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Ubiquitination regulates cellular functions by maintaining homeostasis and responding to various stress stimuli. Therefore, dysregulation of the ubiquitination process can lead to a variety of human diseases, especially cancer. E3 ligases are critical for regulating cellular homeostasis due to their efficient regulation and substrate specificity during the ubiquitination cascade. Obviously, E3 ligase is significantly involved in cancer progression, such as DNA damage and repair, proliferation, invasion, apoptosis, metabolism, immunity and many other aspects. Therefore, E3 ligases may become a promising and effective target for cancer therapy.
Transfected stable cell lines are important research tools for drug discovery, compound screening, and gene therapy research. Creative Biogene has proven its success in the generation and validation of stable cell lines to accelerate your novel drug discoveries and biomedical research. We have generated a unique collection of E3 ubiquitin ligases stable cell lines to provide powerful assay platforms for all research projects.
Ubiquitination is a post-translational modification of proteins that typically requires the sequential action of three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). E3 ubiquitin ligases, which number over 600 in humans, are a highly heterogeneous family of proteins and protein complexes that recruit ubiquitin-loaded E2 enzymes to mediate the transfer of ubiquitin molecules from E2s to protein substrates.
Figure 1. Types of ubiquitination ligases. (Yang Q, et al. 2021)
Ubiquitination by E3 ligases regulates many areas such as DNA repair, cell transport, and signal transduction and is important in cell biology. The dysregulation of E3 ubiquitin ligases has been implicated in numerous diseases, including cancer, neurodegenerative disorders, and autoimmune diseases. Therefore, the development of stable cell lines expressing specific E3 ubiquitin ligases has emerged as a valuable tool for studying their functions and exploring potential therapeutic interventions.
Applications for E3 Ubiquitin Ligase stable cell lines include:
Case Study 1
HERC4 is an identified Ubiqutin ligase E3 that is a member of the UPS. Here, researchers studied the function of HERC4 in liver cancer cells for the first time. Overexpression of HERC4 increases the proliferation and migration capabilities of liver cancer cells and reduces apoptosis; conversely, knockdown of HERC4 reduces the proliferation and migration capabilities of liver cancer cells and increases the apoptosis rate. These results indicate that HERC4 affects the occurrence and development of liver cancer by promoting the proliferation and migration of liver cancer cells and reducing apoptosis, further providing another therapeutic target for intervening in related diseases.
Figure 2. Effects of HERC4 overexpression on cell biological functions. (A) Colony formation assay on HERC4-overexpressing Bel-7402 and LM3 cells and control cells. (B) CCK8 assay was used to evaluate the proliferation of Bel-7402 and LM3 cells after transfection with pcDNA3.0-HERC4 or empty vector (pcDNA3.0) and control cells. (C) Wound healing assay photographs (left) and wound closure distance (right) taken 24 hours after scratching. (D) EdU assay for assessing the proliferative capacity of HERC4-overexpressing Bel-7402 and LM3 cells. (Zheng Y, et al., 2017).
Case Study 2
Tripartite motif-containing protein 45 (TRIM45) belongs to the large RING finger-containing E3 ligase family, which is highly expressed in the brain. Here, we report that TRIM45 expression is significantly reduced in glioma tissue samples. Overexpression of TRIM45 inhibits the proliferation and tumorigenicity of glioblastoma cells in vitro and in vivo. Furthermore, TRIM45 interacts with and stabilizes p53 by promoting K63-linked polyubiquitination of p53, thereby inhibiting subsequent K48-linked polyubiquitination that targets p53 degradation. These findings demonstrate TRIM45 as a novel regulator responsible for maintaining p53 stability in glioma.
Figure 3. TRIM45 promotes glioblastoma cell apoptosis. TRIM45 overexpressing (a) and KO (b) U87 and LN229 cells were incubated with FITC-Annexin V solution and subjected to FACS analysis. Lysates of TRIM45 overexpressing (c) and TRIM45 KO (d) U87 and LN229 cells were immunoblotted with anti-caspase-3 antibody. (Zhang J, et al., 2017)
Q: What are the types of E3 ligases?
A: According to different structures and functions, E3 ligases can be roughly divided into four types: HECT type, U-box type, RING-finger type, and RBR type. Different types of E3 ligases have low sequence homology and large composition differences.
Q: What cellular processes are E3 ubiquitin ligases involved in?
A: E3 ubiquitin ligases are involved in various cellular processes, including DNA repair, cell cycle regulation, signal transduction, and immune responses.
Q: What types of anti-tumor drugs target E3 ligases?
A: Currently, anti-tumor drugs targeting E3 ligase are mainly divided into four categories according to their mechanism of action: E3 ligase-targeted inhibitors, E3 ligase-targeted agonists, proteolytic targeting chimeras (PROTACs), and molecular glues.
Q: How can E3 ubiquitin ligase stable cell lines be utilized in drug discovery and screening?
A: By using stable cell lines expressing disease-relevant E3 ubiquitin ligases, researchers can identify small molecule modulators that enhance or inhibit the degradation of these disease-associated proteins. This approach has been successfully applied in cancer research, where stable cell lines expressing oncogenic E3 ligases have been utilized in high-throughput screening campaigns to identify compounds that selectively degrade cancer-related proteins.
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