Figure 1.
2-BP inhibits acylation and membrane association of newly synthesized N13GAP-43(C3S).
A) Schematic representation of the experimental procedure used in B and C. The CHO-K1 cells were transfected at −8 h with the plasmid encoding N13GAP-43(C3S)-YFP. At 30 min before CHX withdrawal (-CHX), cells were incubated with 25, 50 or 150 µM 2-BP or DMSO (vehicle, Control). At 0 h, CHX was removed and cells were further incubated with 2-BP at the concentrations indicated above, or with DMSO, at 37°C for 9 h. Finally, cells were analyzed by confocal fluorescent microscopy or subjected to biochemical assays. B) Representative images showing the effect of 2-BP or DMSO (Control) on the TGN association of N13GAP-43(C3S). The fluorescent signal from YFP was pseudocoloured gray. The inset shows details of the boxed area at a higher magnification. Scale bars: 5 µm. C) After treatment with 2-BP, CHO-K1 cells transiently expressing N13GAP-43(C3S)-YFP were lysed, ultracentrifuged and the supernatant (S) and pellet (P) fractions were recovered. Proteins from these fractions were western blotted with an antibody to GFP (α-GFP) and α-tubulin (α-tub).
Figure 2.
Deacylation kinetic of N13GAP-43(C3S) at different doses of 2-BP.
A) Schematic representation of the experimental procedure used in B, C and E. The CHO-K1 cells expressing N13GAP-43(C3S)-YFP, 72 h after transfection, were treated at 20°C with 25, 50 or 150 µM 2-BP or DMSO (Control) in the presence of CHX and protein degradation inhibitors which were added 1 h before imaging and during all the experiments. The N13GAP-43(C3S) subcellular distribution was analyzed by live cell confocal microscopy. B) Representative images showing the effect of different doses of 2-BP or DMSO (vehicle, Control) on the TGN-membrane association of N13GAP-43(C3S)-YFP. The fluorescent signal from YFP (pseudocoloured gray) at 0, 5, 15 and 30 min after 2-BP or vehicle addition is shown. The insets show the expression of the TGN marker GalNAc-T-CFP (pseudocolored gray). Cell boundaries (white lines) are indicated. C) Quantification of the images shown in B (for details see Materials and methods). Curves were fitted to the exponential decay function for each data set, and data are expressed as means±SEM for a representative experiment from nine independent ones. D) The half-life for deacylation at each 2-BP dose calculated from the C data (n = 6). (*p<0.05; ***p<0.0001; compared to 25 µM). E) Representative images showing the effect of 50 µM 2-BP on the TGN-membrane association of GalNAc-T-YFP over time. The fluorescent signal from YFP (pseudocoloured gray) at 0, 5, 15 and 30 min after 2-BP addition is shown. Scale bars: 5 µm.
Figure 3.
Deacylation kinetic and membrane association of GAP-43 at different doses of 2-BP.
CHO-K1 cells coexpressing diacylated GAP-43-YFP and GalNAc-T-CFP were treated with 25, 50 or 150 µM 2-BP or vehicle (DMSO, Control), and the GAP-43 subcellular distribution was analyzed by live cell confocal microscopy at the indicated times. CHX and protein degradation inhibitors were added and maintained in the culture media until the end of each experiment. Representative images show the effect of different doses of 2-BP on the membrane association of GAP-43. The fluorescent signals from YFP and CFP were pseudocoloured green and red, respectively. Scale bars: 5 µm.
Figure 4.
Purification and biochemical characterization of recombinant human APT1 and APT2.
A) Coomasie blue staining (left) or western blot (right) of supernatant fractions obtained from E. coli transformed with His-APT1, His-APT2 or the empty vector (C). Recombinant proteins were analyzed using an antibody to Hisx6 tag. B) Coomassie blue staining of recombinant APT1 and APT2 was obtained by affinity chromatography and analyzed by SDS-PAGE. Molecular masses of the markers in kDa are indicated on the right. C) The initial rate of palmitoyl-CoA hydrolase activity was measured with 0.5 µg of recombinant APT1 or APT2. The data shown are representative experiments performed in triplicate. Curves were fitted to the Michaelis-Menten rate equation for each data set, and the kinetic parameters are shown in the table.
Figure 5.
Dose-response curve for APT activities following different concentrations of palmitoyl-CoA and the structural characterization of the substrate by SAXS analysis.
The initial rate of palmitoyl-CoA hydrolase activity was measured with 0.5 µg of recombinant APT1 (A) or APT2 (B), both in control conditions (-CHAPS -Mg2+, left bars) or in the presence of 7.5 mM CHAPS and 2 mM MgCl2 (+CHAPS +Mg2+, right bars) at 150, 300, 600 and 1500 µM. Data show the initial rate of the reaction (V, µM/min) at different concentrations of palmitoyl-CoA (P-CoA), which are from representative experiments performed in triplicate. C) SAXS analysis. Palmitoyl-CoA was resuspended in buffer (50 mM Hepes, pH 8.0) at 50, 300, 600 and 1775 µM, and measurement were carried out as indicated in Materials and methods. The figure shows the SAXS raw data (after subtraction of the buffer background and the concentration normalization) for increasing concentrations of palmitoyl-CoA. As can be seen, no noticeable diffraction peak (due to any strong correlation) is observed in any of the curves. The curve for 50 µM palmitoyl-CoA does not display any obvious tendencies. The curves for 300 and 600 µM show increasing intensity at a very low angle, adopting similar slopes and absolute values. The curve at 1775 shows a different behavior with an increment at a low angle, which reached a plateau below 0.3 nm−1 with a prominent bump centered at 1.6–1.7 nm−1 (very common in bilayers and micelles). In agreement with the wedge-shaped molecular structure, this molecule did not display the global form factor of bilayers, but rather one of the micelles. This is evident from the non-quadratic decay of the intensity as a function of q. The saturation value at low q (Guinieŕs approximation) for the 1775 µM may indicate globular micelles. The clear differences present between the curves at 300–600 µM and the one at 1775 µM is probably due to the fact that the micelles have a different geometry, with the decay at low q values (q<0.5 nm−1) having a finite slope closer to an inverse (first power) behavior, suggesting rod-like structures.
Figure 6.
Analysis of acyl-protein thioesterase expression in CHO-K1 cells and biochemical characterization.
A) CHO-K1 cells transiently expressing APT1-YFP (APT1) or APT2-YFP (APT2) were analyzed by confocal microscopy (pseudocoloured gray). B) CHO-K1 cells expressing APT1-YFP (APT1) or APT2-YFP (APT2) were lysed, ultracentrifuged and the supernatant (S) and pellet (P) fractions were recovered. Proteins from these fractions were western blotted with an antibody to GFP (α-GFP). C) CHO-K1 cells transiently expressing APT1-YFP (APT1) or APT2-YFP (APT2) were lysed, ultracentrifuged, and the supernatant (S) and pellet (P) fractions were isolated. Buffer containing 1% v/v Triton X-114 was added to the samples and phase separation was induced at 37°C. Proteins from the A (aqueous) and D (detergent) phases were western blotted with an antibody to GFP (α-GFP) and α-tubulin (α-tub). The Triton X-114 partition assay was performed as described by [29]. Note that APT1 and APT2 are mainly present in the aqueous phase of the supernatant (S) fraction, clearly demonstrating their hydrophilic character. Scale bars: 5 µm.
Figure 7.
In vitro inhibition of APT1 by 2-BP.
A) The initial rate of palmitoyl-CoA hydrolase activity was measured with 0.5 µg of recombinant APT1 in the presence of 50 or 100 µM 2-BP or DMSO (vehicle), with the APT1 activity of control condition (vehicle) taken as 100%. B) The initial rate of palmitoyl-CoA hydrolase activity (µM/min) was measured with 0.5 µg of recombinant APT1 in the presence of 50 µM 2-BP (+2-BP) or in the presence of DMSO (vehicle, −2-BP). The data shown are representative experiments performed in triplicate. Curves were fitted to the Michaelis-Menten rate equation for each data set. The kinetic parameters (Km and Vmax) are shown in the table below the figure.