We thank the members of our laboratory for critical discussion and helpful advice on the manuscript. We apologize for not having cited valuable contributions due to size limitation. This work is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - SFB 1218 - Projektnumber 269925409 and Cluster of Excellence EXC 229/ CECAD to TH. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany&#180;s Excellence Strategy - EXC 2030 - 390661388 and - SFB 1218 - Projektnumber 269925409 to T.H. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) im Rahmen der deutschen Exzellenzstrategie - EXC 2030 - 390661388 und - SFB 1218 - Projektnummer: 269925409 an T.H. gef&#246;rdert.

1. Preparation of buffers and reagents
NOTE: Buffers and reagents that were manually prepared in the laboratory are listed below. All other buffers and reagents used in the protocols were purchased from different sources and used according to the manufacturers&#39; instructions.
<ol>
	<li>Prepare 10x phosphate-buffered saline (10x PBS). For this purpose, mix 1.37 M sodium chloride (NaCl), 27 mM potassium chloride (KCl), 80 mM of disodium-hydrogen-phosphate dihydrate (Na2HPO4.2 H2O), and 20 mM of potassium-dihydrogen phosphate (KH2PO4) in 1 L of double-distilled H2O (ddH2O). Autoclave the 10x PBS, and prepare a 1x PBS solution in ddH2O.
	<ol>
		<li>Autoclaving is carried out for 15 min at 121 &#176;C and 98.9 kPa. 
		NOTE: Autoclaving is carried out under these conditions for all buffers and solutions. If not indicated otherwise, solutions are stored at room temperature (RT).</li>
	</ol>
	</li>
	<li>Prepare 1 L of PBS-Tween (PBS-T) solution by adding 0.1% Tween to 1 L of 1x PBS.</li>
	<li>Prepare 10 mL of a 0.5 M L-lysine stock solution by dissolving L-lysine in ddH2O. Aliquot L-lysine in 1 mL portions, and store them at -20 &#176;C until further use. 
	NOTE: L-lysine can be frozen and thawed repeatedly.</li>
	<li>Prepare a 0.25 M ethylene diamine tetra acetic acid (EDTA) solution by dissolving EDTA in 200 mL of ddH2O.
	<ol>
		<li>Adjust the pH to 8.0 with sodium hydroxide (NaOH).</li>
		<li>Adjust the volume to 250 mL with ddH2O.</li>
		<li>Autoclave the solution.</li>
	</ol>
	</li>
	<li>Prepare 10 mL of a 20 mg/mL bovine serum albumin (BSA) solution by dissolving BSA in ddH2O. Store at -20 &#176;C in 200 &#181;L aliquots. 
	NOTE: BSA can be frozen and thawed repeatedly.</li>
	<li>Prepare 1 L of 2-(N-morpholino)ethane sulfonic acid (MES) running buffer by dissolving 50 mM MES, 50 mM Tris base, 3.47 mM sodium dodecyl sulfate (SDS), and 1.03 mM EDTA in 0.9 L of ddH2O. After mixing properly, adjust the volume to 1 L.
	<ol>
		<li>The pH of the 1x buffer is 7.6. Do not adjust the pH with acid or base.</li>
	</ol>
	</li>
	<li>Prepare 200 mL of blocking solution (5% milk) by dissolving 10 g milk powder in 1x PBS-T. Store the blocking solution at 4 &#176;C for up to one week.</li>
	<li>Prepare 1 L of transfer buffer (see the Table of Materials) according to the manufacturers&#39; instruction.</li>
	<li>Prepare 2x SDS sample buffer by dissolving 125 mM Tris base, 4% SDS, 4% glycerol, 0.03% bromophenol blue, and 50 &#181;L/mL of &#946;-mercaptoethanol in 50 mL ddH2O. Aliquot in 1 mL portions, and store at -20 &#176;C until use.</li>
</ol>
2. In vitro auto-ubiquitylation assay
<ol>
	<li>Assay preparation and execution
	<ol>
		<li>Calculate the volume of each reagent required per reaction based on the given molarities of the proteins and the protein concentrations of the protein solutions. Per auto-ubiquitylation reaction, use 100 nM E1, 1 &#181;M E2, 1 &#181;M E3, and 50 &#181;M Ub. Adjust the volume of the reaction to 20 &#181;L with ddH2O. 
		NOTE: ATP solution and ubiquitylation buffer were purchased as 10x stock solutions and used at 1x.</li>
		<li>Prepare a pipetting scheme for all reactions. Test nine different E2 enzymes for their ability to function (I) with CHIP, (II) with a catalytically inactive CHIP(H260Q) mutant, and (III) without CHIP in individual ubiquitylation reactions.</li>
		<li>To avoid pipetting errors, prepare a master mix on ice that contains the volume required for all reactions plus one extra reaction. Calculate the amount of master mix required per reaction. 
		NOTE: Master mix components are reagents that are equally required for each reaction, i.e., E1, E3, Ub, ATP, and ubiquitylation buffer.</li>
		<li>Add ddH2O and master mix to the polymerase chain reaction (PCR) tubes. Keep the tubes on ice.</li>
		<li>Before starting the ubiquitylation reactions by adding the E2 enzymes, set up a PCR thermal cycler with the following program: 2 h, 37 &#176;C followed by 4 &#176;C, infinitely.</li>
		<li>Add 1 &#181;M of the E2 enzymes in the respective tubes, and incubate the samples in the PCR thermal cycler for the indicated period of time.</li>
		<li>After 2 h, add SDS sample buffer to each reaction, and mix by pipetting up and down several times. 
		NOTE: The required volume of the sample buffer depends on the stock concentration of the sample buffer. Here, 20 &#181;L of 2x SDS sample buffer were added to each reaction.</li>
		<li>Boil the samples immediately at 95 &#176;C for 5 min, and continue with polyacrylamide gel electrophoresis (PAGE). Store the denatured proteins at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Gel electrophoresis and western blot
	<ol>
		<li>After thawing, spin down the samples for approximately 10 s and load the SDS-PAGE gel. Use a protein ladder as size reference. 
		NOTE: Here, 4-12% Bis-Tris gradient gels and 3 &#181;L of protein ladder were used. Divide the sample volumes and load two gels equally using 20 &#181;L of each sample.</li>
		<li>Run the gel at 160-200 V for approximately 30-45 min so that the sample front reaches the bottom of the gel.</li>
		<li>Transfer each gel to a plastic container filled with semidry blotting buffer, and incubate it for 2-5 min at RT. Remove the stacking gel.</li>
		<li>Prepare two pieces of gel-sized blotting paper and a gel-sized nitrocellulose membrane per SDS gel and pre-soak in semidry blotting buffer. Assemble the western blot (WB) sandwich in a WB chamber from bottom to top in the following order: blotting paper, nitrocellulose membrane, SDS-PAGE gel, blotting paper.</li>
		<li>Remove air bubbles that may have been trapped between the sandwich layers. For this purpose, use a roller to carefully roll over the sandwich a few times.</li>
		<li>Close the chamber, and allow excess blot buffer to drain out.</li>
		<li>Place the chamber in the respective blotting device, and use the following program for semidry blotting: 25 V, 1.0 A, 30 min.</li>
		<li>Transfer the blotted membrane to a plastic container filled with blocking solution, and incubate for 30 min at RT.</li>
		<li>Prepare the primary antibody solution by adding the antibody to 10 mL of PBS-T containing a blocking reagent (see the Table of Materials). 
		NOTE: The working concentration of the antibody is antibody-specific. In this case, a monoclonal mouse anti-ubiquitin antibody was used at a dilution of 1:5,000. For the second gel, a monoclonal rabbit anti-CHIP antibody was used at 1:5,000 in PBS-T/blocking reagent.</li>
		<li>Replace the blocking solution with the primary antibody solution, and incubate overnight at 4 &#176;C on a rocker.</li>
		<li>Wash the membrane three times for 10 min with PBS-T.</li>
		<li>Prepare the secondary antibody solution by adding the respective antibody to 10 mL of PBS-T. 
		&#8203;NOTE: Here, goat anti-mouse and mouse anti-rabbit antibodies are used at a dilution of 1:10,000.</li>
		<li>Incubate the membrane with the secondary antibody for 1 h at RT on a rocker.</li>
		<li>Wash the membrane three times for 5 min with PBS-T.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Add western blot detection reagents for horseradish peroxidase (HRP)-conjugated antibodies to the washed membrane according to the manufacturer&#39;s instructions, and capture the HRP signal by using X-ray films or a charge-coupled device camera (Figure 1).</li>
	</ol>
	</li>
</ol>
3. In vitro substrate ubiquitylation assay
<ol>
	<li>Assay preparation and execution
	<ol>
		<li>Calculate the volume of each reagent required per reaction, based on the given molarities of the proteins and the protein concentrations of the protein solutions. Per substrate ubiquitylation reaction, use 100 nM E1, 1 &#181;M E2, 1 &#181;M E3, 1 &#181;M substrate, and 50 &#181;M Ub. Use 10x ATP solution and 10x ubiquitylation buffer at 1x. Adjust the volume of the substrate ubiquitylation reaction to 20 &#181;L with ddH2O.</li>
		<li>Prepare a pipetting scheme for all reactions. Include proper control reactions verifying that the substrate ubiquitylation is E3-specific. Use the protein UNC-45B as a representative substrate of CHIP and a catalytically inactive CHIP mutant (H260Q) as a control. Prepare the following reactions: E1, E2, Ub mix (including Ub, ATP, ubiquitylation buffer); E1, E2, Ub mix, UNC-45B; E1, E2, Ub mix, CHIP(H260Q), UNC-45B; and E1, E2, Ub mix, CHIP, UNC-45B.</li>
		<li>To avoid pipetting errors, prepare a master mix on ice that contains the volume required for all reactions plus one extra reaction. Calculate the volume of the master mix that is required per reaction. 
		NOTE: Master mix components for this assay include E1, E2, Ub, ATP, and ubiquitylation buffer.</li>
		<li>Add reaction components in the following order: ddH2O, substrate, E3 ligase.</li>
		<li>Before starting the ubiquitylation reaction by addition of the master mix, set up a PCR thermal cycler with the following program: 2 h, 37 &#176;C followed by 4 &#176;C, infinitely.</li>
		<li>Add the master mix to each tube, and incubate the samples in the PCR thermal cycler for the indicated period of time.</li>
		<li>After 2 h, add 2x SDS sample buffer to each reaction, and mix by pipetting up and down several times.</li>
		<li>After the run, boil the samples immediately at 95 &#176;C for 5 min, and continue with PAGE. Alternatively, store the denatured proteins at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Gel electrophoresis and western blot
	<ol>
		<li>Perform gel electrophoresis and western blot as described in section 2.2. Use specific antibodies for the detection of the substrate and the E3 ligase, respectively. 
		NOTE: Here, UNC-45B is fused to a polypeptide protein tag derived from the c-myc gene product allowing for the use of a monoclonal mouse anti-MYC antibody. Anti-MYC is used at 1:10,000.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Perform data analysis as described in section 2.3 (Figure 2).</li>
	</ol>
	</li>
</ol>
4. Lysine discharge assay
<ol>
	<li>Assay preparation and execution
	<ol>
		<li>Charging of the E2 enzyme with Ub by E1
		<ol>
			<li>Calculate the volume of each reagent required per reaction based on the given molarities of the proteins and the protein concentrations of the protein solutions. Per charging reaction, use 2 &#181;M E1, 4 &#181;M E2, and 4 &#181;M lysine-free ubiquitin (Ub K0). Use 10x ATP and 10x ubiquitylation buffer at 1x. Adjust the volume of the charging reaction to 20 &#181;L with ddH20. 
			NOTE: The lysine-free Ub K0 mutant is used to enforce the exclusive production of mono-ubiquitylated E2. Wild-type Ub can also be used; however, this might lead to diverse E2~Ub modifications (e.g., poly-ubiquitylation of E2), which are more difficult to analyze. For first time use or when using a new E2 enzyme, determine the level of Ub charging on E2 that can be achieved by E1. To determine the yield of charging, visualize uncharged vs. charged E2 via Coomassie staining. Here, approximately half of Ub K0 was reliably converted to UBE2D2~Ub when using equimolar ratios of UBE2D2 and Ub K0 (Supplemental Figure S2A).</li>
			<li>Prepare a pipetting scheme for the charging reaction. Adjust the volume of the charging reaction to the subsequent discharge reactions of choice, e.g., use CHIP and CHIP(H260Q) in individual discharge reactions. Thus, prepare double the volume of one charging reaction. 
			NOTE: For first time use, test different concentrations of the E3 ligase of choice to monitor optimal discharge conditions, and analyze them by western blotting. In an ideal condition, the discharge of Ub from E2 will increase over time until the respective E2~Ub protein band disappears.</li>
			<li>Incubate the charging reaction for 15 min at 37 &#176;C.</li>
		</ol>
		</li>
		<li>Termination of the charging reaction
		<ol>
			<li>Stop the charging reaction by addition of apyrase at a final concentration of 1.8 U/mL. Carry out the incubation with apyrase for 5 min at RT, followed by addition of EDTA at a final concentration of 30 mM. Adjust the volume of the charging reaction to 30 &#181;L with ddH2O. 
			NOTE: Apyrase is used to convert ATP molecules to ADP (adenosine diphosphate) molecules. As the activity of the E1 enzyme is ATP-dependent, E1 can no longer charge E2. EDTA is additionally used to ensure that E1 is inhibited by quenching Mg2+ ions that are necessary co-factors for E1. The volume of apyrase that is required to stop the reaction can vary among assay conditions. Although apyrase alone already quenches available ATP molecules, a combination of apyrase and EDTA was more effective at stopping the charging reaction for the E1-UBE2D2 pair.&#160;As stopping the reaction is a critical factor for assay reproducibility, efficient stopping should be tested for new E1-E2 pairs. For this purpose, set up a charging reaction, stop it, and collect samples at specific time points, e.g., after 2, 5, 10, and 15 min. Non-changing levels of E2~Ub indicate that the reaction has stopped, and that the thioester was stable during that period of time (Supplemental Figure S2B).</li>
		</ol>
		</li>
		<li>Discharging of E2~Ub by E3
		<ol>
			<li>Prepare four tubes corresponding to the different time points (t0, t1, t2, t3); add non-reducing sample buffer (6.7 &#181;L of 4x lithium dodecyl sulfate (LDS) buffer) to each tube. 
			NOTE: Do not use reducing agent in the sample buffer.</li>
			<li>Remove 6 &#181;L from the stopped charging reaction for t0,&#160;and adjust the volume to 20 &#181;L with ddH2O.</li>
			<li>Incubate the t0 sample for 10 min at 70 &#176;C. 
			NOTE: Do not boil the sample by extending the incubation time as thioesters are labile at higher temperatures.</li>
			<li>Calculate the volume of each reagent required per discharge reaction. Use the remaining 24 &#181;L of the charged E2, and add 10 mM L-lysine, 1 mg/ml BSA, and 500 nM of the E3 ligase. Use the ubiquitylation buffer at 1x, and adjust the volume to 80 &#181;L with ddH2O.</li>
			<li>Prepare a pipetting scheme for all discharge reactions, and use CHIP(H260Q) as control in addition to CHIP(WT).</li>
			<li>Set up discharge reactions on ice by adding the required components in the following order: ddH2O, ubiquitylation buffer, BSA, lysine, E3 ligase, and the charged E2 to start the reaction.</li>
			<li>Incubate the discharge reaction at RT.</li>
			<li>Take samples after 5, 30, and 60 min by transferring 20 &#181;L of the discharge reaction into the respective sample tubes. 
			NOTE: Suitable time points that allow for proper monitoring of the discharge can vary between E2-E3 pairs.</li>
			<li>Vortex the sample immediately, and incubate it at 70 &#176;C for 10 min.</li>
			<li>Directly proceed with PAGE. 
			NOTE: E2~Ub thioesters can be labile even after denaturing in sample buffer. Thus, gel electrophoresis should be performed directly after assay execution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Gel electrophoresis and western blot
	<ol>
		<li>Perform the gel electrophoresis and the western blotting as described in section 2.2. Use a Ub-specific antibody and, if available, also use an E3-specific antibody that was raised in a different species, allowing for simultaneous detection of the Ub and E3 ligase signal.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Perform data analysis as described in section 2.3 (Figure 3).</li>
	</ol>
	</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-selective+chaperone+CDC-48/p97+links+myosin+assembly+to+human+myopathy.">The ubiquitin-selective chaperone CDC-48/p97 links myosin assembly to human myopathy.</a> Nature Cell Biology. 9 (4), 379-390 (2007).</li><li>Donkervoort, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+variants+in+the+myosin+chaperone+UNC-45B+cause+progressive+myopathy+with+eccentric+cores.">Pathogenic variants in the myosin chaperone UNC-45B cause progressive myopathy with eccentric cores.</a> The American Journal of Human Genetics. 107 (6), 1078-1095 (2020).</li><li>Murata, S., Minami, Y., Minami, M., Chiba, T., Tanaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHIP+is+a+chaperone-dependent+E3+ligase+that+ubiquitylates+unfolded+protein.">CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein.</a> EMBO Reports. 2 (12), 1133-1138 (2001).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHIP+is+a+U-box-dependent+E3+ubiquitin+ligase:+identification+of+Hsc70+as+a+target+for+ubiquitylation.">CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation.</a> Journal of Biological Chemistry. 276 (64), 42938-42944 (2001).</li><li>Yang, Y., Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+apoptosis:+the+ubiquitous+way.">Regulation of apoptosis: the ubiquitous way.</a> The FASEB Journal. 17 (8), 790-799 (2003).</li><li>Lamothe, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRAF6+ubiquitin+ligase+is+essential+for+RANKL+signaling+and+osteoclast+differentiation.">TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.</a> Biochemical and Biophysical Research Communication. 359 (4), 1044-1049 (2007).</li><li>Amemiya, Y., Azmi, P., Seth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoubiquitination+of+BCA2+RING+E3+ligase+regulates+its+own+stability+and+affects+cell+migration.">Autoubiquitination of BCA2 RING E3 ligase regulates its own stability and affects cell migration.</a> Molecular Cancer Research. 6 (9), 1385 (2008).</li><li>Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W., Klevit, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+UbcH5/ubiquitin+noncovalent+complex+is+required+for+processive+BRCA1-directed+ubiquitination.">A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination.</a> Molecular Cell. 21 (6), 873-880 (2006).</li><li>Sakata, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+UbcH5b~ubiquitin+intermediate:+Insight+into+the+formation+of+the+self-assembled+E2~Ub+conjugates.">Crystal structure of UbcH5b~ubiquitin intermediate: Insight into the formation of the self-assembled E2~Ub conjugates.</a> Structure. 18 (1), 138-147 (2010).</li><li>Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M., Wolberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mms2-Ubc13+covalently+bound+to+ubiquitin+reveals+the+structural+basis+of+linkage-specific+polyubiquitin+chain+formation.">Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation.</a> Nature Structural and Molecular Biology. 13 (10), 915-920 (2006).</li><li>Buetow, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+a+primed+RING+E3-E2+ubiquitin+complex+by+non-covalent+ubiquitin.">Activation of a primed RING E3-E2 ubiquitin complex by non-covalent ubiquitin.</a> Molecular Cell. 58 (2), 297-310 (2015).</li><li>Dou, H., Buetow, L., Sibbet, G. J., Cameron, K., Huang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIRC7-E2+ubiquitin+conjugate+structure+reveals+the+mechanism+of+ubiquitin+transfer+by+a+RING+dimer.">BIRC7-E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer.</a> Nature Structural and Molecular Biology. 19 (9), 876-883 (2012).</li><li>McKenna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncovalent+interaction+between+ubiquitin+and+the+human+DNA+repair+protein+Mms2+is+required+for+Ubc13-mediated+polyubiquitination.">Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination.</a> Journal of Biological Chemistry. 276 (43), 40120-40125 (2001).</li><li>Plechanovov&#225;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+ubiquitylation+by+dimeric+RING+ligase+RNF4.">Mechanism of ubiquitylation by dimeric RING ligase RNF4.</a> Nature Structural Biology. 18 (9), 1052-1059 (2011).</li><li>Pierce, N. W., Kleiger, G., Shan, S. O., Deshaies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+sequential+polyubiquitylation+on+a+millisecond+timescale.">Detection of sequential polyubiquitylation on a millisecond timescale.</a> Nature. 462 (7273), 615-619 (2009).</li><li>Jain, A. K., Barton, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+p53:+TRIM24+enters+the+RING.">Regulation of p53: TRIM24 enters the RING.</a> Cell Cycle. 8 (22), 3668-3674 (2009).</li><li>Swatek, K. N., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+modifications.">Ubiquitin modifications.</a> Cell Research. 26, 399-422 (2016).</li><li>Yan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+K63-linked+polyubiquitination+in+cardiac+hypertrophy.">The role of K63-linked polyubiquitination in cardiac hypertrophy.</a> Journal of Cellular and Molecular Medicine. 22 (10), 4558-4567 (2018).</li><li>Dammer, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyubiquitin+linkage+profiles+in+three+models+of+proteolytic+stress+suggest+the+etiology+of+Alzheimer+disease.">Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease.</a> Journal of Biological Chemistry. 286 (12), 10457-10465 (2011).</li></ol>

The authors have no conflicts of interest.

This paper describes basic in vitro ubiquitylation methods for the analysis of E3 ligase function. When performing in vitro ubiquitylation assays, it should be considered that some E2 enzymes can perform auto-ubiquitylation owing to the attack of their active cysteine on their own lysine residues that are located in close proximity to the active site30. To circumvent this problem, the use of an E2 mutant is recommended in which the respective lysine residue is exchanged for argin...

Ubiquitylation is the process by which Ub is covalently linked to a substrate protein1. The Ub modification is catalyzed by successive enzymatic reactions involving the action of three different enzyme classes, i.e., Ub-activating enzymes (E1s), Ub-conjugating enzymes (E2s), Ub ligases (E3s), and possibly Ub chain elongation factors (E4s)2,3,4,5. After adenosine triphosphate (ATP)- and magnesium (Mg2+)-dependent activation of Ub by E1, the active site cysteine of E1 attacks the C-terminal glycine of Ub, forming a thioester complex (Ub~E1). The energy drawn from ATP hydrolysis causes the Ub to attain a high energy transitional state, which is maintained throughout the following enzyme cascade. Next, the E2 enzyme transfers the activated Ub to its internal catalytic cysteine, thereby forming a transient Ub~E2 thioester bond. Subsequently, Ub is transferred to the substrate protein.
This can be done in two ways. Either the E3 ligase may first bind to E2, or the E3 ligase can directly bind Ub. The latter way results in the formation of an E3~Ub intermediate. In either case, Ub is linked to the substrate protein by formation of an isopeptide bond between the C-terminal carboxyl group of Ub and the lysine &#400;-amino group of the substrate6. The human genome encodes two E1s, approximately 40 E2s, and more than 600 putative ubiquitin ligases7. Based on the Ub transfer mechanism of the E3, Ub ligases are divided into three categories involving Homologous to E6AP C-Terminus (HECT)-type, Really Interesting New Gene (RING)/U-box-type, and RING between RING (RBR)-type ligases8. In this study, the U-box containing ligase, Carboxyl Terminus of HSC70-interacting Protein (CHIP), is used as a representative E3 enzyme. In contrast to HECT-type E3 enzymes that form Ub~E3 thioesters, the U-box domain of CHIP binds E2~Ub and promotes the subsequent Ub/substrate transfer directly from the E2 enzyme8,9. Based on the importance of the U-box for enzymatic function, an inactive CHIP U-box mutant, CHIP(H260Q), is utilized as a control. CHIP(H260Q) fails to bind to its cognate E2s, thus losing its E3 ligase activity10.
Protein ubiquitylation plays a crucial role in regulating a multitude of cellular events in eukaryotic cells. The diversity of cellular outcomes that are promoted by the reversible attachment of Ub molecules to substrate proteins can be attributed to the molecular characteristics of Ub. As Ub itself contains seven lysine (K) residues for further ubiquitylation, there is rich variety of Ub chain-types with different sizes and/or topologies11. For example, substrates can be modified by a single Ub molecule at one (mono-ubiquitylation) or multiple lysines (multi mono-ubiquitylation), and even by Ub chains (poly-ubiquitylation)11. Ub chains are either formed homo- or heterotypically via the same or different lysine residues of Ub, which could even result in branched Ub chains9. Thus, protein ubiquitylation leads to diverse arrangements of Ub molecules that provide specific information, e.g., for degradation, activation, or localization of conjugated proteins12,13. These different Ub signals enable fast reprogramming of cellular signalling pathways, which is an important requirement for the cell&#39;s ability to respond to changing environmental needs.
A central aspect of ubiquitylation is related to protein quality control. Misfolded or irreversibly damaged proteins must be degraded and replaced by newly synthesized proteins to maintain protein homeostasis or proteostasis14. The quality control E3 ligase, CHIP, collaborates with molecular chaperones in the Ub-dependent degradation of damaged proteins9,15,16,17. Apart from that, CHIP regulates the stability of the myosin-directed chaperone, UNC-45B (Unc-45 Homolog B), which is tightly coordinated with muscle function and deviations from the optimal levels lead to human myopathy18,19,20,21. Degradation of UNC-45B by the 26S proteasome is mediated by attachment of a K48-linked poly-Ub chain9. In the absence of substrate proteins, CHIP performs auto-ubiquitylation10,22,23, which is characteristic of RING/U-box E3 ubiquitin ligases24,25 and considered to regulate ligase activity26. Application of the in vitro ubiquitylation assay methods described in this paper helped to systematically identify E2 enzymes that team up with CHIP to promote the formation of free poly-Ub chains and/or auto-ubiquitylation of CHIP (protocol section 2). Furthermore, CHIP-dependent ubiquitylation of UNC-45B was observed, which is a known substrate of the E3 ligase18,19 (protocol section 3). Ultimately, CHIP-dependent transfer of activated Ub from the Ub~E2 thioester was monitored (protocol section 4).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amershan Protran 0.1 &#181;m NC</td>
			<td>GE Healthcare</td>
			<td>10600000</td>
			<td>nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>Anti-CHIP</td>
			<td>Cell Signaling</td>
			<td>2080</td>
			<td>Monoclonal rabbit anti-CHIP antibody, clone C3B6</td>
		</tr>
		<tr>
			<td>Anti-MYC</td>
			<td>Roche</td>
			<td>OP10</td>
			<td>Monoclonal mouse anti-MYC antibody, clone 9E10</td>
		</tr>
		<tr>
			<td>Anti-ubiquitin</td>
			<td>Upstate</td>
			<td>05-944</td>
			<td>Monoclonal mouse anti-Ub antibody, clone P4D1-A11</td>
		</tr>
		<tr>
			<td>Apyrase</td>
			<td>Sigma</td>
			<td>A6535-100UN</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP (10x)</td>
			<td>Enzo</td>
			<td>12091903</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A6003-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Roth</td>
			<td>P749.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LDS sample buffer (4x)</td>
			<td>novex</td>
			<td>B0007</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma</td>
			<td>L5501-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Roth</td>
			<td>4256.4</td>
			<td></td>
		</tr>
		<tr>
			<td>MeOH</td>
			<td>VWR Chemicals</td>
			<td>2,08,47,307</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Milchpulver</td>
			<td>Roth</td>
			<td>T145.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Roth</td>
			<td>P029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>invitrogen</td>
			<td>NP0005</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Transfer buffer (20x)</td>
			<td>novex</td>
			<td>NP0006-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Page ruler plus</td>
			<td>Thermo Fisher</td>
			<td>26619</td>
			<td>Protein ladder</td>
		</tr>
		<tr>
			<td>RotiBlock</td>
			<td>Roth</td>
			<td>A151.1</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>SDS (20%)</td>
			<td>Roth</td>
			<td>1057.1</td>
			<td></td>
		</tr>
		<tr>
			<td>S1000 Thermal Cycler</td>
			<td>Bio Rad</td>
			<td>1852196</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo</td>
			<td>Bio Rad</td>
			<td>1704150EDU</td>
			<td>Transfer system</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roth</td>
			<td>4855.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Roth</td>
			<td>9127.2</td>
			<td></td>
		</tr>
		<tr>
			<td>UbcH Enzyme Set</td>
			<td>BostonBiochem</td>
			<td>K-980B</td>
			<td>E2 enzymes</td>
		</tr>
		<tr>
			<td>Ubiquitin</td>
			<td>BostonBiochem</td>
			<td>U-100H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiquitin-activating enzyme E1</td>
			<td>Enzo</td>
			<td>BML-UW941U-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiquitylation buffer (10x)</td>
			<td>Enzo</td>
			<td>BML-KW9885-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman blotting paper</td>
			<td>Bio Rad</td>
			<td>1703969</td>
			<td>Extra thick filter paper</td>
		</tr>
	</tbody>
</table>

in vitro analysis of e3 ubiquitin ligase function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

To identify E2 enzymes that cooperate with the ubiquitin ligase CHIP, a set of E2 candidates was tested in individual in vitro ubiquitylation reactions. Cooperating E2-E3 pairs were monitored by the formation of E3-dependent ubiquitylation products, i.e., auto-ubiquitylation of the E3 ligase and the formation of free Ub polymers. The ubiquitylation products were analyzed by western blotting. Data interpretation is based on the size comparison of the resulting protein bands with molecular weight markers....

Watch this Scientific Journal Video about In Vitro Analysis of E3 Ubiquitin Ligase Function at JoVE.com

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

in-vitro-analysis-of-e3-ubiquitin-ligase-function

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5.0K Views.  University of Cologne. The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

Video: In Vitro Analysis of E3 Ubiquitin Ligase Function

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Methods Article

In Vitro Analysis of E3 Ubiquitin Ligase Function