Can one cellular pathway contribute to many different types of disease? Research has shown that Ubiquitination is one such pathway due to its role in the regulation of protein degradation within homeostasis, cell cycle, and DNA repair. It has been established that at least the following diseases are among those affected from alterations in this pathway: cancer, viral infection and neurodegenerative disorders to muscle wasting, diabetes, and inflammation.1 Obviously, targeting the aberrations that lead to disease in this protein turnover pathway could benefit individuals tremendously. Scientists are making it their mission to find ways to inhibit the damage caused by unnecessary ubiquitination.
The Role of Ubiquitin Ligase in Ubiquitination
Destroying proteins doesn’t always lead to diseases. Consider the immune response. Ubiquitination is a way to clear unwanted foreign proteins from our body. Whether done for good or to cause harm, the pathway is the same. This rather complex system consists of multiple enzymatic reactions in order to allow the ubiquitin protein access to the target protein (the protein that will get degraded). Think of it as a chain of individual reactions leading to the destruction of a protein. Initially, the ubiquitin-activating enzyme (E1) activates ubiquitin via an ATP-dependent reaction.2 Once activated, ubiquitin is transferred to the ubiquitin-conjugating enzyme (E2), forming another intermediate, which is able to bind to the final ubiquitin-protein ligase (E3).2 Finally, the ligase transfers the activated ubiquitin from E2 to the side chain of a lysine residue of the target protein.2 Though this is a simplified version, it shows the multiple steps and molecules involved in order to obliterate a single protein.
How A Ubiquitin Ligase Assay Can Help
There are numerous ubiquitin ligases that regulate important cellular functions such as the case with MDM2, which regulates the tumor suppressor p53 gene.1 If the balance gets offset and MDM2 catalyzes the p53 protein, tumor progression is likely to occur. There are many other examples like this in which proteins responsible for proper cellular function get destroyed leading to disease. Fortunately, scientists have found a way to exploit the natural ubiquitination enzymatic reaction in order to investigate drug targets, ubiquitin ligase inhibitors.
BellBrook Labs has developed a novel assay platform called Transcreener® HTS Assay that detects the production of AMP using fluorescent immunodetection; AMP is produced in the complex ubiquitination pathway since ATP is one of the substrates.3 The assay relies on antibodies that differentiate between nucleotides on the basis of small structural differences, such as a single phosphate group.3 Therefore, it can be used as a ubiquitin ligase assay with the ability to monitor enzymatic activity of ubiquitin ligases and the effects of its inhibitor.
- Petroski MD. The ubiquitin system, disease, and drug discovery. 2008;15:1-15. doi:10.1186/1471-2091-9-S1-S7.
- Jain J, Jain SK, Walker LA, Tekwani BL. Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads. 2017:1-10. doi:10.1186/s40360-017-0147-4.
- Staeben M, Klink TA, Kopp A, Westermeyer TA, Kleman-leyer KM. Interrogation of Protein Phosphorylation , Ubiquitination , and Sumoylation Pathways Using Adenine Nucleotide Detection Assays