Name: acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins
Uploaded: 2020-03-29T14:00:00
Description: Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

The authors thank K. Woolfrey for advice and input on the APEGS assay. These studies were funded by research grants to E.D. from the Whitehall Foundation and the NIH-NIMH (1R01MH119347).

All animal procedures followed guidelines set forth by the National Institutes of Health (NIH) and have been approved by the Institutional Animal Care and Use Committee at the University of California, Davis.
1. Preparation of mouse brain membranes
<ol>
	<li>Decapitate mouse using a guillotine apparatus and dissect brain out rapidly (in &#60;1 min, if possible, to minimize palmitoylation changes that may occur during dissection procedure). Homogenize immediately in 10 mL of homogenization bu...

<ol>
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In our previous work, we utilized the ABE assay to demonstrate that SynDIG1 is palmitoylated at two conserved juxta-transmembrane Cys residues (found in all SynDIG proteins) in an activity-dependent manner to regulate stability, localization, and function6. A limitation is that the ABE assay requires affinity purification with agarose resins conjugated to avidin moieties as the final step in the procedure, resulting in significant loss of signal that complicate quantitative analysis. Furthermore, ...

S-palmitoylation is a reversible post-translational modification of target proteins that regulates stable membrane association, protein trafficking, and protein-protein interactions1. It involves addition of a 16-carbon palmitate moiety to cysteine residues via thioester linkage catalyzed by palmitoyl acyltransferase (PAT) enzymes. Many synaptic proteins in the brain are palmitoylated, including AMPA-Rs and PSD-95, in an activity-dependent manner to regulate stability, localization, and function2,3,4. Alterations in the levels of synaptic AMPARs in the PSD via interaction of auxiliary factors with synaptic scaffolds such as PSD-95 underlies synaptic plasticity; thus, methods to determine the palmitoylation state of synaptic proteins provides important insight into mechanisms of synaptic plasticity.
Previously, we identified the SynDIG family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs5. Overexpression or knock-down of SynDIG1 in dissociated rat hippocampal neurons increases or decreases, respectively, AMPA-R synapse size and number by ~50% as detected using immunocytochemistry and electrophysiology5. We utilized the acyl-biotin exchange (ABE) assay to demonstrate that SynDIG1 is palmitoylated at two conserved juxta-transmembrane Cys residues (found in all SynDIG proteins) in an activity-dependent manner to regulate stability, localization, and function6. The ABE assay relies on exchange of biotin on cysteines protected by modification and subsequent affinity purification7. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay8,9,10,11,12, which does not require affinity purification and instead utilizes changes in gel mobility to determine the number of modifications for a protein of interest. The protocol is described for investigation of endogenous membrane proteins from mouse brain for which suitable antibodies are available.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydroxylamine (HAM)</td>
			<td>ThermoFisher</td>
			<td>26103</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL (mPEG)</td>
			<td>NOF</td>
			<td>ME-050MA</td>
			<td>MW ~5000 kDa</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>N-ethylmalemide (NEM)</td>
			<td>Calbiochem</td>
			<td>34115</td>
			<td>Highly toxic.</td>
		</tr>
		<tr>
			<td>Optical imager for densitometry</td>
			<td>Azure Biosystems</td>
			<td>Sapphire Biomolecular Imager</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Polypropylene tubes with cap</td>
			<td>Fisher Scientific</td>
			<td>14-956-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (glass)</td>
			<td>Fisher Scientific</td>
			<td>13-678-27D</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Beckman</td>
			<td>Allegra X-15R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP)</td>
			<td>EMD Millipore</td>
			<td>580560</td>
			<td></td>
		</tr>
	</tbody>
</table>

acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Immunoblotting with antibodies against the protein of interest reveals the palmitoylated state (non, singly, doubly, etc.) in mouse brain lysates as determined by mobility shift compared with samples in which HAM was not included. Previously, we had demonstrated that SynDIG1 was palmitoylated at two sites using the ABE assay6; however, we could not determine whether both sites were modified in brain tissue. Here we show that SynDIG1, SynDIG4/Prrt1, and Prrt2 are pa...

Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

This biochemical method allows for the determination of the palmitoylation state of any membrane protein expressed in the brain for which a suitable antibody is available. The main advantage of this technique is that it does not require affinity purification and instead utilizes changes in gel mobility to determine the number of modifications of a protein of interest. After dissecting out the brain, homogenize it immediately in 10 milliliters of homogeneization buffer in a glass homogenizer on ice.Using approximately 12 strokes, centrifuge the lysates for 15 minutes at 1400 times G, and four degrees Celsius. Transfer the supernatant to a new tube on ice. Resuspend the pellet in 10 milliliters of homogeneization buffer.Homogenize it using approximately six strokes. Centrifuge the suspension at 710 times G and four degrees Celsius for 10 minutes. Combine the supernatants and centrifuge them at 40, 000 times G and four degrees Celsius for 20 minutes.Discard the supernatant and resuspend the pelleted membrane fraction in a homogeneization buffer by breaking up the pellet with a pipette tip and pipetting up and down. Perform ABCA assay to quantitate protein levels. To perform the APEGS assay, place 470 microliters of buffer A in a 1.5 milliliter tube and add approximately 100 to 200 milligrams of protein.Sonicate the tube and then separate the insoluble protein by centrifuging the tube at 25 degrees Celsius and a minimum of 13, 000 times G for 10 minutes. Transfer the solubalized protein to a new 1.5 milliliter tube. First, disrupt the disulphide bonds by incubating the solubalized protein in 25 microliters of TCEP for 60 minutes at 55 degrees Celsius.Then, block free cysteines by incubating the solution with 12.5 microliters of the stock solution of NEM for three hours at room temperature. Next, begin the process of chloroform methanol precipitation. Transfer the protein solution from the 1.5 milliliter tube to a polypropylene or glass tube that can be centrifuged in a swinging bucket rotor at modest speed.Add two milliliters of methanol and vortex briefly. Add one milliliter of chloroform and vortex briefly. Then add 1.5 milliliters of deionized water and vortex briefly again.If necessary, invert the tube to ensure thorough mixing. Centrifuge the samples at 3000 times G and 25 degrees Celsius for 30 minutes in a swinging bucket rotor. Carefully remove and discard the upper phase of the solution in a tube.Add 1.5 milliliters of methanol. Mix gently but thoroughly by gentle inversion of the tube, being careful not to fragment the opaque protein pancake. Centrifuge the sample at 3000 times G and 25 degrees Celsius for 10 minutes in a swinging bucket rotor.Using a glass serological pipette, remove as much of the top phase of the solution as possible without disturbing the protein pancake. Carefully rinse the pellet with one milliliter of methanol and allow it to air dry for at least 10 minutes. With the chloroform methanol precipitation complete, begin the cleavage of the palmitoyl thioester linkages by resuspending the protein precipitate in 125 microliters of buffer A.Transfer to a new 1.5 milliliter tube.Sonicate the tube briefly and then centrifuge it at 13, 000 times G or higher and 25 degrees Celsius for 10 minutes to remove insoluble material. Transfer the solubalized protein to new 1.5 milliliter tubes and add 375 microliters of buffer H or buffer T.After incubating the samples for 60 minutes at room temperature, repeat the chloroform methanol precipitation. To add m-PEG to unprotected cysteines, begin by resuspending the pellet in 100 microliters of buffer A containing 10 mil or more TCEP.Then transfer the suspension to a 1.5 milliliter tube. Add 25 microliters of stock m-PEG 5K solution and mix by pipetting. Incubate at room temperature with end over end rotation.After 60 minutes of incubation, remove unincorporated m-PEG 5K by performing chloroform methanol precipitation again. Centrifuge at greater than 13, 000 times G and 25 degrees Celsius for 10 minutes. Carefully remove the upper phase as before, avoiding the thick, flocculent pancake.Add one milliliter of methanol and mix gently but thoroughly. Centrifuge at greater than 13, 000 times G and 25 degrees Celsius for 10 minutes. Carefully remove the supernatant and rinse the pellet with one milliliter of methanol.Again, centrifuge at greater than 13, 000 times G and 25 degrees Celsius for 10 minutes. After air drying the pellet, resuspend it in 50 microliters of buffer A without TCEP or any reducing agent. Reserve 5 microliters of the solution for BCA protein quantitation.After quantitation, add an appropriate amount of 4x Laemmli sample buffer. Load the sample onto an SDS page gel. Use standard separation transfer and detection protocols for western blotting.To investigate the palmitoylation state of SynDIG proteins, P2 membrane fractions from homogenized mouse brains were subjected to the APEGS assay. Separation and probing with antibodies demonstrated that SynDIG1, SynDIG4 Prrt1, and Prrt2 are palmitoylated in the mouse brain. The purpose of the chloroform methanol precipitation is to prevent one chemical, such as NEM, interfering with the next step of the protocol.A proper chloroform methanol precipitation will remove undesirable chemicals while minimizing protein loss. This method can be applied to brain lysates from mice of different ages or from different brain regions to investigate the spatial-temporal regulations of palmitoylation.

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