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Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function

['Erica L. Gorenberg', 'Departments Of Neurology', 'Neuroscience', 'Yale University', 'New Haven', 'Connecticut', 'United States Of America', 'Interdepartmental Neuroscience Program', 'Sofia Massaro Tieze', 'Betül Yücel']

Date: 2022-04

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein–associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Funding: This work was supported by the National Institutes of Health (R01 NS064963, S.S.C.; R01 NS110354, S.S.C.; R01 NS083846, S.S.C.; R21 NS094971, S.S.C.; R01 MH077939, S.T.; T32 NS007224, E.L.G. & S.M.T.; T32 NS041228, E.L.G. & S.M.T.; https://www.nih.gov ), and the United States Department of Defense (W81XWH-17-1-0564, S.S.C.; https://www.defense.gov ). The proteomic experiments were supported by the Yale/National Institute on Drug Abuse (NIDA) Neuroproteomic Center (NIH DA018343, S.S.C. & T.T.L.; https://medicine.yale.edu/keck/nida/general/mission/ ). The Q-Exactive Plus mass spectrometer was funded in part by National Institutes of Health Shared Instrumentation Grant from the Of?ce of The Director, National Institutes of Health (S10OD018034, T.T.L; https://grants.nih.gov/grants/guide/pa-files/par-20-113.html ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Gorenberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we therefore undertook a 2-step proteomic approach to identify PPT1 substrates and elucidate how depalmitoylation contributes to synaptic function and phenotypes of NCL. Our screen greatly expands the repertoire of known palmitoylated proteins in the brain. We detected PPT1 in all synaptic subcompartments and found high levels of PPT1 enzyme activity in the synaptic cytosol. We identified approximately 10% of palmitoylated synaptic proteins as PPT1 substrates. These proteins display increased palmitoylation independently of protein expression in vivo. The validated substrates fall into 9 classes, which, strikingly, are related to phenotypes observed in PPT1 knockout (KO) mice and CLN1 patients, including seizures, decreased synapse density, mitochondrial dysfunction, synaptic vesicle endocytic deficits, impaired long-term potentiation (LTP), and retinal degeneration [ 22 – 27 ]. Notably, PPT1 depalmitoylation sites on validated substrates are frequently cysteine residues that participate in disulfide bonds, suggesting that a novel function of palmitoylation may be to mediate these interactions. Our classification of PPT1 substrates provides a resource to enhance our understanding of depalmitoylation and its contributions to synaptic function, neuronal health, and the molecular basis of NCL.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) lead to deficient depalmitoylation and synaptic function and result in the neurodegenerative disease NCL type 1 (CLN1) [ 6 , 18 ]. PPT1 therefore plays a critical role in the brain. However, the substrates of PPT1 are almost entirely unknown, with the exceptions of the presynaptic chaperone CSPα, the G-protein Goα, and the mitochondrial F1 ATP synthase subunit O [ 19 – 21 ], hindering the dissection of CLN1 disease mechanisms. Identification of the repertoire of PPT1 substrates is needed to understand how depalmitoylation deficits impact neuronal health.

Systematic and unbiased proteomic studies of the mammalian brain have identified over 600 palmitoylated proteins, a high percentage of which are localized to synapses [ 11 – 13 ]. These large-scale screens suggest the importance of palmitoylation for synaptic function and neurotransmission. Studies have begun to identify the synaptic substrates of specific PATs [ 14 – 17 ]. However, it is still unclear for most palmitoylated proteins which enzyme facilitates depalmitoylation in the brain and at the synapse.

Palmitoylation is the posttranslational addition of a C16:0 fatty acid chain to proteins, typically via thioester bond to cysteine residues. Palmitoylation is unique among lipid posttranslational modifications (PTMs) in its reversibility—other lipid PTMs typically last a protein’s entire life span [ 1 ]. Palmitoyl groups are added by a family of 23 palmitoyl acyl transferases (PATs or ZDHHCs) [ 2 , 3 ] and are removed by distinct depalmitoylating enzymes—palmitoyl protein thioesterases (PPTs), acyl protein thioesterases (APTs), and α/β-hydrolase domain-containing proteins (ABHDs) [ 4 – 6 ]. The reversible nature of palmitate PTMs suggests that tight regulation of the palmitoylation/depalmitoylation cycle is necessary for proper protein function. Indeed, palmitoylation dynamics influence protein stability, function, membrane trafficking, and subcellular localization [ 7 – 10 ].

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001590

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