This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.
Post-translational modifications (PTM)
Post-translational modifications (PTMs) refer to a number of covalent modifications on proteins after they are translated. Some types of PTMs include "phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis" [1]. PTMs increase diversity of the proteome because they alter amino acid properties, resulting in altered protein activity, and can also be reversible to allow the proteome to respond to the environment [1]. Phosphorylation, the addition of a phosphate group to protein by a kinase, is an important PTM that is used to regulate protein diversity and plays a major role in signal transduction; however, abnormal phosphorylation can cause disease when a site cannot be phosphorylated or when a phosphate cannot be removed [2]. Phosphorylation most commonly occurs on serine, threonine, and tyrosine residues [3].
Predicted Phosphorylation sites on AIRE
A program called NetPhos can be used to predict phosphorylation sites in a protein based on its sequence. NetPhos also gives a confidence score to each potential phosphorylation site, and any site below the pink horizontal line is below the threshold, meaning its confidence score was too low [4]. Below are images of both the human and mouse NetPhos outputs showing predicted phosphorylation sites. By comparing these two outputs, it can be seen that there are differences in amino acid types, locations, and confidence at the predicted phosphorylation sites. Click on the image to go to the original NetPhos output for each protein.
The NetPhos outputs have phosphorylation potential on the y-axis and the amino acid on the x--axis. The image to the left shows the SMART + pfam output (see domains) of the human AIRE protein. Referencing the location of the potential phosphorylation sites can be helpful to know whether they are in known domains or not.
Human AIRE predicted phosphorylation sites.
545 aa
Mouse AIRE predicted phosphorylation sites.
552 aa
Analysis
The predicted phosphorylation sites from NetPhos suggest that there are a number of highly likely (meaning over the threshold) phosphorylation sites in AIRE. A study analyzing phosphorylation of AIRE in the thymus of mice confirmed that AIRE was indeed phosphorylated in vivo [5]. The study also showed that Protein kinase c and cAMP-dependent protein kinase A were able to phosphorylate recombinant human AIRE in prokaryotes [5]. Further studies could be done to look at which predicted phosphorylation sites are actually phosphorylated in AIRE, and what cellular conditions give rise to phosphorylation.
References:
[1] https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-post-translational-modification.html
[2] Prabakaran et al. 2012. Post-translational modification: nature’s escape from genetic imprisonment and the basis for dynamic information encoding. http://vcp.med.harvard.edu/papers/jg-wire-ptm.pdf
[3] Sigma-Aldrich. Post Translational Modification. http://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/phosphorylation/post-translational-modification.html
[4] NetPhos. Output Format. http://www.cbs.dtu.dk/services/NetPhos-2.0/output.php
[5] Kumar et al. 2001. The Autoimmune Regulator (AIRE) Is a DNA-binding Protein. http://www.jbc.org/content/276/44/41357.long
[1] https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-post-translational-modification.html
[2] Prabakaran et al. 2012. Post-translational modification: nature’s escape from genetic imprisonment and the basis for dynamic information encoding. http://vcp.med.harvard.edu/papers/jg-wire-ptm.pdf
[3] Sigma-Aldrich. Post Translational Modification. http://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/phosphorylation/post-translational-modification.html
[4] NetPhos. Output Format. http://www.cbs.dtu.dk/services/NetPhos-2.0/output.php
[5] Kumar et al. 2001. The Autoimmune Regulator (AIRE) Is a DNA-binding Protein. http://www.jbc.org/content/276/44/41357.long