Biology Case Study
Advanced Cell Biology II
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
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
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