ABHD17 proteins are novel protein depalmitoylases that regulate N-Ras palmitate turnover and subcellular localization
- University of British Columbia, Canada
Figures
Figure 1
Dual-click chemistry labeling reveals differences in protein depalmitoylation dynamics.
(A) Pulse-chase analysis of established palmitoyl-proteins (N-Ras, SNAP25, GAD65, PSD95) by dual-click chemistry in the presence of DMSO (-) or 10 μM PalmB (+). Representative in-gel fluorescence …
Figure 2 with 1 supplement
Downregulation of APT1 and APT2 inhibits HTT depalmitoylation but does not affect palmitate turnover on PSD95 or N-Ras.
(A) Pulse-chase analysis of N-HTT palmitoylation in the presence of DMSO, 10 μM PalmB, 10 μM APT1-selective inhibitor C83, and/or 10 μM APT2-selective inhibitor C115, as described in Figure 1. n = …
Figure 2—figure supplement 1
Downregulation of APT1 and APT2 inhibits GAD65 depalmitoylation but does not affect palmitate turnover on PSD95 or N-Ras.
(A-C) Pulse-chase analysis of (A) GAD65, (B) PSD95, and (C) N-Ras palmitoylation in the presence of DMSO, 10 μM PalmB, 10 μM APT1-selective inhibitor C83, and/or 10 μM APT2-selective inhibitor C115, …
Figure 3 with 1 supplement
Shared targets of Palmostatin B and HDFP identified by competitive activity-based protein profiling.
(A-C) Pulse-chase analysis of (A) N-Ras, (B) PSD95, and (C) N-HTT in the presence of DMSO, 10 μM PalmB or 20 μM lipase inhibitor HDFP as described in Figure 1. n = 3 (DMSO and PalmB) or 2 (HDFP), …
Figure 3—figure supplement 1
Treatment with serine hydrolase inhibitors WWL70, C75, and RHC-80267 does not affect PSD95 palmitate turnover.
(A-B) Competitive ABPP of 10 μM PalmB and (A) 10 μM WWL70 or (B) 20 μM RHC-80267 against candidate depalmitoylases and ACOT1. Percent inhibition of each enzyme is relative to DMSO. (C-D) Pulse-chase …
Figure 4 with 2 supplements
ABHD17A expression promotes N-Ras depalmitoylation and alters N-Ras subcellular localization.
(A) Pulse-chase analysis of N-Ras co-expressed with candidate mSHs as described in Figure 1. n = 3, mean ± SEM. (B) Schematic of the ABHD17A wild type, catalytically-inactive (S211A), and N-terminal …
Figure 4—figure supplement 1
ABHD17 expression promotes PSD95 depalmitoylation.
(A) Pulse-chase analysis of PSD95 co-expressed with candidate mSHs as described in Figure 4A. n = 3, mean ± SEM. (B) Pulse-chase analysis of PSD95 co-expressed with ABHD17A wild type and mutant …
Figure 4—figure supplement 2
ABHD17A is localized to the plasma membrane and endosomal compartments.
(A) Localization of ABHD17A wild-type protein with markers of early endosomes (Rab5), late endosomes (Rab7), recycling endosomes (Rab11), and the Golgi apparatus (GM130) in COS-7 cells as determined …
Figure 5
Simultaneous knockdown of ABHD17 isoforms inhibits N-Ras palmitate turnover.
(A) RT-qPCR of ABHD17A, ABHD17B, and ABHD17C transcript levels in HEK 293T cells treated with Non-Targeting siRNA (”NT”, black), ABHD17A siRNA alone (”A KD”, gray), or ABHD17A/ ABHD17B/ ABHD17C …
Author Response Image 1
Additional files
-
Supplementary File 1
List of Metabolic serine hydrolases inhibited by HDFP.
A summary table compiling the 29 serine hydrolases targeted by HDFP (>25% activity inhibition) as determined by cABPP-SILAC (Stable isotope labeling of amino acids in culture) in (Martin et al., 2011). LYPLAL1 (APT1L) was added to this list as a candidate enzyme for Palmostatin B testing (Tian et al., 2012).
- https://doi.org/10.7554/eLife.11306.012
-
Supplementary File 2
List of cloning oligos used in this study.
A table listing PCR primers used to subclone candidate serine hydrolases for cABPP, pulse-chase/click chemistry, and confocal imaging studies.
- https://doi.org/10.7554/eLife.11306.013
-
Supplementary File 3
List of gene-specific RT-qPCR primer pairs used in this study.
A table listing gene-specific primer pairs for verification of transcript levels in HEK293T cells by RT-qPCR in Figure 5A.
- https://doi.org/10.7554/eLife.11306.014
