Activity-Based Sensing of S-Depalmitoylases: Chemical Technologies and Biological Discovery

Abstract

While lipids were first appreciated as a critical hydrophobic barrier, our understanding of their roles at the cellular and organismal levels continues to grow. Not only are they important independent operators, providing a platform for both static and dynamic organization and communication within the cell, they also exert significant effects via the chemical modification of proteins. Addition of a lipid post-translational modification (PTM) alters protein hydrophobicity and behavior, with distinct consequences for subcellular trafficking, localization, intra- and intermolecular interactions, and stability. One of the most abundant and widespread protein lipidation events is S-acylation, installation of a long-chain lipid to the thiol of a cysteine side chain through a thioester linkage. S-Acylation is often referred to as S-palmitoylation, due to the prevalence of palmitate as the lipid modification. Unlike many lipid PTMs, S-acylation is enzymatically reversible, enabling the cell to tune proteome-wide properties through dynamic alterations in protein lipidation status. While much has been uncovered about the molecular effects of S-acylation and its implications for physiology, current biochemical and chemical methods only assess substrate lipidation levels or steady-state levels of enzyme activity. Yet, the writer protein acyl transferases (PATs) and eraser acyl protein thioesterases (APTs) are dynamically active, responsible for sometimes-rapid changes in S-palmitoylation status of target proteins. Thus, to understand the full scope, significance, and subtlety of S-deacylation and its regulation in the cell, it is necessary to observe the timing and cellular geography of regulatory enzyme activities. In this Account, we review the chemical tools developed by our group to selectively visualize and perturb the activity of APTs in live cells, highlighting the biological insights gained from their application. To visualize APT activity, we masked fluorogenic molecules with thioacylated, peptide-based APT substrate mimetics; APT activity and thus thiol deprotection releases a fluorescent product in the turn-on depalmitoylation probes (DPPs), while in ratiometric depalmitoylation probes (RDPs) the emission of the parent fluorophore is altered. Application of these probes in live cells reveals that APT activity is sensitive to cell signaling events and metabolic disturbances. Additionally, as indicated above, the location of regulatory enzymes is critical in lipid signaling, and one organelle of particular interest, due to its role in maintaining cellular homeostasis and its legion of lipidated proteins, is the mitochondria. Therefore, we developed a class of spatially constrained mitoDPPs to visualize mitochondrial APT activity as well as a selective inhibitor of mitochondrial deacylation activity, mitoFP. With these tools, we identify two mitochondrial S-depalmitoylases and connect mitochondrial S-depalmitoylation to redox buffering capacity. Moreover, some of the changes in activity observed are specific to the mitochondria, confirming spatial as well as temporal regulation of eraser protein activity. Overall, this chemical toolkit for S-depalmitoylase activity, imaging reagents and a targeted inhibitor, will continue to illuminate the regulatory mechanisms and roles of S-depalmitoylation within the complex homeostatic networks of the cell.

Figure 1.

Overview of S-palmitoylation and its broad relevance in cell and organismal physiology.

Figure 2.

Chronological progression of chemical and biochemical methods to assess and probe protein lipidation in the decades since its initial observation.

Figure 3.

Overview of Depalmitoylation Probes (DPPs). (A) Schematic representing the mechanism of DPP fluorescence upon deacylase activity. Thiol acyl modification (R₂), the peptide substrate (R₁), or the xanthene scaffold (R₃) can be varied (see Table 1) to modulate the physical and biological properties of DPPs. (B) In vitro characterization of DPP-2 (1 μM) in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) with or without 50 nM APT1 (λ_ex 490/20 nm; λ_em 545/20 nm). (C) Incubation of HEK293T cells with DPP-2 (1 μM) results in a robust fluorescence signal that is abrogated by PalmB (5 μM), validating that DPPs respond to enzymatic S-deacylation in live cells. 50 μm scale bar shown.

Figure 4.

Overview of Ratiometric Depalmitoylation Probes (RDPs). (A) Schematic of RDP design and the mechanism of blue-shifted emission upon deacylase activity. (B) UV-Vis absorption spectrum of 15 μM RDP-1 (see Table 1) and deacylated RDP-1 in buffer (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, pH 7.4). (C) Cells loaded with RDP-1 (1 μM) after treatment with either DMSO carrier or PalmB (20 μM) and then analyzed by ratiometric imaging, demonstrating the blue-shift in RDP-1 fluorescent signal upon enzymatic S-deacylation in live cells. 20 μm scale bar shown.

Figure 5.

Overview of mitoFP. (A) Chemical structure and salient features of mitoFP, a selective, mitochondrial-targeted APT inhibitor. (B) Fluorescence microscopy images of HepG2 cells demonstrating inhibition of mitochondrial APTs by mitoFP, as assessed by mitoDPP-2. (C) Quantification of mitoDPP-2 signal shown in (B). (D) Fluorescence microscopy images of HepG2 cells demonstrating no inhibition of cytosolic APTs by mitoFP, as assessed by DPP-2. (E) Quantification of DPP-2 signal shown in (D). 20 μm scale bar shown.

Figure 6.

ABHD10 is a mitochondrial APT. (A) Fluorescence microscopy images of HEK293T cells demonstrating ABHD10 overexpression (OE) enhances mitoDPP-2 signal. Scale bar: 20 μm. (B) Quantification of mitoDPP-2 signal shown in (A). (C) Crystal structure of mitochondrial presequence-cleaved murine ABHD10, highlighting the catalytic triad (PDB: 6NY9).

Figure 7.

APT activities respond dynamically to growth factors and lipid stress. (A) Fluorescence microscopy images of A431 cells demonstrating EGF stimulation deactivates APTs, as assessed by DPP-3. 20 μm scale bar shown. (B) Quantification of DPP-3 signal shown in (A). (C) Fluorescence microscopy images of HepG2 cells demonstrating exogenous palmitate activates APTs, as assessed by DPP-5. 20 μm scale bar shown. (D) Quantification of DPP-5 signal shown in (C).