Name: in vitro ubiquitination and deubiquitination assays of nucleosomal histones
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We thank Diana Adjaoud for technical assistance. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (2015-2020), Genome Quebec (2016-2019) and Genome Canada (2016-2019) to E.B.A. E.B.A. is a senior scholar of the Fonds de la Recherche du Qu&#233;bec-Sant&#233; (FRQ-S). L.M and N.S.K. have a PhD scholarship from the FRQ-S. H.B had a PhD scholarship from the Ministry of Higher Education and from Scientific Research of Tunisia and the Cole Foundation.

1. GSH-agarose Affinity Purification of the GST-RING1B(1-159)-BMI1(1-109) E3 Ubiquitin Ligase Complex
<ol>
	<li>Use the pGEX6p2rbs-GST-RING1B (1-159 aa) - BMI1 (1 -109aa) bacteria expression construct to transform BL21 (DE3) bacteria (see Table of Materials)23. This construct allows the expression of murine RING1B domain 1-159 fused to BMI1 domain 1-109 with GST tag in pGEX-6P-2 backbone.</li>
	<li>Perform an overnight starter culture by inoculating RIL-bacteria expressing GST-R...

<ol>
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There are several advantages of establishing robust in vitro ubiquitination and deubiquitination assays for proteins of interest. These assays can be used to: (i) establish optimal conditions and define minimal requirement for these reactions, (ii) determine enzymatic kinetic and biochemical constants, (iii) define the roles of cofactors or inhibitors that can impact these reactions, (iv) identify interaction interfaces, (v) test the impact of artificial or disease-associated mutations and (vi) establish assay conditions...

Ubiquitination is one of the most conserved post-translational modifications and is critical for a wide variety of organisms including yeast, plants and vertebrates. Ubiquitination consists of the covalent attachment of ubiquitin, a highly conserved 76 amino acid polypeptide, to target proteins and occurs in three sequential steps involving three enzymes, i.e., E1-activating, E2-conjugating and E3 ligase1,2,3. This post-translational modification plays central roles in a wide spectrum of biological processes. Indeed, the E3 ligases, which provide the specificity of the reaction, constitute a large superfamily of enzymes and are the most abundant enzymes of the ubiquitin system4,5,6. The downstream effects of protein ubiquitination depend on nature of the modification: monoubiquitination, multi-monoubiquitination, and linear or branched polyubiquitination. Monoubiquitination is rarely associated with proteasomal degradation, but instead this modification is involved in mediating various signaling events. Polyubiquitination involves the N-terminal or the lysine residues in ubiquitin molecule itself, and the destiny of a polyubiquitinated protein depends on which residue is involved in ubiquitin chain extension. It has long been known that polyubiquitination mediated by lysine 48 of ubiquitin induces proteasomal degradation. On the contrary, polyubiquitination via lysine 63 of ubiquitin is often associated with protein activation7,8,9. Similar to other important post-translational modifications, ubiquitination is reversible and ubiquitin removal from proteins is ensured by specific proteases termed deubiquitinases (DUBs), which have emerged as important regulators of cellular processes2,10. Importantly, many DUBs are highly specialized, and regulate, through deubiquitination, specific substrates, indicating that a fine balance between ubiquitination and deubiquitination is critical for protein function. E3s and DUBs, along with the proteasome degradation machinery and accessory factors, form the Ubiquitin Proteasome System (UPS, with &#62;1200 genes) which regulates major signaling pathways, several of which are associated with cell growth and proliferation, cell fate determination, differentiation, cell migration, and cell death. Importantly, deregulation of several signaling cascades involving ubiquitination promotes tumorigenesis and neurodegeneration diseases5,11,12,13,14.
Ubiquitination plays pervasive roles in chromatin biology and DNA-dependent processes15,16,17. For instance, monoubiquitination of histone H2A on lysine 119 (hereafter H2A K119ub) is a critical post-translational modification involved in transcriptional repression and DNA repair18,19,20,21,22. H2A K119ub is catalyzed by the Polycomb Repressive Complex 1 (PRC1), which plays a key role in the maintenance of epigenetic information and is highly conserved from Drosophila to human. Canonical PRC1 is constituted notably by the RING1B and BMI1, which are the core E3 ubiquitin ligase complex responsible for the above-mentioned ubiquitination event22,23. In Drosophila, H2A monoubiquitination (H2A K118ub which corresponds to H2A K119ub in mammalians) is reversed by the DUB Calypso, which interacts with Additional Sex Comb (ASX) forming the Polycomb-repressive DUB (PR-DUB) complex24. The mammalian ortholog of calypso, BAP1, is a tumor suppressor deleted or inactivated in various human malignancies25,26,27,28,29,30,31,32,33. BAP1 regulates DNA-dependent processes in the nucleus and Calcium-signaling-mediated apoptosis at the endoplasmic reticulum33,34,35,36,37,38,39,40,41,42. BAP1 assembles multi-subunit protein complexes containing transcription regulators notably ASXL1, ASXL2 and ASXL3 (ASXLs), three orthologues of ASX38,43. ASXLs use the DEUBiquitinase ADaptor (DEUBAD) domain, also termed ASXM domain, to stimulate BAP1 DUB activity35,36,44. Hence, ASXLs play important roles in coordinating BAP1 DUB activity at chromatin and more broadly its tumor suppressor function.
Several methods exist to study ubiquitination and deubiquitination processes. Notably, biochemical assays using proteins purified from bacteria remain very powerful in demonstrating direct ubiquitination of, or removal of ubiquitin from, specific substrates. These experiments can be conducted to investigate a range of parameters such as the determining the requirement of minimal complexes, determining reactions kinetics, defining structure/function relationships, and understanding the impact of pathological gene mutations. Here, we provide protocols to conduct ubiquitination and deubiquitination reactions on chromatin substrates with purified components. As a model system, in vitro ubiquitination and deubiquitination of nucleosomal H2A protein is presented. Bacteria-purified proteins assembled in minimal complexes of RING1B/BMI1 and BAP1/DEUBAD are used for ubiquitination or deubiquitination of nucleosomal H2A, respectively.

<table>
	<tbody>
		<tr>
			<td>Amylose agarose beads</td>
			<td>New England Biolabs</td>
			<td>#E8021</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 ml centrifugal filters 10K</td>
			<td>Sigma-Aldrich</td>
			<td>#UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AK119ub (H2Aub)</td>
			<td>Cell Signaling Technology</td>
			<td>#8240</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flag-agarose beads</td>
			<td>Sigma-Aldrich</td>
			<td>#A4596</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-protease cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) CodonPlus-RIL bacteria</td>
			<td>Agilent technologies</td>
			<td>#230240</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>#319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty chromatography column</td>
			<td>Biorad</td>
			<td>#731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flag peptide</td>
			<td>Sigma-Aldrich</td>
			<td>#F3290</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-agarose beads</td>
			<td>Sigma-Aldrich</td>
			<td>#G4510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>ATCC</td>
			<td>#CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>#I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrococcal nuclease (MNase)</td>
			<td>Sigma-Aldrich</td>
			<td>#N3755</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose beads</td>
			<td>ThermoFisher Scientific</td>
			<td>#88221</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methylmaleimide (NEM)</td>
			<td>Bioshop</td>
			<td>#ETM222</td>
			<td></td>
		</tr>
		<tr>
			<td>Pore syringe filter 0.45 &#956;m</td>
			<td>Sarstedt</td>
			<td>#83.1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Polysciences Inc</td>
			<td>#23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX6p2rbs-GST-RING1B(1-159)-Bmi1(1-109)</td>
			<td>Addgene</td>
			<td>#63139</td>
			<td></td>
		</tr>
		<tr>
			<td>Ub Activating Enzyme (UBE1)</td>
			<td>Boston Biochem</td>
			<td>#E-305</td>
			<td></td>
		</tr>
		<tr>
			<td>UBCH5C (UBE2D3)</td>
			<td>Boston Biochem</td>
			<td>#E2-627</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro ubiquitination and deubiquitination assays of nucleosomal histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

GST-BMI1 and RING1B proteins are well produced in bacteria and can be readily extracted in the soluble fraction. Figure 1A shows a Coomassie blue staining for a typical purification of the GST-BMI1-RING1B complex. The GST-BMI1 and RING1B bands migrate at the expected molecular weight, ~45 kDa and ~13 kDa respectively. Notably the E3 ligase complex is highly homogenous with very low levels of bacteria proteins contaminants and/or degradation products. Moreover...

Watch this Scientific Journal Video about In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones at JoVE.com

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Given the complexity of the ubiquitination and deubiquitination reactions inside the cells it is important to establish in vitro assays with defined purified components. This helps to understand the mechanisms of ubiquitination and deubiquitination and also to determine how mutations associated with genes that encode the enzymes responsible for this reaction can cause human disease. We can identify the domains and motifs in these components that drive these reactions.We can also develop high throughput screen assays that can be used to screen for small molecule inhibitors that can be developed as potential therapeutics. To begin bacteria lysis, add PMSF and antiprotease cocktail into the ice cold lysis buffer A.Add 25 millilters of the lysis buffer A into the bottle containing bacteria pellet and resuspend the pellet. Transfer the lysate containing bacteria in a tube for sonication.Use a probe sonicator to sonicate the lysate at 70%output amplitude for 30 seconds, four to five times. Then centrifuge at 21, 000 times g for 20 minutes at four degrees Celsius. After incubating the bacteria lysate with GSH beads for five hours according to the manuscript, transfer the beads along with 10 millilters of buffer into an empty chromatography column.Then, add 1.5 millilters of buffer A containing 25 millimolar glutathione into the column and collect the elution by gravity into 1.5 milliliter microtubes. Repeat the elution procedure four times with fresh buffer A an continue treatment of the eluted fractions according to the manuscript. To begin, resuspend the His-BAP1 and the MBP-DEUBAD bacteria pellets in 25 millilters of ice cold lysis buffer B.Mix 25 millilters of each lysate and incubate on ice for 15 minutes.Sonicate and then centrifuge the lysate at 21, 000 times g for 20 minutes. After lysate centrifugation, filter the lysate through a 0.45 micrometer pore syringe filter into a bottle and mix it with the Nickel NTA beads. Incubate at four degrees Celsius with shaking for five hours.After that, spin down the beads at 25, 000 times g for three minutes. Use a pipette to save the supernatant in a 50 milliliter tube as the flow through. Wash the beads with five millilters of buffer B, containing 20 millimolar imidazole, six times with three minute centrifuge.After the last wash, transfer the beads with 10 millilters of buffer B into an empty chromatography column and add one milliliter of buffer B containing 200 millimolar imidazole. Collect the elution by gravity into 1.5 milliliter microtubes containing DTT and EDTA. Repeat the elution procedure four times and pool the desired eluted fractions into a 15 milliliter tube.Add buffer C to dilute the eluted complex three times. Then transfer the diluted elution to the prepared amylose beads in tubes and incubate overnight at four degrees Celsius with shaking. In the morning, centrifuge the tubes at 2, 500 times g for three minutes and keep the supernatant in a 15 milliliter tube as flow through.Wash the protein bound beads with five millilters of buffer C for five to six times. Then add 500 microliters of buffer D to the purified His-BAP1/MBP-DEUBAD complex on the amylose agarose beads to make a 50%bead solution. Transfer 20 microliters of the 50%bead solution to a microcentrifuge tube and add 20 microliters of two times Laemmli Sample Buffer for SDS-PAGE and Coomassie blue analysis.Store the remaining bead solution at minus 80 degrees Celsius for future usage. After culture the HEK293T cells, plate 12 million cells in 20 millilters of complete media in a 15 centimeter culture dish, one day before the transfection. After one one day, under the cell culture hood, aspirate media and supply with 12 millilters of serum free media.Transfect the cells with 21 micrograms of pCDNA flat-H2A using 63 microliters of PEI at one milligram per milliliter. After 12 hours, change to complete medium. After harvesting the cells according the manuscript, resuspend the cell pellet in about 10 volumes of buffer E containing NEM to inhibit deubiquitinases and you incubate on ice for 20 minutes.After centrifugation at 3, 000 times g for five minutes, discard the supernatant. Wash the chromatin pellet twice with 10 volumes of buffer E first and then wash twice with buffer F.Resuspend the chromatin in five millilters of buffer F and treat the pellet with micrococcal nuclease at the concentration of three units per milliliter for 10 minutes at room temperature. After incubation, transfer a 40 microliter aliquot of the mixture to a 1.5 milliliter tube for analysis of nucleosomal DNA fragments.Add 40 microliters of phenol-chloroform and 20 microliters of six times DNA loading buffer. Vortex and spin at 18, 000 times g for two minutes. Then transfer 15 microliters of the aqueous phase in a 2%agarose gel to load the DNA.After stopping the reaction and centrifugation according to the manuscript, incubate the soluble chromatin fraction with anti-flag resin beads in a 15 milliliter tube overnight at four degrees Celsius with shaking. Wash the beads six times with buffer G.Transfer the beads with five millilters of buffer G into an empty chromatography column. Dilute the bead's bound nucleosomes with 260 microliters of buffer G containing 200 micrograms per milliliter of flag elution peptide and one to five one molar Tris at pH eight.Elute the nucleosomes three times, one hour in the cold room. To perform ubiquitination assay, first add one microgram of the purified nucleosomes in 40 microliters of buffer H.After that, add an enzyme solution mix containing 250 nanograms of UBE1, 672 nanograms of UB and E2 UB conjugating enzymes and one microgram of BMI1-RING1B E3 ubiquitin ligase complex into the tube. Incubate the reaction for three hours at 37 degrees Celsius with occasional shaking.Stop the reaction by adding 40 microliters of two times Laemmli Sample Buffer and analyze histone H2AK119 ubiquitination by western blotting. To perform the in vitro deubiquitination assay resuspend the purified nucleosomes in 40 microliters of buffer I and one microgram of bacteria purified His-BAP1/MBP-DEUBAD beads. Place the tube in an incubator at 37 degrees Celsius for three hours to carry out the deubiquitination reaction with occasional shaking.Then stop the reaction by adding 40 microliters of two times Laemmli buffer and analyze by immunoblotting. In this experiment, Coomassie blue staining for a typical purification of the GST-BM1-RING1B complex using GST affinity beads, shows that the bands migrate at expected molar weight around 45 kilodaltons and 13 kilodaltons respectively. Similarly the purification of His-BAP1 MBP-DEUBAD complex also resulted in relatively high homogenous preparations with bands around 90 kilodaltons and 55 kilodaltons respectively.On the other hand the purified nucleosomal fraction is essentially composed by a 147 base pair band indicating the presence of highly enriched mononucleosomal fraction. Coomassie blue staining of purified nucleosomes shows a typical band pattern of the four histone subunits with stoichiometric amounts. Ubiquitination of histone H2A with the BMI1-RING1B complex shows a time dependent increase of the ubiquitinated form of the protein while the levels of the nonmodified form are concomitantly decreased.The ubiquitination reaction is total as virtually all H2A proteins are transformed to H2A K119ub. On the other hand, deubiquitination assay was conducted native nucleosomes. Following incubation of these nucleosomes with BAP1-DEUBAD a time dependent reduction of H2A K119ub signal was observed.The success of ubiquitination and deubiquitination reactions depend on the manner the protein are purified. We need a good yield and the buffer use for protein resuspension and purification should be compatible with biochemical reactions. When this ubiquitination and deubiquitination assays are well established we can test the affect activators and inhibitors as well as the impact of disease associated gene mutations.This method have been established by the contribution of several laboratories and are very useful to understand the mechanisms of ubiquitination and deubiquitination. Several chemicals are hazardous and need to be manipulated in the hood. This include phenol-chloroform, acids, bases, and reducing agents.

in-vitro-ubiquitination-deubiquitination-assays-nucleosomal

Research

JoVE Journal

Biochemistry

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

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 ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...