the membrane form of DHHC5. Membrane DHHC5 interacted with more proteins overall than the cytoplasmic form, especially those involved in cell adhesion. The authors then showed that cell adhesion is reduced in cells depleted of DHHC5. Finally, the authors showed that a cadherin, desmogelin-2, found to interact with and be palmitoylated by DHHC5, is removed from cell adhesions in the absence of either DHHC5 or Golga7b. This suggests an overall model where a Golga7b/DHHC5 complex, regulated by palmitoylation, may be localizing to the membrane and palmitoylating the appropriate adhesion proteins (such as desmogelin-2) therefore facilitating cell to cell adhesion.

Why I like this preprint:

These results suggest that DHHC5 may be the PAT that is responsible for palmitoylating membrane proteins involved in cell adhesion. I like this preprint because:

1) I study electrical synapses which are a form of gap junction. Cell adhesion at electrical synapses is still poorly understood, but papers such as this that elucidate the regulators of cell adhesion inform my work on how electrical synapses could be developing; and

2) More research is showing the importance of palmitoylation as a post-translational modification. Specifically in neuroscience, palmitoylation of synaptic scaffolding proteins regulates chemical synapse formation and plasticity. Therefore, it is important for us to understand the regulation of PATs responsible for palmitoylation in different circumstances.

Future directions:

The primary future directions are to identify other adhesion proteins interacting with this complex and to test this overall model in vivo.

Questions for the author:

  • Is the interaction between Golga7b and DHHC5 direct?
  • Is this interaction the same in different cell types?
  • DHHC5 can also be phosphorylated. How might this fit into the model?
  • Does DHHC5’s potential role in cell adhesion explain its prior described functions in tumors, cardiac tissue, and the nervous system?
  • Do you have any predictions regarding how/if this process might occur in vivo?
    • Is DHHC5 actually palmitoylating Golga7b in an animal?
    • Can this complex be visualized?
    • Does Golga7b palmitoylation target DHHC5 to the cell membrane in vivo?

Tags: cell adhesion, desmosome, dhhc5, golga7b, palmitoylation, post-translational modification, protein s- acyltransferase

Posted on: 28th January 2019 , updated on: 29th January 2019

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  • Author's response

    Keith Woodley and Mark Collins shared

    Dear Abagael

    Thank you for your interest in our paper.

    Is the interaction between Golga7b direct?

    The interaction between Golga7b and DHHC5 is likely to be direct, but this is unconfirmed. DHHC5 and Golga7b co-immunoprecipitate and this interaction isabolished upon mutation of palmitoylation sites in the C-terminus of DHHC5. Therefore, we can say that they can form a complex that is regulated by structural features in the C-terminus of DHHC5. Another technique such as cross-linking-Mass Spectrometry would confirm that the interaction is direct and would have the added benefit of allowing the interaction site(s) to be mapped.

    Is this interaction the same in different cell types?

    The interaction appears to be the same in a number of cell types. Pulldowns have been performed in HEK293 and HeLa cell lines and with endogenous proteins from mouse forebrain lysates, so the interaction certainly seems to be the same in multiple cell types and the effects of Golga7b on DHHC5 are the same in all the cell lines mentioned.

    DHHC5 can also phosphorylate proteins. How might this fit into the model?

    The Bamji lab hasshown that DHHC5 can be phosphorylated by Fyn kinase in neurons and the function of this phosphorylation is to stabilise DHHC5 with PSD-95 at the postsynaptic membrane. Neuronal activity disrupts the interaction between DHHC5 and Fyn and leads to the endocytosis of DHHC5. This activity dependent movement of DHHC5 allows it to then palmitoylate another substrate (δ-catenin) in the dendritic shaft which then leads to changes in synaptic structure and glutamate receptor stabilisation in the postsynaptic membrane. Just how this phosphorylation-dependent cycling of DHHC5 at synapses integrates with our model of how DHHC5 plasma membrane localisation is regulated by Golga7b binding and palmitoylation, remains to be resolved. Given that we can detect a robust interaction between DHHC5 and Golga7b in brain lysates, this would suggest that it may also regulate DHHC5 localisation at synapses. If this is the case, it is likely that phosphorylation and palmitoylation could work in concert to modulate the endocytosis of DHHC5.

    It is also worth pointing out that DHHC5 is heavily modified by phosphorylation as well as other PTMs (at over 100 sites, https://www.phosphosite.org/proteinAction?id=5460) such as acetylation and ubiquitination. Many of these PTM sites are located on the particularly long cytoplasmic domain of DHHC5 and may contribute to the regulation of DHHC5 activity, localisation and substrate specificity.

    Does DHHC5’s potential role in cell adhesion explain its prior described functions in tumors, cardiac tissue, and the nervous system?

    The effects of DHHC5 on adhesion could go some way to explaining the role of DHHC5 in neurons, as it could regulate adhesion at synapses. In tumours, the loss of adhesion would likely lead to epithelial to mesenchymal transition and subsequent metastasis, but this role hasn’t been assigned to DHHC5 in the past. DHHC5 regulates a number of process in the heart such as massive endocytosis (MEND) and the cardiac sodium pump but a specific role of DHHC5 in the regulation of adhesion in the heart has not been reported.

    Do you have any predictions regarding how/if this process might occur in vivo?

    It is likely that DHHC5 is the major Golga7b palmitoylating enzyme in vivo although a knockout animal would need to be generated to confirm this. No full knockout has been generated to our knowledge, and this could prove difficult as DHHC5 does seem to have a hand in many important processes so a knockout could be embryonically lethal. A gene trap mouse that had 5% of endogenous expression showed a 50% embryonic lethality in mice. Our data suggestthat DHHC5 is targeted to the plasma membrane without Golga7b, but that Golga7b is needed to stabilise it there, but this process could be cell type specific. It is possible that there are other factors in vivoor in other cell types that could contribute to the localisation of DHHC5 at the plasma membrane. For example, loss of DHHC5 phosphorylation promotes endocytosis in neurons, so unless this process involves a cascade that results in the loss of Golga7b palmitoylation, this event could be the main determinant of DHHC5 endocytosis in this cell type.

    Thank you again for your interest, hopefully,we’ve answered your questions and that this work will lead to a greater understanding of DHHC5 and how it acts in different cell types and tissues.

    Keith and Mark

    2 comments

    5 months

    Dale Martin

    Dear Abigail, I liked your take on this paper. If you are interested in palmitoylation at the synapse, then you might like this paper too, where we looked at what pathways and diseases palmitoylation is enriched in https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004405

    We also found that ~40% of the synaptic proteome has a palmitoylated proteo form.

    Best,
    Dale

    3

    5 months

    Abagael Lasseigne

    Dale,
    Thank you for this recommendation! I will definitely take a look at this paper!
    Best,
    Abbey

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