Biology Case Study Argumentative

Step 1: How will you identify the “vital” cellular protein that the virus targets for degradation? (Hint: think proteomics). (3 pts.)

First, since we know the viral RNA sequence and its targeting protein, we can investigate it in bioinformatics database, and can acquire some clues or hints about the target proteins. From the bioinformatics database, we might be able to find its structure, similarity with other proteins, functions, and binding domains. In other words, we can get some partial or complete amino sequences of the targeting proteins or information about likeness.

Second, in order to further confirm the information about characteristics and function of the targeting protein that we have obtained from the bioinformatics database, we can actually introduce the virus into the cell, comparing it with a non-infected cell. SDS-PAGE or 2-dimentional electrophoresis can be used to detect the differences between the two: targeting proteins will exist in the non-infected cell but will not exist in the infected cell. Two-dimensional gel electrophoresis will separate proteins on the basis of charge and mass.

Finally, we can obtain the pure targeting protein, and final step is to identify the amino sequence of the protein in order to determine its type and name. Since many proteins in vital cell survival has been identified and listed in the web database, all we need is to identify the amino sequence of the protein, and match this sequence in the database. We can also reconfirm the finding by using immunoassays in order to confirm whether the protein is actually degraded by the virus.

Step 2: How will you identify the ubiquitin ligase responsible for ubiquitinylation of the “vital” protein? (3 pts.)

Ubiquitin ligases combine with an ubiquitin-containing E2 Ub-carrier protein, and targets specific protein substrates for degradation. Its important function is to determine specificity. Therefore, if it were not to function correctly, the targeting protein would not be degraded.

In order to determine which ubiquitin ligase is responsible for the ubiquitination of the protein, we can obstacle the function of each type of ubiquitin ligases in different cells. Since we have already known the amino sequence of the protein in previous step, we can narrow down the targeting ubiquitin ligase based on existing research data such as papers, NCBI data.

There are many types of ubiquitin ligases in cells. However, we can make some candidate groups of targeting E3 based on the bioinformatics database.

We will use antibodies which specifically bind to each type of ubiquitin ligase and impede its function. For example, the antibodies may covalently bind to the targeting ubiquitin ligase, and therefore, impede its function. Then, we will measure the amount of targeting protein. If we find that the amount of targeting protein is not changed in a cell, we can identify the target ubiquitin ligase. This is because only when the function of the target ubiquitin ligase is impeded, the degradation of the targeting protein will not be occurred.

Step 3: What protein will be your drug target? What property of that protein will you target? Design an assay/approach to identify an antidote for “degron”. (4 pts.)

Since “degron” targets a vital cellular protein for ubiquitin-dependent degradation, if we block its process of degradation, we can effectively turn off its effect. As mentioned earlier, ubiquitin ligase brings specificity. Since we have already known which ubiquitin ligase is responsible for degrading the targeting protein, a drug that targets the ligase will be a good antidote.

I will target the ubiquitin ligase which brings specificity for reducing side effects of the antidote. In order to design the antidote:
First, it should specifically bind to the target ligase. Protein structure and binding domain of the target ligase will be carefully determined to design the antidote.

Second, the antidote effectively impedes the essential function of the ligase. Since the critical role of the E3 ligase is to combine with E2 and responsible for targeting ubiquitination to specific substrate proteins, its binding region is very crucial. By covalently binding these crucial binding regions, E3 will not properly function in a cell, and as a result, the “degron” will not show its effect anymore.

References:
Molecular Cell Biology, 7th Edition, 2013, Lodish, Berk, Kaiser, Krieger, Bretscher. Ploegh, Amon, and Scott. W.H. Freeman and Company (ISBN-13: 978-1-4292-3413-9) Pictures:
http://en.wikipedia.org/wiki/File:Ubiquitylation.svg