Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Summary

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.

Abstract

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.

Introduction

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 acids^3,

Protocol

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

1. Prepare lysis buffer as described in Table 1. To 10 mL of PBS, add 0.1 g of n-dodecyl β-D-maltoside detergent (DDM) and rotate to dissolve. Add 100 µL of phosphatase inhibitor cocktail 2, ML211 (10 µM), PMSF (10 mM) and protease inhibitor cocktail (1x) and chill the buffer on ice before use.
2. Transfer required media containing cells from the incubator into 15 mL or 50 mL conical....

Results

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-acylated^9,28,29 were chosen to demonstrate the utility of this method. As shown in Figure 2.......

Discussion

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.......

Materials

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