Editorial Feature

A Novel Method for Targeted Protein Analysis of Linear Ubiquitin Substrates

Ubiquitin is a prototype of a family of isostructural conserved proteins that are responsible for a variety of processes in eukaryotic cells1. These structurally-conserved proteins covalently bind to other cellular proteins to change the stability, localization and activity of target proteins.

The mature forms of these proteins terminate with a signature diglycine sequence, which is only exposed after proteolytic processing1. Ubiquitin-like modifier proteins (Ubl) share a common biochemical mechanism where they form an isopeptide bond between the glycine residue of the Ubls and an amino group, which is usually contributed by the lysine residues of the target proteins1. Ubiquitin can also be linked to cysteine through thioester bonds, as well as to serine and threonine through ester bonds, or to the amino group of the N-terminus of proteins via peptide bonds1.

The addition of ubiquitin moieties to a substrate protein is called ubiquitination, which is a process that can affect target proteins in several ways. Of these include the signaling for degradation by proteasomes, alteration of the cellular location, as well as promotion or prevention of other protein interactions. Ubiquitination usually occurs in three main steps; (1) activation by ubiquitin activating enzymes (E1s), (2) conjugation by ubiquitin conjugation enzymes (E2s) and (3)  ligation by ubiquitin ligases (E3s).

As a small protein of only 8.5 kDa, ubiquitin is ubiquitously present in almost all tissues of eukaryotic organisms, in which they can bind to other cellular proteins in many different forms, either as a single molecule, or by forming a distinct chain leading to different cellular outcomes according to the formation of diverse conformations1,2. These linear chains are formed by a series of ubiquitin molecules, where the head of one ubiquitin molecule is linked to the tail of the next one. A number of diseases such as cancer and neurodegenerative disorders like Parkinson's disease, as well as the development and progression of infections and inflammatory diseases, can occur as a result of errors in the ubiquitin system2.

The secret ubiquitin code that is responsible for determining the fate of several cellular processes is not fully decoded yet2.  Until now, a single E3 ligase complex (LUBAC) and a specific deubiquitinase (OTULIN) are the only known enzymes capable of synthesizing and degrading such ubiquitin chains3. The target proteins of linear ubiquitin and their fate of cellular function is not yet clear2. Unsuitability of the existing methods to study lysine-based poly ubiquitination combined with the lack of appropriate methods for proteomic analysis of these linear chains of ubiquitin are thought to be the main reasons for the slow progress of this research3,4.

Researchers at the Goethe University, in collaboration with the University of Tubingen, Francis Crick Institute and Queen Mary University recently developed a novel technology to decipher this secret ubiquitin code. Koraljka Husnjak’s team achieved this by internally modifying the ubiquitin molecule and combining such lysine-less internally tagged ubiquitin (INT-UB.7KR) with SILAC-based mass spectroscopy. Surprisingly, this modification did not affect the cellular functions of the normal ubiquitin, while instead it enabled the further analysis of linear ubiquitin targets by mass spectroscopy3,4.

Husnjak’s team further validated the method by identifying several new protein targets of the linear ubiquitin chains including the components of a major proinflammatory pathway in the cells. The team applied their method to TNFa-stimulated T-Rex HEK293T cells3. Furthermore, they also found that linear polyubiquitination of the novel LUBAC substrate TRAF6 is crucial for NFkB signaling. Using this method, researchers were able to demonstrate that the linear ubiquitin chains play a key role in relaying signals as well as maintaining the regulation of the immune responses in pathogen defenses and immunologic disorders3.

These researchers are hopeful that this new method could pave the way for the future identification of new target proteins modified by linear ubiquitin chains, while also allowing us to know the exact position in the protein where the linear ubiquitin chain is attached. This highly sensitive approach is believed to be a great breakthrough in improving the understanding of the functions of linear ubiquitination and its role in the manifestation of various diseases.

References:

  1. Pickart, Cecile M., and Michael J. Eddins. "Ubiquitin: Structures, Functions, Mechanisms." Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1695.1-3 (2004): 55-72. Web.
  2. "Tracing down Linear Ubiquitination." ScienceDirect.com. 20 March 2017. Web. https://www.sciencedaily.com/releases/2017/03/170320122846.htm.
  3. Kliza, Katarzyna, Christoph Taumer, Irene Pinzuti, Mirita Franz-Wachtel, Simone Kunzelmann, Benjamin Stieglitz, Boris Macek, and Koraljka Husnjak. "Internally Tagged Ubiquitin: A Tool to Identify Linear Polyubiquitin-modified Proteins by Mass Spectrometry." Nature Methods (2017). Web.
  4. "Tracing down Linear Ubiquitination: New Technology Enables Detailed Analysis of Target Proteins." Phys.org. 20 Mar. 2017. Web. https://phys.org/news/2017-03-linear-ubiquitination-technology-enables-analysis.html.
  5. Image Credit: shutterstock.com/sciencephoto

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Benedette Cuffari

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Benedette Cuffari

After completing her Bachelor of Science in Toxicology with two minors in Spanish and Chemistry in 2016, Benedette continued her studies to complete her Master of Science in Toxicology in May of 2018. During graduate school, Benedette investigated the dermatotoxicity of mechlorethamine and bendamustine, which are two nitrogen mustard alkylating agents that are currently used in anticancer therapy.

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