Dataset Information

Identification of palmitoyl protein thioesterase 1 substrates defines roles for synaptic depalmitoylation

ABSTRACT: We report here the identification of substrates of the depalmitoylating enzyme PPT1 by quantitative mass spectrometry. We utilized a stringent two-step Acyl Resin-Assisted Capture (Acyl RAC) screen in which increased palmitoylation in vivo in PPT1 knockout is the first selection criteria for substrates. We validated hits by direct depalmitoylation with PPT1 to identify bona fide substrates. Most proteins identified are previously reported to be palmitoylated and the few known PPT1 substrates were validated. Significantly, the screen identified >100 proteins not previously known to be depalmitoylated by PPT1, including a wide range of endocytic, signaling, synaptic adhesion, mitochondrial, and lysosomal proteins. These proteins collectively explain the many facets of CLN1 disease caused by the loss of PPT1 function. We determined that an Ig domain in synaptic adhesion molecules may be a PPT1 recognition site for depalmitoylation. Finally, we provide evidence that PPT1 participates in neuronal activity-dependent palmitoylation-depalmitoylation cycles at the synapse.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: TuKiet Lam

LAB HEAD: TuKiet Lam

PROVIDER: PXD017270 | Pride | 2022-05-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	LF16-032_Chandra_HA_re-process.mgf	Mgf
	LF16-032_Chandra_HA_re-process_F518494.xml	Xml
	LF17-017_Chandra_totalPPT1_re-do_082216_ttl.mgf	Mgf
	LF17-017_Chandra_totalPPT1_re-do_082216_ttl_F503200.xml	Xml
	LF18-002_Gorenberg_HA_syn_Bio1.mgf	Mgf

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Publications

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

Gorenberg Erica L EL Massaro Tieze Sofia S Yücel Betül B Zhao Helen R HR Chou Vicky V Wirak Gregory S GS Tomita Susumu S Lam TuKiet T TT Chandra Sreeganga S SS

PLoS biology 20220331 3

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 increas ...[more]

PMID: 35358180

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Project description:Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1 result in early onset neurodegeneration, therefore, we hypothesized that the levels of proteins extracted from purified brain membranes of newborn mice will differ. One of the main effects of palmitoylation is changing the hydrophobic index of proteins and thus affecting these proteins interactions with different membranal domains. Assuming that PPT1 substrates are over palmitoylated in Ppt1-/- brains it is possible that its localization to specific membranal domain is different than in the wild-type brains. We isolated Triton X-100 soluble membranes from three wild-type and three Ppt1-/- brains and identified the proteins by PalmPISC. Many of the identified proteins were found to be palmitoylated. A second feature, which was noted is that many of the differential proteins (62 out of 88, 70%) can be detected in the cilia proteome (http://v3.ciliaproteome.org/cgi-bin/index.php). This observation led to test whether ciliary proteins differ between wild-type and Ppt1-/-. Cilia were prepared from newborn dissociated brain cell cultures and analyzed by MS