Elucidating the role of GODZ-mediated palmitoylation in controlling GABAergic inhibition
Open Access
- Author:
- Kilpatrick, Casey Leigh
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 24, 2016
- Committee Members:
- Bernhard Luscher, Dissertation Advisor/Co-Advisor
Bernhard Luscher, Committee Chair/Co-Chair
Melissa Rolls, Committee Member
Curtis John Omiecinski, Special Member
Yanming Wang, Committee Member - Keywords:
- GODZ
palmitoylation
GABA - Abstract:
- Palmitoylation involves the addition of a 16-carbon fatty acid chain to cysteine residues, which increases the hydrophobicity of the target protein. This modification functionally influences membrane targeting, protein conformational state, complex formation and protein-protein interactions. Palmitoylation is a rapidly reversible and dynamically regulated post-translational modification that affects the function or localization of many neuronal proteins including GABAA receptors (GABAARs). GABAARs are heteropentameric chloride channels responsible for mediating phasic inhibitory neurotransmission in the brain. The γ2 subunit of GABAARs is critically important for trafficking of these receptors to the synapse and for normal GABAergic synaptic transmission. GABAAR γ2 heterozygous knockout mice have moderate functional deficits in GABAergic neurotransmission that result in behavioral and other alterations that are analogous to depression in humans. Diverse post-translational modifications of the γ2 subunit intracellular loop affect the membrane dynamics of GABAARs and therefore the function of inhibitory synapses. The γ2 subunit is subject to palmitoylation by Golgi-specific DHHC-type zinc finger protein (GODZ, DHHC3) and its paralog, Sertoli cell gene with a zinc finger domain (SERZ-β, DHHC7). Knockdown of GODZ by shRNA or a dominant negative construct indicated that GODZ-mediated palmitoylation regulates the accumulation of GABAARs at synapses, as well as GABAergic innervation. Neurons derived from GODZ knock-out (KO) and GODZ/SERZ-β double KO mice grown in co-culture with wild-type (WT) neurons, exhibit a loss of punctate immunostaining for presynaptic glutamate decarboxylase (GAD) and postsynaptic γ2 indicating a loss of GABAergic innervation. The loss of synapses could potentially be a result of GODZ palmitoylating other proteins involved in synapse formation and maintenance such as the postsynaptic adhesion molecule, neuroligin 2 (NL2). The main objective of this dissertation was to further elucidate the mechanism by which GODZ-mediated palmitoylation controls GABAergic inhibition in vivo, by utilizing GODZ KO mice. I screened for candidate novel substrates of GODZ using a combined acyl-biotin exchange assay and quantitative proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) approach. Proteomic analyses of putative palmitoylated proteins revealed that there were a wide variety of proteins that were differentially palmitoylated or expressed in brain tissue of WT compared to GODZ KO mice. This result is consistent with previous evidence that GODZ has broad substrate specificity. Through proteomics, growth associated protein (GAP-43) was identified as a palmitoylated protein that is less abundant or less palmitoylated in brain of KO compared to WT mice. We confirmed that GAP-43 could be palmitoylated by GODZ in transfected HEK 293T cells. Moreover, steady state palmitoylation of the γ2 subunit and GAP-43 was significantly reduced in brain extracts of GODZ KO vs. WT mice, thereby identifying both proteins as important substrates of GODZ in vivo. In contrast, palmitoylation of PSD-95, which can be palmitoylated by GODZ in transfected HEK 293T cells, was unaffected. The next objective was to assess the precise localization of GODZ in the Golgi apparatus using cis- and trans-Golgi markers in HEK 293T cells to gain further insights into the subcellular role of GODZ-mediated palmitoylation. GODZ was more strongly associated with the cis- than with the trans-Golgi network, suggesting a role in controlling the exit of substrate proteins from the endoplasmic reticulum (ER) to the Golgi. In addition to Golgi membranes in the soma, GODZ was also found localized to Golgi outposts in dendrites. These results suggest that GODZ-mediated palmitoylation occurs on de novo synthesized proteins and contributes to trafficking of its substrates from the ER to the Golgi complex. Finally, synaptic deficits seen in GODZ KO neurons could also be explained by the loss of NL2 palmitoylation. NL2 is localized preferentially to inhibitory synapses where it is important for synapse maturation. Moreover, knockdown of NL2 leads to defects in inhibitory synapse formation similar to those of GODZ KO neurons where deficits are only seen selectively when mutant neurons compete with WT neurons. Indeed, we find that GODZ and SERZ-β can palmitoylate NL2 in transfected HEK 293T cells. However, we could not ascertain a measurable difference in NL2 palmitoylation between WT and GODZ KO mice. A screen of the 23-member DHHC family of PATs in transfected HEK 293T cells identified GODZ and SERZ-β as two of several PATs that can act on NL2. A unique Cys residue (C710) present in the cytoplasmic domain of NL2 but is absent in the other neuroligin isoforms has been identified as the primary site of palmitoylation by site directed mutagenesis. Palmitoylated NL2 was observed in cultured neurons and in the cerebellum of transgenic Histidine-Flag-YFP-NL2 tagged mice. Native NL2 is likely to be palmitoylated as the protein was detected in the proteomics screen of palmitoylated proteins. Both putative palmitoylation sites of NL2 are also subject to nitrosylation, a potential competitive post-translational modification. Nitrosylated NL2 was also isolated from whole brain extracts. These results suggest the interplay between palmitoylation and nitrosylation of NL2 likely influences the synaptic localization of NL2 and could contribute to the GABAergic synaptic deficits seen in GODZ KO mice.