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Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.
Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.
S-acylation is a reversible post-translational modification involving addition of a fatty acyl chain to an internal cysteine residue on a target protein via a labile thioester bond1. It was first reported as a modification of proteins with palmitate, a saturated 16-carbon fatty acid2, and therefore this modification is often referred to as S-palmitoylation. In addition to palmitate, proteins can be reversibly modified by a variety of longer and shorter saturated (myristate and stearate), monounsaturated (oleate) and polyunsaturated (arachidonate and eicosapentanoate) fatty acids3,4,5,6,7. In eukaryotic cells, S-acylation is catalyzed by a family of enzymes known as DHHC protein acyltransferases and the reverse reaction of cysteine deacylation is catalyzed by protein thioesterases, most of which still remain enigmatic8.
The lability of the thioester bond makes this lipid modification reversible, allowing it to dynamically regulate protein clustering, plasma membrane localization, intracellular trafficking, protein-protein interactions and protein stability9,10. Consequently, S-acylation has been linked to several disorders including Huntington’s disease, Alzheimer’s disease and several types of cancer (prostate, gastric, bladder, lung, colorectal), which necessitates development of reliable methods to detect this post-translational protein modification11.
Metabolic labeling with radioactive ([3H], [14C] or [125I]) palmitate was one of the first approaches developed to assay protein S-acylation12,13,14. However, radiolabeling-based methods present health concerns, are not very sensitive, time consuming, and only detect lipidation of highly abundant proteins15. A faster and nonradioactive alternative to radiolabeling is metabolic labeling with bioorthogonal fatty acid probes, which is used routinely to assay dynamics of protein S-acylation16. In this method, a fatty acid with a chemical reporter (alkyne or azide group) is incorporated into the S-acylated protein by a protein acyltransferase. Azide-alkyne Huisgen cycloaddition reaction (click chemistry) can then be used to attach a functionalized group, such as a fluorophore or biotin, to the integrated fatty acid allowing for detection of the S-acylated protein17,18,19.
Acyl-biotin exchange (ABE) is one of the extensively used biochemical methods for capture and identification of S-acylated proteins that bypasses some of the shortcomings of metabolic labeling such as unsuitability for tissue samples15. This method can be applied for analysis of S-acylation in a diverse range of biological samples, including tissues and frozen cell samples20,21. This method is based on selective cleavage of the thioester bond between the acyl group and the cysteine residue by neutral hydroxylamine. The liberated thiol groups are then captured with a thiol-reactive biotin derivative. The generated biotinylated proteins are then affinity-purified using streptavidin agarose and analyzed by immunoblotting.
An alternative approach termed acyl-resin assisted capture (Acyl-RAC) was later introduced to replace the biotinylation step with direct conjugation of free cysteines by a thiol-reactive resin22,23. This method has fewer steps compared to ABE and similarly can be used to detect protein S-acylation in a wide range of samples1.
Acyl-RAC consists of 4 main steps (Figure 1),
1. Blocking of free thiol groups;
2. Selective cleavage of the cysteine-acyl thioester bond with neutral hydroxylamine (HAM) to expose cysteine thiol groups;
3. Capturing of the lipidated cysteines with a thiol-reactive resin;
4. Selective enrichment of the S-acylated proteins after elution with reducing buffer.
The captured proteins can then be analyzed by immunoblotting or subjected to mass spectrometry (MS) based proteomics to assess the S-acylated proteome in a varied range of species and tissues22,24,25. Individual S-acylation sites can also be identified by trypsin digestion of the captured proteins and analysis of the resulting peptides by LC-MS/MS22. Here, we demonstrate how acyl-RAC can be used for simultaneous detection of S-acylation of multiple proteins in both a cell line and a tissue sample.
Mice used in this protocol were euthanized according to NIH guidelines. The Animal Welfare Committee at University of Texas Health Science Center in Houston approved all animal work.
1. Preparation of cell lysates
2. Acyl-RAC: Blocking of free thiol groups
NOTE: All subsequent steps can be performed at room temperature (RT).
3. Acyl-RAC: Hydroxylamine (HAM) cleavage and capture of S-acylated proteins
4. Elution and detection of S-acylated proteins
Following the protocol described above, we first used acyl-RAC to simultaneously detect S-acylation of several proteins in Jurkat cells, an immortalized T cell line originally derived from the peripheral blood of a T cell leukemia patient27. Regulatory T cell proteins previously identified as S-acylated9,28,29 were chosen to demonstrate the utility of this method. As shown in Figure 2...
Here, we successfully utilized the acyl-RAC assay to detect S-acylation of selected proteins in both cultured human cells and primary cells derived from mouse tissue. This method is simple, sensitive, and can be easily performed with minimal equipment requirements using standard biochemistry techniques. This method has been shown to successfully identify novel S-acylated proteins such as the β-subunit of the protein translocating system (Sec61b), ribosomal protein S11 (Rps11), and microsomal glutathione-S-t...
No conflicts of interest declared.
This work was supported by the National Institutes of Health grants 5R01GM115446 and 1R01GM130840.
Name | Company | Catalog Number | Comments |
cOmplete Protease Inhibitor Cocktail tablets | Sigma | 11836170001 | |
Eppendorf Centrifuge 5424 | Eppendorf | 22620444 | |
Hydroxylamine (HAM) | Sigma | 159417 | |
Methyl methanethiosulfonate (MMTS) | Sigma | 64306 | |
Mini tube rotator | LabForce | ||
ML211 | Cayman | 17630 | |
Multi-Therm Cool-Heat-Shake | Benchmark Scientific | H5000-HC | |
n-Dodecyl β-D-maltoside (DDM) | Sigma | D641 | |
Phosphatase Inhibitor Cocktail 2 | Sigma | P5726 | |
Thiopropyl-Sepharose 6B (TS) | Sigma | T8387 | |
Ultrasonics Quantrex Sonicator | L & R |
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