Identifying the Potential Substrates of the Depalmitoylation Enzyme Acyl-protein Thioesterase 1

Abstract

Background: The homeostasis of palmitoylation and depalmitoylation is involved in various cellular processes, the disruption of which induces severe physiological consequences. Acyl-protein thioesterase (APT) and palmitoyl-protein thioesterases (PPT) catalyze the depalmitoylation process. The natural mutation in human PPT1 caused neurodegenerative disease, yet the understanding of APT1 remains to be elucidated. While the deletion of APT1 in mice turned out to be potentially embryonically lethal, the decoding of its function strictly relied on the identification of its substrates. Objective: To determine the potential substrates of APT1 by using the generated human APT1 knockout cell line. Methods : The combined techniques of palmitoyl-protein enrichment and massspectrometry were used to analyze the different proteins. Palmitoyl-proteins both in HEK293T and APT1-KO cells were extracted by resin-assisted capture (RAC) and data independent acquisition (DIA) quantitative method of proteomics for data collection. Results: In total, 382 proteins were identified. The gene ontology classification segregated these proteins into diverse biological pathways e.g. endoplasmic reticulum process and ubiquitin-mediated proteolysis. A few potential substrates were selected for verification; indeed, major proteins were palmitoylated. Importantly, their levels of palmitoylation were clearly changed in APT1-KO cells. Interestingly, the proliferation of APT1-KO cells escalated dramatically as compared to that of the WT cells, which could be rescued by APT1 overexpression. Conclusion: Our study provides a large scale of potential substrates of APT1, thus facilitating the understanding of its intervened molecular functions.

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

“…Validated proteins also include all known PPT1 substrates—CSPα, Goα, and F1 subunit O of mitochondrial ATP synthase—and exclude substrates of other depalmitoylating enzymes, such as PSD-95, an abundant synaptic protein that is exclusively depalmitoylated by ABHD17A, 17B, and 17C [ 40 ]. Putative APT1 substrates such as RAB9A, RAB6A, N-RAS, R-RAS, GAP-43, and SNAP-23 are also excluded [ 20 , 41 , 42 ]. We also screened our results against a PPT1 interactome dataset ( n = 64) [ 24 ] and found 4 high-confidence substrates (DYL2, LDHB, STXB1, and THY1) and 4 medium-confidence substrates (DYN1, GBB2, TERA, and VA0D1) to be present.…”

In a mouse model of INCL reduced S‐palmitoylation of cytosolic thioesterase APT1 contributes to microglia proliferation and neuroinflammation

“…The generation of human LYPLA1 (APT1) gene ablation in HEK293T cells by CRISPR/Cas9 has previously been reported. 29 Homogeneous clones of APT1 −/− cells were derived by single cell sorting of eGFP-and DsRed2-positive populations into 96-well microplates. The expanded monoclonal mutant cells were screened by Western blot analysis using rat anti-human APT1 polyclonal antibody, which showed that LPLA1 (APT1)protein was not expressed.…”

“…We reasoned that if Ppt1‐deficiency decreases APT1 levels it may suppress depalmitoylation of H‐Ras, thereby increasing its level on the plasma membrane where the activation of H‐Ras signaling pathway may stimulate the proliferation and activation of the innate immune cells in the CNS leading to neuroinflammation. Since Cln1 /Ppt1 is expressed in all cells and tissues, we used both Ppt1‐deficient Cln1 −/− mice, 24 which mimic INCL, 25 HEK293T cells in which the CLN1/PPT1 gene is ablated by CRISPR/Cas9, APT1‐KO HEK293T cells, 29 cultured INCL fibroblasts as well as those from Cln1 −/− mice as models. Finally, we sought to understand the neuroprotective mechanism of a Ppt1‐mimetic small molecule, N‐tert (Butyl) hydroxylamine (NtBuHA) 30 …”

Identification of synaptic PPT1 substrates highlight roles of depalmitoylation in disulfide bond formation and synaptic function

“…It also excludes proteins that have been identified as substrates of other depalmitoylating enzymes; for example, PSD-95, an abundant synaptic protein that is exclusively depalmitoylated by ABHD17 (Yokoi et al, 2016). Putative APT1 substrates such as RAB9A, RAB6A, N-RAS, R-RAS, GAP-43, and SNAP-23 are also excluded (Duncan and Gilman, 1998;Kong et al, 2013;Liu et al, 2019).…”

“…Validated proteins also include all known PPT1 substrates-CSPα, Goα, and F1 subunit O of mitochondrial ATP synthase-and exclude substrates of other depalmitoylating enzymes, such as PSD-95, an abundant synaptic protein that is exclusively depalmitoylated by ABHD17A, 17B, and 17C (Yokoi et al, 2016). Putative APT1 substrates such as RAB9A, RAB6A, N-RAS, R-RAS, GAP-43, and SNAP-23 are also excluded (Duncan and Gilman, 1998;Kong et al, 2013;Liu et al, 2019). We also screened our results against a PPT1 interactome dataset (Sapir et al, 2019) and found 4 high-confidence substrates (DYL2, LDHB, STXB1, THY1) and 7 medium-confidence substrates (DYN1, KIF5C, SSDH, SYT2, TERA, UBA1, VA0D1) to be present.…”