Palmitoylation of Gephyrin
Found a study that shows importance of the modification of Gephyrin by attaching palmitic acid (palmitoylation) for clustering of GABA receptors.
Here, we identified palmitoylation of gephyrin as an important mechanism of strengthening GABAergic synaptic transmission, which is regulated by GABAAR activity. We mapped palmitoylation to Cys212 and Cys284, which are critical for both association of gephyrin with the postsynaptic membrane and gephyrin clustering.
We identified DHHC-12 as the principal palmitoyl acyltransferase that palmitoylates gephyrin.
Furthermore, gephyrin pamitoylation potentiated GABAergic synaptic transmission, as evidenced by an increased amplitude of miniature inhibitory postsynaptic currents. Consistently, inhibiting gephyrin palmitoylation either pharmacologically or by expression of palmitoylation-deficient gephyrin reduced the gephyrin cluster size. (R1)
The enzyme that does palmitoylation is DHHC-12 (Gene ZDHHC12).
The same enzyme also palmitoylates NLRP3 which results in chaperone-mediated autophagy of the inflammasome, effectively stopping the inflammation program (R4).
Palmitoylation
Palmitoylation is an essential and is the most prevalent form of protein lipidation.
Palmitoylation is regulated by two classes of enzymes, the DHHC domain containing protein acyl-transferases (PAT) which mediate the addition of palmitate to target substrates, and the acyl-protein thioesterases (APT) which remove palmitate.
Twenty three PATs have been identified in mammals and alterations in the expression and function of several PATs have been observed in cancer. (R12)
There are three types of palmitoylation:
Palmitoylation is categorized into S-palmitoylation or the less frequently occurring O-palmitoylation and N-palmitoylation.
O-palmitoylation is the addition of fatty acyl group to serine residues, while N-palmitoylation is the addition of fatty acyl group to the N-terminus.
To date, a few oncoproteins are identified to be O- or N-palmitoylated where two well-known examples of O-palmitoylated proteins are Wnt and Histone H4, while Hedgehog proteins are N-palmitoylated (R15)
Some examples include:
- Palmitoylation of serotonin receptors (R10): required for proper clustering and localization of serotonin receptors.
- EGFR (R12)
- transferrin receptor (R15)
How it works
In the first step of catalysis, zDHHCs undergo autoacylation by palmitoyl-CoA, thereby generating an acylated enzyme, which, in a subsequent transpalmitoylation step, transfers the palmitoyl group to the substrate. Considering the 23 zDHHC enzymes and the approximately 5000 substrates identified in proteomic studies, each zDHHC must act on multiple protein substrates. Yet, it has been shown that many substrates can be palmitoylated by more than one zDHHC enzyme. (R11)
ZDHHC12 gene
Full names: Zinc finger DHHC domain-containing protein 12 / Palmitoyltransferase ZDHHC12
Located in Golgi apparatus membrane and in Endoplasmic reticulum membrane.
Targets of ZDHHC12
Claudin-3 (CLDN3)
Here, we report that CLDN3 is positively correlated with ovarian cancer progression both in vitro and in vivo. Of interest, CLDN3 undergoes S-palmitoylation on three juxtamembrane cysteine residues, which contribute to the accurate plasma membrane localization and protein stability of CLDN3.
Moreover, the deprivation of S-palmitoylation in CLDN3 significantly abolishes its tumorigenic promotion effect in ovarian cancer cells. By utilizing the co-immunoprecipitation assay, we further identify ZDHHC12 as a CLDN3-targating palmitoyltransferase from 23 ZDHHC family proteins. (R7)
What is interesting, serum level of Claudin-5 was found to be elevated in patients with OCD (R8, R9)
Other Palmitoyltransferases
ZDHHC19
ZDHHC19 is a palmitoyltransferase that adds palmitate groups to proteins like RRAS and SQSTM1. It palmitoylates RRAS, enhancing cell survival. It also promotes autophagy by palmitoylating SQSTM1, which strengthens SQSTM1’s interaction with ATG8 proteins and facilitates the recruitment of ubiquitinated cargo to autophagosomes.
p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets.
Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1).
S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes.
Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins.
Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins. (R17)
ZDHHC20
- Silencing DHHC20 increases EGFR mediated cell responses
- Inhibition of DHHC20-mediated palmitoylation increases EGF-induced EGFR activation
Inhibition of DHHC20 induced a change in cell morphology from an elongated spindle shape to a more spread morphology with extensive membrane ruffling in MDA-MB-231 cells (R12)
ZDHHC21
Mechanistically, ZDHHC21 specifically catalyzed the palmitoylation of mitochondrial adenylate kinase 2 (AK2) and further activated OXPHOS in leukemic blasts.
Inhibition of ZDHHC21 arrested the in vivo growth of AML cells and extended the survival of mice inoculated with AML cell lines and patient derived xenograft AML blasts. Moreover, targeting ZDHHC21 to suppress OXPHOS markedly eradicated AML blasts and enhanced chemotherapy efficacy in relapsed/refractory leukemia. (R5)
Zdhhc13 (in mice)
Here, for the first time, we show that Zdhhc13 plays a key role in anxiety-related behaviors and motor function, as well as brain bioenergetics, in a mouse model (luc) carrying a spontaneous Zdhhc13 recessive mutation.
At 3 m of age, mutant mice displayed increased sensorimotor gating, anxiety, hypoactivity, and decreased motor coordination, compared to littermate controls. Loss of Zdhhc13 in cortex and cerebellum from 3- and 24 m old hetero- and homozygous male mutant mice resulted in lower levels of Drp1 S-palmitoylation accompanied by altered mitochondrial dynamics, increased glycolysis, glutaminolysis and lactic acidosis, and neurotransmitter imbalances.
Our results indicated that Zdhhc13, acting via S-palmitoylation of Drp1, alone or acting in concert, is critical for sustaining cortex and cerebellum mitochondrial dynamics and function resulting in deficits in motor- and non-motor skills. This study, for the first time, shows that S-palmitoylation constitutes a critical component of the post-translational modifications of mitochondrial targets in the brain, affecting basic mitochondrial functions and behavior. (R6)
Mitochondrial function, MCAT, CTNND1
400 (31%) of S-palmitoylation sites on 254 proteins were down-regulated in Zdhhc13-deficient mice, representing potential ZDHHC13 substrates.
Among these, lipid metabolism and mitochondrial dysfunction proteins were overrepresented. MCAT and CTNND1 were confirmed to be specific ZDHHC13 substrates.
Furthermore, we found impaired mitochondrial function in hepatocytes of Zdhhc13-deficient mice and Zdhhc13-knockdown Hep1–6 cells.
These results indicate that ZDHHC13 is an important regulator of mitochondrial activity. Collectively, our study allows for a systematic view of S-palmitoylation for identification of ZDHHC13 substrates and demonstrates the role of ZDHHC13 in mitochondrial function and metabolism in liver.
The spare respiratory capacity was lower in Zdhhc13-knockdown cells than in the scramble control in the presence of 0.5 μM FCCP. Silencing of Zdhhc13 led to lower mitochondrial membrane potential compared with that of the scrambled control (Fig. 7c).
As expected, knockdown of Zdhhc13 also led to the production of more mitochondrial ROS (Fig. 7d). In summary, these data support that knockdown of Zdhhc13 disrupts mitochondrial function in Hep 1–6 cells. (R16)
AMPK activates ZDHHC13 by phosphorylation
Here, we report that AMP-activated protein kinase (AMPK) phosphorylates ZDHHC13 at S208 to strengthen the interaction between ZDHHC13 and MC1R-RHC, leading to enhanced MC1R palmitoylation in redheads.
Consequently, phosphorylation of ZDHHC13 by AMPK increased MC1R-RHC downstream signaling. AMPK activation and MC1R palmitoylation repressed UVB-induced transformation of human melanocytes in vitro and delayed melanomagenesis in vivo in C57BL/6J-MC1R-RHC mice. (R13)
Huntingtin
huntingtin is palmitoylated by ZDHHC17 and ZDHHC13 (R16)
Zdhhc17 (in mice)
Deficits in Zdhhc17 (one of the 23 or 24 PATs from human and rat or mouse, respectively) are associated with behavioral, memory and synaptic defects based on the palmitoylation of substrates relevant to neurogenesis and neurotransmission (e.g. SNAP-25, synaptotagmin I, and huntingtin (Htt)4,6). (R6)