Conclusions
For this project we had to use many online bioinformatics tools to understand how mutations in the prkag2 gene attributed to Wolff-Parkinson-White syndrome. When I looked at the homologous genes and proteins in other organisms, it was shown that the prkag2 gene and AAKG2 protein was highly conserved among a wide variety of species. The highly conserved nature of the homologous gene and protein would mean that studies in many different model organisms would be relevant, even in C. elegans.
When using these bioinformatic tools, I noticed that prkag2 is involved in many different biological life processes. This makes sense because homologous genes and proteins were observed in plant species as well as mammals. The high conservation of this gene and protein most likely stems from the four domains found in the AAKG2 protein. These domains, known as CBS domains, were even found in ancient archaic algae. Ultimately, the identification of these CBS domains in algae suggest that they are very vital in many of the central functions of life, not just heart function.
Many tools have been used to analyze prkag2 gene's function, expression, and explore possible treatment options for mutations here. These molecular tools have given great insights into many facets of prkag2 and it's involvement in tissues other than the heart. These tools have lead to the discovery of small molecules that are often able to treat the symptoms that occur in WPW syndrome.
Overall, Wolff-Parkinson-White syndrome is a well managed condition that has many effective treatments that allow patients to easily regulate their symptoms or cure them altogether. Throughout this exploration, it was easy to see that the prkag2 gene and WPW syndrome have been well characterized and extensively studied.
When using these bioinformatic tools, I noticed that prkag2 is involved in many different biological life processes. This makes sense because homologous genes and proteins were observed in plant species as well as mammals. The high conservation of this gene and protein most likely stems from the four domains found in the AAKG2 protein. These domains, known as CBS domains, were even found in ancient archaic algae. Ultimately, the identification of these CBS domains in algae suggest that they are very vital in many of the central functions of life, not just heart function.
Many tools have been used to analyze prkag2 gene's function, expression, and explore possible treatment options for mutations here. These molecular tools have given great insights into many facets of prkag2 and it's involvement in tissues other than the heart. These tools have lead to the discovery of small molecules that are often able to treat the symptoms that occur in WPW syndrome.
Overall, Wolff-Parkinson-White syndrome is a well managed condition that has many effective treatments that allow patients to easily regulate their symptoms or cure them altogether. Throughout this exploration, it was easy to see that the prkag2 gene and WPW syndrome have been well characterized and extensively studied.
Future Directions
When doing research, I realized that the four CBS domains found in AAKG2 were extremely conserved in many organisms, all the way down to an archaic algae--making them significant in all three domains of life. I was interested in exploring these CBS domains because six of the seven known missense mutations to the prkag2 gene, causing WPW syndrome, occur within one of these domains. I discovered that one of the main functions of a CBS domain was to help restore cellular ATP balance. I came across a paper by Xiao (et al. 2007) that had located the specific ATP binding sites in these CBS domains. They researched the affect that the six known CBS missense mutations had on these sites. Five of these missense mutations had evidence that, when mutated, they reduced AMP/ATP binding and were structurally close or interacting with these binding sites in rats [1]. Knowing this, I wanted to look into how these same missense mutations affected organisms that did not have a heart.
Question: Will these missense mutations have the same affect in a model organism without a heart?
Hypothesis: I expect that the insertion of a human missense mutation causing WPW syndrome into a heartless C. elegans would exhibit the phenotypic dauer formation due to a lack of ATP binding and energy imbalance within cells [2].
Proposed Experiment: The C. elegan missense mutation that is homologous to the human R302Q would be inserted into 100 C. elegans that are in the L2 developmental stage. The stock of C. elegans would be kept at 20°C in a liquid culture. Because of the shorter lifespan of this organism (2-3 weeks at 20°C), the presence of phenotypic dauer formation will be screened for everyday [3].
Another thing that I found quite interesting when doing my bioinformatic research, was the protein network that I observed for AAKG2. When researching the protein networks for humans, a few other mammalians, and a frog, the protein serine/threonine kinase 11 (STK11) was seen interacting with AAKG2 in all of these organisms' networks. Because the protein interaction between AAKG2 and STK11 was conserved all the way down to a frog, I wanted to look more into it. The STK11 protein, also known as liver kinase B1 (LKB1), has a role in G1 cell cycle arrest and tumor suppression [4]. In relationship to AAKG2, I discovered that STK11 activates AMPK (the larger protein that AAKG2 is a part of) via phosphorylation [5].
Question: What is the function of STK11 in ATP regulation?
Hypothesis: Mutations in STK11 would inhibit the AMPK protein from performing ATP binding and would thus alter development of the heart.
Proposed Experiment: First I would propose knocking out this STK11 protein in adult mice. I would then perform a microarray in order to screen for the genetic expression of prkag2 in a wild type, prkag2 mutated, and stk11 mutated mouse. I would screen for the expression of prkag2 in cardiac, skeletal, brain, and liver tissue because this is where this gene is localized. I expect to see prkag2 more highly expressed in these specific tissues in a wild type mouse, while I would expect to see decreased prkag2 expression in both prkag2 and stk11 mutated mice.
In the future it might be insightful to look further into the highly conserved nature of prkag2's CBS domains and determine how and where they evolved from. It also might be interesting to look further into the AMPK protein in order to potentially develop treatments for STK11 mutated diseases. As the collection of information for protein network increases, it would be relevant to see if there are any more proteins being affected by mutation to the prkag2 gene.
Question: Will these missense mutations have the same affect in a model organism without a heart?
Hypothesis: I expect that the insertion of a human missense mutation causing WPW syndrome into a heartless C. elegans would exhibit the phenotypic dauer formation due to a lack of ATP binding and energy imbalance within cells [2].
Proposed Experiment: The C. elegan missense mutation that is homologous to the human R302Q would be inserted into 100 C. elegans that are in the L2 developmental stage. The stock of C. elegans would be kept at 20°C in a liquid culture. Because of the shorter lifespan of this organism (2-3 weeks at 20°C), the presence of phenotypic dauer formation will be screened for everyday [3].
Another thing that I found quite interesting when doing my bioinformatic research, was the protein network that I observed for AAKG2. When researching the protein networks for humans, a few other mammalians, and a frog, the protein serine/threonine kinase 11 (STK11) was seen interacting with AAKG2 in all of these organisms' networks. Because the protein interaction between AAKG2 and STK11 was conserved all the way down to a frog, I wanted to look more into it. The STK11 protein, also known as liver kinase B1 (LKB1), has a role in G1 cell cycle arrest and tumor suppression [4]. In relationship to AAKG2, I discovered that STK11 activates AMPK (the larger protein that AAKG2 is a part of) via phosphorylation [5].
Question: What is the function of STK11 in ATP regulation?
Hypothesis: Mutations in STK11 would inhibit the AMPK protein from performing ATP binding and would thus alter development of the heart.
Proposed Experiment: First I would propose knocking out this STK11 protein in adult mice. I would then perform a microarray in order to screen for the genetic expression of prkag2 in a wild type, prkag2 mutated, and stk11 mutated mouse. I would screen for the expression of prkag2 in cardiac, skeletal, brain, and liver tissue because this is where this gene is localized. I expect to see prkag2 more highly expressed in these specific tissues in a wild type mouse, while I would expect to see decreased prkag2 expression in both prkag2 and stk11 mutated mice.
In the future it might be insightful to look further into the highly conserved nature of prkag2's CBS domains and determine how and where they evolved from. It also might be interesting to look further into the AMPK protein in order to potentially develop treatments for STK11 mutated diseases. As the collection of information for protein network increases, it would be relevant to see if there are any more proteins being affected by mutation to the prkag2 gene.
Press the button below to download a copy of my final presentation on findings and proposed experiments.
References
[1] Xiao, B., Heath, R., Saiu, P., et al. (2007) Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature 449:496-500
[2] Hu, P.J. Dauer (August 08, 2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.144.1
[3] Stiernagle, T. Maintenance of C. elegans (May 07, 2005), WormBook, ed. The C. elegans Research Community, Wormbook, doi/10.1895/wormbook.1.101.1
[4] Baas, A.F., Boudeau J., Sapkota, G.P., et al. (2003) Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Embo Journal. 22:3062-72. doi: 10.1093/emboj/cdg292
[5] Jessen, N., Koh, H-J., Folmes, C.D., et al. (2010) Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 7-8:593-600.
[2] Hu, P.J. Dauer (August 08, 2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.144.1
[3] Stiernagle, T. Maintenance of C. elegans (May 07, 2005), WormBook, ed. The C. elegans Research Community, Wormbook, doi/10.1895/wormbook.1.101.1
[4] Baas, A.F., Boudeau J., Sapkota, G.P., et al. (2003) Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Embo Journal. 22:3062-72. doi: 10.1093/emboj/cdg292
[5] Jessen, N., Koh, H-J., Folmes, C.D., et al. (2010) Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 7-8:593-600.
Margaret Beatka ([email protected])
Page Last Updated: 5/21/13
This web page was produced as an assignment for Genetics 677, as an undergraduate course at UW-Madison.
Page Last Updated: 5/21/13
This web page was produced as an assignment for Genetics 677, as an undergraduate course at UW-Madison.