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E3 Ubiquitin Ligases
As much as 5% of human genes encode E3 Ubiquitin Ligases (E3’s), with the total number of these enzymes being estimated at 600 or more. E3 Ligases are important components of the Ubiquitin Proteasome System (UPS), the master regulator of protein homeostasis that is essential for proper cellular function. As the last component of a enzymatic cascade that also consists of a ubiquitin activating enzyme (E1) and ubiquitin conjugating enzymes (E2’s), E3 Ligases selectively modify proteins by covalently attaching ubiquitin to lysine, serine, threonine or cysteine residues within the particular E3’s cognate substrate. Attaching ubiquitin to a protein (known as “ubiquitination” or “ubiquitylation”) can have profound effects on the protein’s cellular localization, protein-protein interactions and/or stability. Perhaps the most well-studied consequence of protein ubiquitination is the ATP-dependent degradation of polyubiquitinated proteins via the 26S proteasome. The biological importance of E3 Ubiquitin ligases cannot be overstated, as protein ubiquitination is crucial in signal transduction, mitophagy, DNA damage repair, inflammatory pathways, endocytosis and cell-cycle progression.
Targeted Protein Degradation is a rapidly emerging field wherein small molecule degraders (e.g. PROTAC® molecules) are used to redirect or co-opt E3 Ubiquitin Ligases to new targets (“neosubstrates”) for polyubiquitination and subsequent proteasome-mediated degradation. By redirecting the Ubiquitin-Proteasome System in this manner, disease-driving proteins that were once considered undruggable are receiving new attention.
Single-subunit E3 Ligases
These single subunit E3 ligases are capable of ubiquitinating protein substrates in vitro when supplied with ubiquitin, E1, an appropriate E2, and ATP. These include enzymes from the RING, U-box, HECT and RBR class E3’s.
Many E3 ligases are assembled from multiple subunits, in many cases consisting of a substrate receptor, an adaptor(s), a cullin and a RING. These include enzymes from Skp-Cul1-Fbox, Elongin-Cul2/5-SOCS box, DCAF-Cul4 and others E3 classes. Some products are capable of ubiquitinating protein substrates in vitro when supplied with ubiquitin, E1, an appropriate E2, and ATP. Others are purified substrate receptors for use in binding studies.
E3 Activity Kits supply everything needed to perform in vitro ubiquitination reactions with a customer supplied substrate. Most kits include a control substrate with primary detection antibody for Western Blot analysis of results.
TUBEs, Tandem Ubiquitin Binding Entities, are used for isolation and identification of ubiquitinated proteins. TUBEs display increased affinity for poly-ubiquitin moieties over the single Ubiquitin Binding Associated domain (UBA). TUBEs also display a protective effect on poly-ubiquitinated proteins, allowing for detection at relatively low levels.