Description
Key facts
Sample types:Purified protein,
Assay type:Enzyme activity
Product details:
E3 Ligase Auto-Ubiquitylation Assay Kit (ab139469) enables proteins to be tested for ubiquitin E3 ligase activity through assessment of their ability to undergo auto-ubiquitylation (also known as ubiquitination). Utilizing the first three steps in the ubiquitin cascade the kit facilitates ubiquitylation of known or putative E3 ligase enzymes followed by Western blot analysis using the highly sensitive reagents provided or using antibodies to the specific protein of interest (user supplied). Hdm2 ubiquitin E3 ligase enzyme is also provided for use as a positive control.
The Kit provides sufficient material for approximately 10 auto-ubiquitinylation assays.
Suggested uses for this kit include:
1. Qualitative assessment of an Ub E3 ligase enzyme's activity through its ability to auto-ubiquitylate.
2. Testing of proteins for auto-ubiquitylation activity allowing their identification as putative ubiquitin E3 ligases.
3. Ubiquitylation of substrate proteins (user provided) specific to a particular ubiquitin E3 ligase.
Other Notes
The covalent attachment of ubiquitin to proteins (ubiquitylation or ubiquitination) plays a fundamental role in the regulation of cellular function through biological events involving cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis.
Ubiquitylation is achieved through three enzymatic steps. In an ATP-dependent process, the ubiquitin activating enzyme (E1) catalyzes the formation of a reactive thioester bond with ubiquitin, followed by its subsequent transfer to the active site cysteine of a ubiquitin carrier protein (E2). The selectivity of the ubiquitin cascade for a particular substrate protein relies on the interaction between the E2 conjugating enzyme (of which a cell contains relatively few) and a ubiquitin-protein ligase (E3), of which over 600 have been identified to date.
The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn
-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitinylation, RING and U-box E3s facilitate protein ubiquitinylation. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitinylation. Although many RING-type E3s, such as MDM2 and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex.
Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms utilising the ubiquitin-proteasome system. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.
E3 ligases also undergo auto-ubiquitinylation, through modification of specific lysine residues within an individual ligase, providing a mechanism thought to be responsible for the regulation of the E3 enzyme itself.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--80°C, Appropriate long-term storage conditions--80°C, Storage information--80°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
The Ubiquitin E3 Ligase also known by several names such as E3 ligase and E3 ubiquitin ligase is an important element in the ubiquitination process. Mechanically E3 ubiquitin ligases facilitate the transfer of ubiquitin from an E2 enzyme to a substrate protein determining the specificity of the substrate by recognizing and binding to it for subsequent ubiquitination. It plays a central role in the ubiquitin-proteasome pathway which regulates protein degradation. The expression of E3 ubiquitin ligases is widespread across various tissues and they exhibit a wide range of molecular weights due to the numerous types that exist typically between 20 kDa and 200 kDa.
Biological function summary
E3 ubiquitin ligases operate as part of larger ubiquitin ligase complexes often working with E1 activating enzymes and E2 conjugating enzymes. This ligase activity is responsible for the regulation of protein turnover cell cycle progression and DNA repair processes by targeting proteins for degradation. The specificity of the ubiquitination process is what allows E3 ligases to orchestrate a diverse range of cellular functions impacting cell signaling transcriptional regulation and receptor modulation.
Pathways
E3 ubiquitin ligases significantly influence the Wnt signaling and NF-kB pathways. In the Wnt signaling pathway they regulate beta-catenin levels by tagging it for degradation affecting gene transcription associated with cell proliferation and differentiation. In the NF-kB pathway E3 ligases modulate the stability of key proteins like IkB thereby controlling NF-kB activation which is vital for immune and inflammatory responses. Proteins such as SKP1-Cullin-F-box (SCF) complexes are integral to these pathways linking the function of E3 ligases to significant cellular outcomes.
Malfunctions of E3 ubiquitin ligases have connections to cancer and neurodegenerative diseases. Dysregulation of E3 ligase activity can lead to the accumulation of damaged proteins contributing to pathologies such as Parkinson's disease where the PARKIN E3 ligase is implicated. In cancer altered expression or mutations in E3 ligases such as Mdm2 lead to the inappropriate degradation of tumor suppressor proteins like p53 promoting tumorigenesis. These insights highlight the importance of E3 ligases in maintaining cellular homeostasis and their potential as therapeutic targets.
