Site-specific analysis of protein S-acylation by resin-assisted capture

Abstract

impact protein function and localization, largely via modulating membrane affi nity and protein stability (7)(8)(9). In contrast to the stable thioether linkage of S -prenylation, the thioester linkage of S -acylation confers a reversible and dynamic nature on this modifi cation, and many recent efforts are shedding light on how this modifi cation is regulated ( 8-11 ).There are a variety of methodologies to detect protein S -acylation/palmitoylation in intact cells. A well-established method involves incubating cells with 3 H-labeled palmitate, followed by autoradiography to visualize the degree of isotopic incorporation. However, this approach requires high levels of [ 3 H]palmitate (as many as several mCi per sample) and exposure times on the order of weeks ( 12, 13 ). More recent methods have cleverly circumvented these issues by using nonradioactive derivatives of palmitate, which can be enriched or detected via cycloaddition reactions ( "click chemistry") ( 14-17 ). Nonetheless, these "palmitate-centric" approaches are encumbered by i ) the need for radioactive or chemically modifi ed palmitate analogs; ii ) the likely bias for proteins that undergo rapid palmitate turnover versus proteins that are more stably palmitoylated; iii ) diffi culty in detecting individual S -acylated proteins or their specifi c sites of S -acylation; and iv ) the inability to detect proteins that are acylated with moieties other than palmitate (e.g., shorter, longer, or unsaturated lipid chains).Recently, a "cysteine-centric" approach to identify S -acylated proteins was introduced that uses the conversion of the protein thioester to a disulfi de-linked biotin ( 18, 19 ). This assay, known as acyl-biotin exchange (ABE), is readily Abstract Protein S -acylation is a major posttranslational modifi cation whereby a cysteine thiol is converted to a thioester. A prototype is S -palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modifi cation is a well-recognized determinant of protein function and localization, current techniques to study cellular S -acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S -nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S -acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S -acylation. Acyl-RAC should therefore fi nd general applicability in studies of both global and individual protein S -acylation in mammalian cells. Supplementary key words acylation • H-Ras • lipid • palmitoylation • proteomicsProtein cysteine residues undergo a wide variety of chemical reactions owing to thiol nucleophilicity and redox reactivity. These reactions include S -nitrosylation ( 1, 2 ), S -prenylation ( 3, 4 ), and S -acylation ( 5, 6 ), wh...

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

“…3 H]palmitate and moving on to acylbiotin exchange protocols, acyl-resin-assisted capture with thiopropyl-Sepharose and click chemistry based on metabolic labeling with bioorthogonal palmitate analogs combined with quantitative mass spectroscopy (45,46,49,50).…”

In Silico Screening for Palmitoyl Substrates Reveals a Role for DHHC1/3/10 (zDHHC1/3/11)-mediated Neurochondrin Palmitoylation in Its Targeting to Rab5-positive Endosomes

“…false-positive results). Last, identifying palmitoylation sites by mass spectrometry is not easily accessible to every laboratory (19). Our method used in this study, in silico prediction of palmitoylation sites of proteins, is able to complementally overcome those problems and identify novel palmitoyl substrates.…”

Palmitoylation of caspase-6 by HIP14 regulates its activation

“…42 Indeed, the palmitoylation of CASP6 was significantly decreased by~15% in the YAC128 brains (Figure 2a). In addition, using the acyl resin-assisted capture (ARAC) of palmitoylated proteins, 43 we found a 53% decrease in CASP6 palmitoylation in the HD STHdhQ111 cell line compared with the control STHdhQ7 (Figure 2b). Thus palmitoylation of CASP6 is decreased both in the absence of HIP14 (Hip14 −/− mice) and when HIP14 is dysfunctional in the presence of mutant HTT.…”

“…43 Cells were harvested and immediately frozen and stored at − 80°C. Samples were resuspended in 1 ml RIPA Buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 0.5% sodium deoxycholate, 1% Igepal CA-630, 0.1% SDS, 1 × Roche Complete protease inhibitor tablets, 5 μM ZVAD (Calbiochem, Caspase Inhibitor I)) with 100 mM NEM, sonicated, cleared by centrifugation at 14 000 r.p.m.…”