Ubiquitin cell signalling

One of the more rapidly expanding fields in cell signalling is the current characterisation of proteins conjugated to ubiquitin. The reversible covalent attachment of these small peptides remodels the target protein, providing new protein-protein interaction interfaces that can be dynamically regulated with a given set of enzymes for conjugation and deconjugation.

Ubiquitin is attached to target proteins either as a monomer or as a polyubiqtuiin chain linked via one of the seven lysine residues within the sequence or via the N-terminal methionine residue. Depending upon which of the seven lysines, (K6, K11, K27, K29, K33, K48, K63), are conjugated a variety of branching patterns can be generated in the polyubiquitin chain. This versatility is highly regulated and increases the possible signalling functions of these post-translational modifications.
While it is generally accepted that K48-linked chains are involved in protein degradation via the proteasome and K63-linked chains function within DNA-damage responses, the roles that the other lysine-linked polyubiquitin chains may play in intracellular signalling are still not fully defined. K6-linked polyubiqiutin chains may be involved in DNA repair, while K33-linked chains have been associated with post-Golgi transport and T-cell receptor signalling. Additionally, while the role of K48-linked polyubiquitin chains in targeting proteins to the proteasome is fairly well established aberrant function in the UPS has been associated with disease, including neurological disorders, cancer and inflammatory conditions such as rheumatoid arthritis.
The killer combination of an historical lack of foundational tools and technologies available to study the ubiquitin pathway in detail and the significant complexity inherent in the system has meant that we are just not sure about the signals resulting from a target protein being tagged with each of the different polyubiquitin chains or how to specifically target these in disease. Avacta Life Sciences now offers our engineered affinity protein Affimer products for use in this key research field. Using this technology we have been able to develop affinity reagents to targets such as the K33 diubiquitin linkage, for which is there is no equivalent antibody or aptamer available. With targets ranging from the specific diubiquitin linkages of K6 and K48 and the unique K33 to the ubiquitin peptide itself we hope to overturn this trend and help you to make sense of these signals.
The process by which proteins are ubiquitinated comprises three main steps:
  • Activation of ubiquitin at its C-terminus by an E1 ubiquitin-activating enzyme
  • Conjugation of ubiquitin to an E2 ubiquitin-conjugating enzyme
  • Transfer of ubiquitin to the substrate protein by an E3 ubiquitin ligase

With two E1 enzymes, approximately 60 E2 enzymes and 600-800 different E3 enzymes in the human cell, there is a huge 90,000 different possible enzyme combinations by which target proteins can be ubiquitinated. The huge diversity that this system offers makes the precise regulation of this highly specific post-translational modification within the cell essential and the untangling of this web of specificity a highly complex task.

Due to a previous lack of antibodies or aptamers that would enable the exploration of this system in detail, a deep understanding of the nuances of the ubiquitin signalling system – the hierarchy, interdependence and potential redundancy among system components – is still in its infancy. While the effect of each of the polyubiquitin chains on different physiological processes is only starting to be understood the potential opportunities afforded by this unexploited pathway in terms of drug development targets show much promise.
Affimer reagents are designed to help you discover the intricacies of these pathways. Should you want to determine the profile of polyubiquitin linkages, examine the localisation of these within your cell or tissue or perform a pull-down to examine the array of ubiquitinated proteins, Affimer technology can help. Of course if you would like an Affimer against a target that you can’t see in our catalogue we can quickly generate custom Affimer molecules in just seven weeks.