Name: in vitro analysis of e3 ubiquitin ligase function
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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<...

<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. 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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 ...

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