Identification of PSD-95 Depalmitoylating Enzymes

Abstract

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. ␣/␤-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, G␣ q , and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function.

Protein palmitoylation: Palmitoyltransferases and their specificity

“…67 Similarly, enhanced palmitate removal by ABHD17 was observed in the case of overexpressed N-Ras in HEK293T cells, but not when endogenous N-Ras in the neuronal cells was studied. 67,68 The amino acid residues which are indispensable for the catalytic activity of ABHD17 included amino acid S170, together with residues D235 and H264, as their mutation resulted in inactive protein. 67 Similarly to APT1 and APT2, the palmitoylation of ABHD17 proteins is required for their targeting to the plasma membrane 61 and for conferring their depalmitoylating activity toward PSD-95.…”

“…67,68 The amino acid residues which are indispensable for the catalytic activity of ABHD17 included amino acid S170, together with residues D235 and H264, as their mutation resulted in inactive protein. 67 Similarly to APT1 and APT2, the palmitoylation of ABHD17 proteins is required for their targeting to the plasma membrane 61 and for conferring their depalmitoylating activity toward PSD-95. 67 The discovery of new depalmitoylating enzymes suggest the existence of a new layer of complexity in the palmitoylation/depalmitoylation network.…”

“…67 Similarly to APT1 and APT2, the palmitoylation of ABHD17 proteins is required for their targeting to the plasma membrane 61 and for conferring their depalmitoylating activity toward PSD-95. 67 The discovery of new depalmitoylating enzymes suggest the existence of a new layer of complexity in the palmitoylation/depalmitoylation network. Considering that the enzymes that mediate palmitate removal are also the substrates for PATs, it is obvious that specific interplay exists between these two processes.…”

Mass‐Tag Labeling Using Acyl‐PEG Exchange for the Determination of Endogenous Protein S‐Fatty Acylation

“…We speculate this might be due to differences in transfer to the western blot membrane, or PEG-mediated differences in exposure of the protein to antibodies or HRP. While the signal differences challenge the accuracy of the S-fatty acylation ratios, the APE still provides a reliable method to determine the qualitative measurement of S-fatty acylation states (Roberts et al, 2016), as well as a semi-quantitative comparative assay between different conditions (Nathan P. Westcott, In Press; Yokoi et al, 2016). …”

Palmitoylation mechanisms in dopamine transporter regulation

“…Palmitoyl protein thioesterase 1 and 2 (PPT1, PPT2) are associated with lysosomes and are linked with control of protein stability by catalyzing protein depalmitoylation prior to lysosomal degradation (Fukata and Fukata, 2010; Vesa et al , 1995). Recently members of a subfamily of α/β-hydrolase domain-containing serine hydrolases not belonging to either of these families were found to depalmitoylate the neuronal protein PSD-95 (Yokoi et al , 2016), suggesting that additional proteins involved in depalmitoylation remain to be discovered.…”