#ubiquitination

[cancer-researcher]

[cytoscape-publications]

Multiomics approach reveals the ubiquitination-specific processes hijacked by SARS-CoV-2. Xu, et al., Signal transduction and targeted therapy doi: 10.1038/s41392-022-01156-y.

[centralhub01]

Specifically Targeting Proteins Using Engineered "Ubiquibodies"

Understanding protein interaction in the body is crucial for drug discovery process. To understand the role of a particular protein in the body, scientists utilize protein knockout techniques to bind the proteins onto something so that it cannot react, or just remove it altogether, and study the effects of its absence.

The techniques are not fool-proof; there are downsides for each that need to be addressed, and thus, scientists continuously look for ways to evolve and improve upon it. The reverse genetics technique, which eliminates a target protein by blocking or removing its sequence at the DNA or RNA level so that it cannot be expressed in the body, has the downside of low resolution and high level risk. The genetic sequence encoding for the target protein may have analogues, a genetic code that is essentially very similar in reactivity with the target gene, and it is possible that both codes may be cut. This poses a huge problem--what if the analogue encode for an essential protein and skew the results? Additionally, gene manipulation has a waiting time for biosynthesis to occur and does not solve the problem of knocking out already existing target proteins in the body.

To answer these issues, scientists came up with another technique that operates post-translationally, which means that instead of targeting at the genetic level, the gene is allowed to be translated and transcribed into proteins. For this, antibodies are recruited for their high specificity toward their targets, whether they be proteins, cell membranes, virus, etc. A downside is that these antibodies start degrading and unfolding in the intracellular environment, making protein targets inside cells inaccessible. To address this issue, scientists engineered designer binding proteins (DBP) in the form of antibody mimics that have the capability to keep its structure together even inside the cells. 

So far use of DBPs seem to be the answer. The downside to this, however, is that when the DBP binds to the target protein, they create a DBP-protein complex that is unnatural in the body, and thus has no natural elimination pathway. Once these complexes form, they just float around in the body, accumulating in higher concentration as more DBP binds to its target. 

DBP + protein <--> DBP-protein

When DBP specific to the protein is introduced, reaction will shift toward the right to create DBP-protein complex. Since this cannot be eliminated, the high concentration of the complex will begin to affect the rate of reaction and shift the reaction back to the left. Depending on the stability of DBP-protein complex, they may simply let go of each other and go back to equilibrium as freely floating DBP and protein. If they are stable enough so that they cannot break free from each other, the reaction will simply slow down and the rest of the DBP floating around in the body will no longer seek its protein target. The only way to get around this issue is to overflood the body with DBP so that the reaction go toward the right. This technique is effective at silencing the target protein by binding it to the DBP and not allowing it to react with anything. However, to some, this solution is not satisfactory.

A team of scientists from Cornell University, led by Alysse Portnoff, came up with a clever way to not just silence, but to completely eliminate these target proteins from the body. They modified the E3 enzyme used by the Ubiquitin-Proteasome Pathway (UPP) by attaching DBP to it, which will then bind to their target protein. This enzyme tags the target protein with a ubiquitin chain that will signal a proteasome to come up and break the protein down.

Protein degradation means to break down any proteins into its constituent amino acid blocks so that it can be reused in the body. Mainly this breakdown occurs when a proteasome, a type of enzyme, attacks damaged proteins and breaks the peptide bonds between these amino acids. These proteasome need to be very selective with their targeting, otherwise it might end up breaking down all the protein it comes across. To achieve this selectivity, it needs to have a sign that it can read on the protein surface to let it know whether it should break the protein down, or to leave it alone. For the protein degradation process via UPP, the sign that these proteasome read on the protein surface is a ubiquitin chain.

UPP has several steps and recruits 3 different enzymes (E1, E2, and E3):

When the E1 enzyme sees the ubiquitin, it binds to it, a process which requires the use of ATP. This activates ubiquitin and the active site on E1.

E1's active site seeks out E2, then ubiquitin transfer commences. 

E3 has two active sites. One site binds to the E2+ubiquitin complex, which then activates its other binding site to go and bind to its substrate (like proteins). The close proximity of E2+ubiquitin complex to the tail of the substrate makes it easy for ubiquitin to jump ship and transfer to the protein substrate.

In a cascade reaction, the ubiquitin tail on the substrate creates a chain. Eventually the proteasome picks up on this signal and digests the substrate by breaking down its bonds.

Figure:

The new technique takes the E3 enzyme and attaches a DBP in its substrate active site. The DBP can be engineered to specifically attack any protein desired. Following the UPP, the target protein can then be ubiquitinated and tagged for proteasome degradation.

Newer advances and manipulation of the E3 enzyme even takes out the accessory factors (E1 and E2) and simply let E3 directly ubiquitinate its substrate.

As with the other techniques, this one is not its final form. The researchers have admitted that complete target elimination was not achieved using this technique and currently, they do not know exactly what the reasons are. Additionally, it is possible for the E3 enzymes to ubiquitinate each other and tag each other for elimination. Nevertheless, this is a powerful new tool for understanding protein interactions in the body, and future research could also provide a new kind of biologic medicine to eliminate otherwise healthy cells (like in cancer). The good thing is that Portnoff and her colleagues already have an idea to improve upon this technique, and it would be interesting to see the new solutions they come up with next.

 doi:10.1074/jbc.M113.544825jbc.M113.544825.

[labness]

The Database of Ubiquitinating and Deubiquitinating Enzymes

also known as DUDE

[shortsharpshock]

So I'll be 'characterising potential new drug targets in multiple myeloma' for my research project this year..eek

[lifeologycomic-blog]

Came up with this in my sleep last night...probably due to the stress of a biochem final today. Ubiquitinated protein get degraded by the proteosome...However, a protein not ubiquitinated will not be degraded. The phrase came from "Fear me, if you dear." from Puss in Boots in Shrek 2. So picture Antonio Banderas' Voice when reading this! © 2010 Bethany Tiner