PMID- 35358180 OWN - NLM STAT- MEDLINE DCOM- 20220414 LR - 20220518 IS - 1545-7885 (Electronic) IS - 1544-9173 (Print) IS - 1544-9173 (Linking) VI - 20 IP - 3 DP - 2022 Mar TI - Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function. PG - e3001590 LID - 10.1371/journal.pbio.3001590 [doi] LID - e3001590 AB - 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. FAU - Gorenberg, Erica L AU - Gorenberg EL AUID- ORCID: 0000-0003-0542-3793 AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. AD - Interdepartmental Neuroscience Program, Yale University, New Haven, Connecticut, United States of America. FAU - Massaro Tieze, Sofia AU - Massaro Tieze S AUID- ORCID: 0000-0001-7545-411X AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. AD - Interdepartmental Neuroscience Program, Yale University, New Haven, Connecticut, United States of America. FAU - Yucel, Betul AU - Yucel B AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. FAU - Zhao, Helen R AU - Zhao HR AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. FAU - Chou, Vicky AU - Chou V AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. FAU - Wirak, Gregory S AU - Wirak GS AUID- ORCID: 0000-0001-9645-1882 AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. FAU - Tomita, Susumu AU - Tomita S AD - Departments of Neuroscience and of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, United States of America. FAU - Lam, TuKiet T AU - Lam TT AD - Departments of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America. AD - Keck MS & Proteomics Resource, WM Keck Biotechnology Resource Laboratory, New Haven, Connecticut, United States of America. FAU - Chandra, Sreeganga S AU - Chandra SS AD - Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, United States of America. LA - eng GR - T32 NS007224/NS/NINDS NIH HHS/United States GR - R01 NS083846/NS/NINDS NIH HHS/United States GR - T32 NS041228/NS/NINDS NIH HHS/United States GR - P30 DA018343/DA/NIDA NIH HHS/United States GR - R21 NS094971/NS/NINDS NIH HHS/United States GR - R01 MH077939/MH/NIMH NIH HHS/United States GR - R01 NS064963/NS/NINDS NIH HHS/United States GR - S10 OD018034/OD/NIH HHS/United States GR - RF1 NS110354/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20220331 PL - United States TA - PLoS Biol JT - PLoS biology JID - 101183755 RN - 0 (Disulfides) SB - IM MH - Animals MH - Disulfides/metabolism MH - Lipoylation MH - Mice MH - Mice, Knockout MH - *Neuronal Ceroid-Lipofuscinoses/genetics/metabolism MH - Synapses/metabolism PMC - PMC9004782 COIS- The authors have declared that no competing interests exist. EDAT- 2022/04/01 06:00 MHDA- 2022/04/15 06:00 PMCR- 2022/03/31 CRDT- 2022/03/31 17:14 PHST- 2021/09/20 00:00 [received] PHST- 2022/03/02 00:00 [accepted] PHST- 2022/04/12 00:00 [revised] PHST- 2022/04/01 06:00 [pubmed] PHST- 2022/04/15 06:00 [medline] PHST- 2022/03/31 17:14 [entrez] PHST- 2022/03/31 00:00 [pmc-release] AID - PBIOLOGY-D-21-02508 [pii] AID - 10.1371/journal.pbio.3001590 [doi] PST - epublish SO - PLoS Biol. 2022 Mar 31;20(3):e3001590. doi: 10.1371/journal.pbio.3001590. eCollection 2022 Mar.