Name: evaluation of substrate ubiquitylation by e3 ubiquitin-ligase in mammalian cell lysates
Uploaded: 2022-05-10T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates at JoVE.com

F.R.T is supported by FAPESP grant number 2020/15771-6 and CNPq Universal 405836/2018-0. P.M.S.P and V.S are supported by CAPES. C.R.S.T.B.C was supported by FAPESP scholarship number 2019/23466-1. We thank Sandra R. C. Maruyama (FAPESP 2016/20258-0) for the material support.

NOTE: An overview of ubiquitylation assay protocol in mammalian cells is represented in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63561/63561fig01.jpg" /> 
Figure 1. Overview of the ubiquitylation assay procedure. <a href="https://www.jove.com/files/ftp_upload/63561/63561fig01large.jpg" target="_blank">Please click here to view a larger version...

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Ubiquitylation is an essential post-translational modification that regulates the levels of several proteins and plays a crucial role in many signaling pathways and biological processes, ensuring a healthy intracellular environment. The ubiquitin-proteasome system (UPS) is one of the main focuses of recent pharmaceutical research, providing the possibility of stabilizing tumor suppressors or inducing the degradation of oncogenic products22. For instance, the aberrant proliferation of plasma cell n...

Post-translational modifications (PTMs) are an important mechanism regarding protein regulation, which is essential for cell homeostasis. Protein ubiquitylation is a dynamic and intricate modification that creates an assortment of different signals resulting in several cellular outcomes in eukaryotic organisms. Ubiquitylation is a reversible process consisting in the attachment of a ubiquitin protein containing 76 amino acids to the substrate, occurring in an enzymatic cascade composed by three distinct reactions1. The first step is characterized by ubiquitin activation, which depends on an ATP hydrolysis to form a high-energy thioester-linked ubiquitin between the ubiquitin C-terminus and the cysteine residue present in the active site of the E1 enzyme. Subsequently, the ubiquitin is transferred to the E2 enzyme forming a thioester-liked complex with the ubiquitin. Afterward, the ubiquitin is covalently attached to the substrate by the E2, or more often, by the E3 enzyme, which recognizes and interacts with the substrate2,3. Occasionally, E4 enzymes (Ubiquitin-chain elongation factors) are necessary to promote multiubiquitin chain assembly3.
Ubiquitin has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), allowing the formation of polyubiquitin chains that generate distinct linkages to produce different tridimensional structures that are going to be recognized by several effector proteins4,5. Hence, the kind of polyubiquitin chain introduced in the substrate is essential to decide its cell fate6,7,8. Moreover, the substrate could also be ubiquitinated through its N-terminal residues called N-degrons. Specific E3 ubiquitin-ligases are responsible for N-degron recognition, allowing the polyubiquitylation of nearby lysine residue9.
Nowadays, there are more than 40 different SCF-specific substrates characterized. Among those, key regulators of several biological pathways, including cell differentiation and development as well as cell survival and death, can be found10,11,12,13. Thus, the identification of specific substrates of each E3 ubiquitin-ligase is essential to design a comprehensive map of various biological events. Even though the identification of true substrates is biochemically challenging, the use of biochemistry-based methods is very suitable to evaluate chain specificity and the distinction between mono- and polyubiquitylation14. This study describes a complete protocol for ubiquitylation assay using the mammalian cell line HEK293T overexpressing the substrate UXT-V2 (Ubiquitously expressed prefoldin-like chaperone isoform 2) with the E3 ubiquitin-ligase complex SCF(Fbxo7). UXT-V2 is an essential co-factor for NF-&#954;B signaling, and once this protein is knocked down in cells, it inhibits TNF-&#945;-induced NF-&#954;B activation11. Thus, to detect polyubiquitylated UXT-V2, the proteasome inhibitor MG132 is used since it has the ability to block the proteolytic activity of the 26S subunit of the proteasome complex15. Furthermore, the cell extract is submitted to a small-scale IP to purify the substrate, utilizing a specific antibody immobilized to agarose resin for subsequent detection by WB using selected antibodies. This protocol is very useful to validate substrate ubiquitylation in the cellular environment, and it can also be adapted for different types of mammalian cells and other E3 ubiquitin-ligase complexes. However, it is necessary to validate the substrate tested through an in vitro ubiquitylation assay as well, since both protocols complement each other regarding the identification of true substrates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microtube</td>
			<td>Axygen</td>
			<td>PMI110-06A</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-treated culture dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Corning</td>
			<td>430766</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Cralplast</td>
			<td>655111</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose-anti-HA beads</td>
			<td>Sigma-Aldrich</td>
			<td>E6779</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Mouse antibody</td>
			<td>Seracare</td>
			<td>5220-0341</td>
			<td>Goat anti-Mouse IgG</td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody</td>
			<td>Seracare</td>
			<td>5220-0337</td>
			<td>Goat anti-Rabbit IgG</td>
		</tr>
		<tr>
			<td>Anti-Actin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A3853</td>
			<td>Dilution used: 1:2000</td>
		</tr>
		<tr>
			<td>Anti-Fbxo7 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB1407251</td>
			<td>Dilution used: 1:1000</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Sigma-Aldrich</td>
			<td>H3663</td>
			<td>Dilution used: 1:1000</td>
		</tr>
		<tr>
			<td>Anti-Myc antibody</td>
			<td>Cell Signalling</td>
			<td>2272</td>
			<td>Dilution used: 1:1000</td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-100G</td>
			<td>Bovine Serum Albumin</td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Nuaire</td>
			<td>NU-4850</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>500 x g for 5 min</td>
		</tr>
		<tr>
			<td>ChemiDoc</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital pH meter</td>
			<td>Kasvi</td>
			<td>K39-2014B</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Corning</td>
			<td>10-017-CRV</td>
			<td>High glucose</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>F4135</td>
			<td>Filtrate prior use</td>
		</tr>
		<tr>
			<td>HA peptide</td>
			<td>Sigma-Aldrich</td>
			<td>I2149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR Life Science</td>
			<td>0365-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kline rotator</td>
			<td>Global Trade Technology</td>
			<td>GT-2OIBD</td>
			<td></td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>Boston Biochem</td>
			<td>I-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5418R</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4 (Ortovanadato)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose blotting membrane</td>
			<td>GE Healthcare</td>
			<td>10600016</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40 (IGEPAL CA-630)</td>
			<td>Sigma-Aldrich</td>
			<td>I8896-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>OPTIKA microscopes</td>
			<td>SN510768</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3</td>
			<td>Invitrogen</td>
			<td>V79020</td>
			<td>For mammalian expression</td>
		</tr>
		<tr>
			<td>pcDNA3-2xFlag-Fbxo7</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI</td>
		</tr>
		<tr>
			<td>pcDNA3-2xFlag-Fbxo7-&#916;F-box</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI. &#916;335-367</td>
		</tr>
		<tr>
			<td>pcDNA3-UXTV2-HA</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag HA (C-terminal). Restriction enzymes: EcoRI and XhoI</td>
		</tr>
		<tr>
			<td>pCMV-6xHis-Myc-Ubiquitin</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag 6x-His-Myc (N-terminal). Restriction enzymes: EcoRI and KpnI</td>
		</tr>
		<tr>
			<td>Pen Strep Glutamine 100x</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 10x</td>
			<td>AccuGENE</td>
			<td>51226</td>
			<td>To obtain a 1x PBS, dilute the 10x PBS into ultrapure water</td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>9002-98-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>VWR Life Science</td>
			<td>0860-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail SIGMAFAST</td>
			<td>Sigma-Aldrich</td>
			<td>S8820</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker</td>
			<td>Kasvi</td>
			<td>19010005</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BioRad</td>
			<td>165-8004</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution Homogenizer</td>
			<td>Phoenix Luferco</td>
			<td>AP-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6066-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine (TrypLe Express)</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blotting Luminol Reagent</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2048</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of substrate ubiquitylation by e3 ubiquitin-ligase in mammalian cell lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

UXT (ubiquitously expressed transcript) is a prefoldin-like protein that forms ubiquitously expressed protein-folding complexes in mouse and human tissues such as heart, brain, skeletal muscle, placenta, pancreas, kidney, and liver18. Two splicing isoforms of UXT, which are named UXT-V1 and UXT-V2, have been described performing distinct functions and subcellular locations. UXT-V1 is predominantly localized in the cytoplasm and inside the mitochondria, and it is implicated in TNF-&#945;-induced ap...

Watch this Scientific Journal Video about Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates at JoVE.com

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

This protocol allows to evaluate the ubiquitylation of a specific substrate by an E3 ligase. We know that the characterization of ligase-substrate interaction is crucial to understanding their function in cells. This technique does not require expensive reagents or sophisticated equipment.It needs plasmids and coding the genes of interest, developing an immunoprecipitation assay with cell lysates, and a Western blot to detect substrate ubiquitylation. Several human disease like cancer and neurological disorders are associated with dysregulation of E3 ligase. Hence, the identification of substrate ubiquitylation by specific E3 ligase could assist in treating or diagnosing those diseases.Demonstrating the procedure will be by Valentine Spagnol. To begin, grow HEK293T cell line to 80 to 90%confluence in Dulbecco's modified Eagles medium supplemented with 10%fetal bovine serum and 100 units penicillin, 100 micrograms streptomycin, and 0.292 milligrams per milliliters L-glutamine. Then, incubate this culture at 37 degrees Celsius in a humidified cell culture incubator at 5%carbon dioxide.Passage the cells by aspirating the media from the culture dish using a serological pipette and wash them once with one milliliter of sterilized 1X PBS. Next, detach the cells by adding one milliliter of trypsin ethylenediaminetetraacetic acid solution and after five minutes of incubation at 37 degrees Celsius, resuspend these cells in two milliliters of growth media using a serological pipette. Transfer the cell suspension to a fresh and clean 15-milliliter tube and centrifuge at 500 times G for five minutes at room temperature.Then, gently remove the supernatant and resuspend the cell pellet in three milliliters of growth media by pipetting up and down to obtain a homogenous cell suspension. Then, transfer one milliliter of this cell suspension to a 100-millimeter TC-treated culture dish containing nine milliliters of growth media. Before transfection, certify if the cells are free from contamination and at an adequate confluence for transient transfections.For each transfection sample, prepare the DNA-polyethyleneimine complexes by diluting three micrograms of each plasmid in 100 microliters of Opti-MEM 1 reduced serum medium without supplementation and mixing it gently by pipetting the solution up and down. Next, thaw the DNA-polyethyleneimine at room temperature and add it into the solution following a proportion of three microliters of DNA-polyethyleneimine per one microgram of DNA. Homogenize the solution by pipetting up and down.Then, incubate it for 15 minutes at room temperature to allow the formation of DNA-polyethyleneimine complexes. Add the total volume of DNA-polyethyleneimine complexes to each dish containing the cell culture and mix it gently by rocking the plate back and forth. Then, incubate these cells at 37 degrees Celsius in a humidified cell culture incubator.After the incubation and six hours prior to cell lysis, treat the transfected cells with 10-micromolar proteasome inhibitor MG132 and incubate the cells in the humidified cell culture incubator. Next, aspirate the media from each culture dish using a serological pipette and wash the cells once with one milliliter of 1X PBS. Then, detach the cells by adding one milliliter trypsin and incubate the dish at 37 degrees Celsius for five minutes.Resuspend the cells in one milliliter of growth media and transfer the cell suspension into a fresh and clean two-milliliter microtube and centrifuge it at 500 times G for five minutes at room temperature. Once centrifugation is over, remove the supernatant by pouring it out carefully and gently resuspend the cell pellet in 200 microliters of ice-cold MP40 lysis buffer supplemented with the protease and phosphatase inhibitors cocktail. Then transfer this solution into a clean 1.5-milliliter microtube.Incubate this cell lysate for 30 minutes on ice, and after the incubation, centrifuge the cell lysates at 16, 900 times G for 20 minutes at four degrees Celsius. Meanwhile, equilibrate the agarose anti-HA beads with ice-cold MP40 lysis buffer. Use 15 microliters of agarose anti-HA beads for each sample.Wash the beads with 200 microliters of MP40 lysis buffer by pulsing them in a micro-centrifuge tube at 3, 000 times G for one minute at four degrees Celsius. Next, aspirate and discard the supernatant carefully with a pipette and repeat this process three times. Afterward, keep the beads equilibrated on ice until use.After centrifuging the cell lysates, recover the supernatant and quantify the protein content in the total lysate using the Bradford method to ensure that each sample subjected to immunoprecipitation presents an equal amount of protein. Incubate the necessary volume of cell lysate with the equilibrated agarose anti-HA beads for four hours, gently rotating in a rotating incubator at four degrees Celsius, which allows the UXT-V2-HA to bind to the agarose anti-HA beads. Collect the agarose anti-HA beads by pulsing them in a microcentrifuge tube at 3, 000 times G for one minute at four degrees Celsius.Carefully aspirate and discard the supernatant. Wash the beads three times with ice-cold MP40 cell lysis buffer and twice with ice-cold FLAG HA buffer. After the final wash, remove all the supernatant carefully using a pipette and elute the poly-ubiquitylated protein with 300 micrograms per milliliter of HA peptide diluted on FLAG HA buffer.Incubate the agarose anti-HA beads with HA peptide for one hour at four degrees Celsius in a rocking shaker platform. Next, spin down the beads at 3, 000 times G for two minutes at four degrees Celsius and carefully remove the supernatant containing the poly-ubiquitinated proteins. If necessary, store the eluent in a fresh and clean microtube at minus 20 degree Celsius.Resolve the eluents and the cell lysates in 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. To perform a wet transfer Western blotting, place the gel in a transfer sandwich composed of filter paper, gel membrane filter paper, cushion it with pads, and press it together by a support grid. Then, place this system vertically in a tank filled with transfer buffer in between stainless steel or platinum wire electrodes.The transfer occurs for 90 minutes at 150 volts in a wet transfer buffer. Finally, probe the immunoblot membrane using anti-myc antibody. The specific poly-ubiquitylation of UXT-V2-HA mediated by F-box protein 7 in cells was observed after probing the eluent from anti-HA immunoprecipitation with an anti-myc antibody, which detects the myc-ub conjugated to the substrate.The specificity of anti-myc for poly-ubiquitylated substrate was visualized in lanes one and two, wherein a smear of poly-ubiquitylated proteins was not detected when cells were co-transfected with F-box protein 7 and myc-ub in the absence of UXT-V2-HA plasmid. Additionally, no smear corresponding to protein poly-ubiquitination was visualized in the combination of F-box protein 7 and UXT-V2-HA without the myc-ub plasmid. When an empty-vector wild-type F-box protein 7 or F-box protein 7 mutant unable to assemble active SCF was transected in combination with UXT-V2-HA and myc-ub, a strong smear signal of poly-ubiquitylated UXT-V2 was observed only for wild-type F-box protein 7, suggesting that UXT-V2 was poly-ubiquitylated by SCF F-box protein 7 complex and cells compared to the controls.The mono-precipitation steps, including incubation of cell lysates with the equilibrated beads and elution of immunoprecipitated proteins are essential to achieve the aiming of this protocol. After this procedure, in vitro ubiquitylation assay should be performed to confirm the specificity of the substrate ubiquitylation by the E3 ligase.

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Research

JoVE Journal

Biochemistry

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson&#039;s Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...