Modification Protocols | Protein | Microbial biochemistry | Microbiology

Detection and Analysis of S-Acylated Proteins via Acyl Resin–Assisted Capture (Acyl-RAC)

DA Dina A. Abdulrahman

MV Michael Veit

1993 Views

Apr 5, 2025

Protein palmitoylation is a lipid modification where a palmitoyl group is covalently attached via a thioester linkage to one or more cysteines on a substrate protein. This modification, catalyzed by a group of enzymes named DHHC enzymes after their conserved Asp-His-His-Cys motif, plays a significant role in regulating the localization, stability, and function of a wide range of cellular and viral proteins. By influencing how and where proteins interact within the cell, palmitoylation is essential for various cellular processes, including signaling pathways, membrane dynamics, and protein–protein interactions. Here, we describe the acyl-RAC assay, a biochemical technique designed to specifically enrich and analyze palmitoylated proteins from complex biological samples, such as cell lysates or tissue extracts. The assay begins by reducing and blocking free cysteine thiol groups on proteins, ensuring that only those thiols involved in thioester bonds with palmitates are accessible for downstream analysis. These thioester bonds are then cleaved to release the fatty acids from the cysteines, which are subsequently captured using thiopropyl Sepharose beads that bind to the newly exposed thiol groups. The captured proteins are eluted from the beads by breaking the bond between the thiol and the resin with reducing agents, and the proteins are then analyzed by SDS-PAGE followed by western blotting to identify and quantify them. The acyl-RAC assay's specificity for S-palmitoylated proteins makes it an invaluable tool for exploring this modification. It not only allows for the identification of previously unknown palmitoylated proteins, thereby deepening our understanding of palmitoylation in cellular processes and viral infections, but it also enables quantitative comparisons of protein palmitoylation under different experimental conditions or treatments.

In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors

AS Alan G. Sulpizio

MM Marena E. Minelli

YM Yuxin Mao

0 Q&A

4003 Views

Nov 5, 2020

Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA’s activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA’s modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA’s substrate Rab33b. Prior to the identification and publication of SdeA’s activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA’s activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.

Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ

AS Alan G. Sulpizio

JS Jung-Ho Shin

MM Marena E. Minelli

YM Yuxin Mao

0 Q&A

3888 Views

Oct 5, 2020

The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of post-translational modification, where the amino group of a free glutamate amino acid is covalently linked to the γ-carboxyl group of a glutamate sidechain in a substrate protein. This protocol describes the SidJ glutamylation reaction using radioactive [U-¹⁴C] glutamate and its substrate SdeA, the separation of proteins by gel electrophoresis, preparation of gels for radioactive exposure, and relative quantification of glutamylation activity. This procedure is useful for the identification of substrates for glutamylation, characterization of substrate and glutamylase activities due to mutations, and identification of proteins with glutamylation activity. Some studies have assayed glutamylation with the use of [³H] glutamate (Regnard et al., 1998) and the use of the GT335 antibody (Wolff et al., 1992). However, the use of [U-¹⁴C] glutamate requires a shorter radioactive exposure time with no dependence on antibody specificity.

In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria

KS Krishna K. Singh

DS Devendra P. Singh

RS Rambir Singh

DS Deepak K. Saini

0 Q&A

5707 Views

Jul 5, 2019

Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic intermediates. In this protocol, using response regulator proteins as targets we describe the experimental strategy for probing the occurrence of acetylation using purified recombinant proteins in an in vitro setup. Further using M. smegmatis strains overexpressing the wild type or mutant response regulator protein, we also describe how in vivo acetylation can be validated in Mycobacterial proteins. The described approach can be used for analyzing acetylation of any mycobacterial protein under both in vitro and in vivo conditions.

Characterization of Protein Domain Function via in vitro DNA Shuffling

KP Kathy Hiu Laam Po

EC Edward Wai Chi Chan

SC Sheng Chen

0 Q&A

6696 Views

Jun 5, 2018

We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the bla_CTX-M-14 and bla_CTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective approach to identify and characterize important functional domains in bacterial proteins.

Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System

KL Kuancheng Liu

JH Jianming Hu

0 Q&A

7168 Views

Apr 20, 2018

The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.

ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors

Céline Michard

LL Lawrence L. LeClaire

PD Patricia Doublet

0 Q&A

9244 Views

Apr 5, 2017

The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011; LeClaire et al., 2015). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation (Narayanan et al., 2011; LeClaire et al., 2015). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm (Michard et al., 2015). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.

Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae

David Canadell

Samuel Bru

Josep Clotet

Joaquín Ariño

0 Q&A

12420 Views

Jul 20, 2016

Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the polyP values obtained.

Figure 1. Enzymatic hydrolysis of polyP

In vitro Deneddylation Assay

Anna M. Köhler

Cindy Meister

Gerhard H. Braus

0 Q&A

10209 Views

Mar 20, 2016

Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western hybridization experiments using anti-CulA or anti-Nedd8 antibodies could show the ratio of neddylated vs. deneddylated CulA. Using the deneddylation assay, we could show that CsnE is the last subunit joining a 7-subunit pre-assembled CSN in vitro and only then CSN can perform cullin deneddylation by the metalloprotease subunit CsnE. This assay is a fast and non-expensive method, which visualizes enzyme activity for deneddylating proteins. It might be also useful for testing the activity of other isopeptidases removing post-translational modifications from substrates in Aspergillus nidulans (A. nidulans) or other organisms.

Detection of HBV C Protein Phosphorylation in the Cell

JJ Jaesung Jung

Kyongmin Kim

0 Q&A

8657 Views

Aug 5, 2015

Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with ³²P-orthophosphate for 14 h, and then lysed. The ³²Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the ³²Pi-labeled immunoprecipitated C proteins were detected by autoradiography.