Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay

Kanadome, Takashi; Yokoi, Norihiko; Fukata, Yuko; Fukata, Masaki

doi:10.1007/978-1-4939-9532-5_7

Cited by 19 publications

(23 citation statements)

References 21 publications

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“…7 I ). Since lipolytic metabolites, such as FA, may affect protein stability through s-palmitoylation ( 40 ), we measured palmitoylation of STX1A using the APEGS assay that differentiates the degree of palmitoylation as mobility shift in immunoblotting ( 29 ). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Adipose Triglyceride Lipase Is a Key Lipase for the Mobilization of Lipid Droplets in Human β-Cells and Critical for the Maintenance of Syntaxin 1a Levels in β-Cells

et al. 2020

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Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse β-cells, LDs are prominent in human β-cells. However, the regulation of LD mobilization (lipolysis) in human β-cells remains unclear. We found that glucose increases lipolysis in nondiabetic human islets but not in islets in patients with type 2 diabetes (T2D), indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets with shRNA targeting ATGL (shATGL) increased triglycerides (TGs) and the number and size of LDs, indicating that ATGL is the principal lipase in human β-cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS), and insulin secretion to 3-isobutyl-1-methylxanthine and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose-responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL-deficient INS1 cells and human pseudoislets showed reduced SNARE protein syntaxin 1a (STX1A), a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL-deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human β-cells and supports insulin secretion by stabilizing STX1A. The dysregulated lipolysis may contribute to LD accumulation and β-cell dysfunction in T2D islets.

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Section: Resultsmentioning

confidence: 99%

“…Acyl-PeGyl exchange gel shift (APEGS) assay of scr or siATGL transfected INS1 cell followed a published protocol ( 29 ). In brief, 190 µg of protein lysate reduced by 25 mmol/L Tris(2-carboxyethyl)phosphine was treated with 50 mmol/L N-ethylmaleimide to block free thiols.…”

Section: Methodsmentioning

confidence: 99%

Adipose Triglyceride Lipase Is a Key Lipase for the Mobilization of Lipid Droplets in Human β-Cells and Critical for the Maintenance of Syntaxin 1a Levels in β-Cells

et al. 2020

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“…The ABE results imply that the depalmitoylation of excitatory glutamate receptors is not mediated by PPT1. It is also noteworthy that the mRNA expression of ABDH17B, another depalmitoylase in the central nervous system [23,[70][71][72], is lower in the hippocampi of PPT1-KI mice (Supplementary Fig. 4i).…”

Section: Discussionmentioning

confidence: 99%

PPT1 Deficiency-Induced GABAAR Hyperpalmitoylation Impairs Synaptic Transmission And Memory Formation

Tong

Feng

Gao

et al. 2021

Preprint

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Background: Palmitoylation is a reversible and dynamic process involving addition of palmitic acid to cysteine residues of proteins. Studies have indicated that a variety of neuronal receptors, including glutamate receptors such as AMPAR, NMDAR, and GABAAR, are palmitoylated, which contributes to the dynamic modulation of synaptic strength in response to neuronal activity. However, little is known about the depalmitoylation of these receptors. Methods: A mouse model with a lost function mutation knock-in of palmitoyl protein thioesterase 1 (PPT1), an important enzyme for depalmitoylation, was employed to mimic human disease of infantile neuronal ceroid lipofuscinosis (INCLs). Immunofluorescent staining, Western blotting, biochemical assays, electrophysiological recording and behavioural tests were used to measure the effects of PPT1 deficiency.Results: We identified for the first time that the GABAARα1 subunit rather than AMPAR is the substrate of PPT1. In PPT1-deficient mice, excessive palmitoylation and extended membrane location of GABAAR were detected. Simultaneously, spatial learning and memory deficits with dysregulation of neuronal network γ oscillation and impairment of long-term plasticity were shown in the mice at as early as 2-month-old. Application of N-tert-butylhydroxylamine hydrochloride, a thioesterase mimetic, attenuated PPT1 mutation-induced GABAAR hyper-palmitoylation and its membrane accumulation with improved neuronal transmission and memory functions in the mice. Conclusions: These data provide new insights into the mechanisms of neuronal disorder caused by depalmitoylation deficiency and offer a clue for further intervention for INCLs and other neurodegenerative diseases.

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“…The ABE results imply that the depalmitoylation of excitatory glutamate receptors is not mediated by PPT1. It is also noteworthy that the mRNA expression of ABDH17B, another depalmitoylase in the central nervous system[23, [70][71][72], is lower in the hippocampi of PPT1-KI mice (Supplementary Fig. 4i).…”

Section: Discussionmentioning

confidence: 99%

PPT1 Deficiency-Induced GABA <sub>A</sub>R Hyperpalmitoylation Impairs Synaptic Transmission and Memory Formation

et al. 2021

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Background: Palmitoylation is a reversible and dynamic process involving addition of palmitic acid to cysteine residues of proteins. Studies have indicated that a variety of neuronal receptors, including glutamate receptors such as AMPAR, NMDAR, and GABA A R, are palmitoylated, which contributes to the dynamic modulation of synaptic strength in response to neuronal activity. However, little is known about the depalmitoylation of these receptors.Methods: A mouse model with a lost function mutation knock-in of palmitoyl protein thioesterase 1 (PPT1), an important enzyme for depalmitoylation, was employed to mimic human disease of infantile neuronal ceroid lipofuscinosis (INCLs). Immuno uorescent staining, Western blotting, biochemical assays, electrophysiological recording and behavioural tests were used to measure the effects of PPT1 de ciency.Results: We identi ed for the rst time that the GABA A Rα1 subunit rather than AMPAR is the substrate of PPT1. In PPT1-de cient mice, excessive palmitoylation and extended membrane location of GABA A R were detected. Simultaneously, spatial learning and memory de cits with dysregulation of neuronal network γ oscillation and impairment of long-term plasticity were shown in the mice at as early as 2month-old. Application of N-tert-butylhydroxylamine hydrochloride, a thioesterase mimetic, attenuated PPT1 mutation-induced GABA A R hyper-palmitoylation and its membrane accumulation with improved neuronal transmission and memory functions in the mice.Conclusions: These data provide new insights into the mechanisms of neuronal disorder caused by depalmitoylation de ciency and offer a clue for further intervention for INCLs and other neurodegenerative diseases.

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Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay

Cited by 19 publications

References 21 publications

Adipose Triglyceride Lipase Is a Key Lipase for the Mobilization of Lipid Droplets in Human β-Cells and Critical for the Maintenance of Syntaxin 1a Levels in β-Cells

Adipose Triglyceride Lipase Is a Key Lipase for the Mobilization of Lipid Droplets in Human β-Cells and Critical for the Maintenance of Syntaxin 1a Levels in β-Cells

PPT1 Deficiency-Induced GABAAR Hyperpalmitoylation Impairs Synaptic Transmission And Memory Formation

PPT1 Deficiency-Induced GABA <sub>A</sub>R Hyperpalmitoylation Impairs Synaptic Transmission and Memory Formation

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