This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Conclusions
The BTB and BACK domains are conserved in all of the model organisms I looked at, so I concluded these are the most important domains and the ones I wanted to look further into. A study of fruit flies found the BTBD9 protein homolog to localize with another protein Cullin3, which is a ubiquitin protein [1]. A look at the protein interaction network of human BTBD9 revealed an association with two other ubiquitin-related proteins, strengthening my interest of the link between BTBD9 and ubiquitination. To reiterate, ubiquitination is a process in which a protein is tagged by a ubiquin ligase with a strand of ubiquitin, this targets the protein for degradation [2]. To follow below are some of my questions, experiments I did and think are important for the future, as well as further conclusions.
Question 1: Where are the ubiquitination sites in BTBD9?Experiment 1: Putting the BTBD9 FASTA sequence into the website UbPred.org, I found four ubiquitination sites. My results are shown in the image on the right: ubiquitation sites at amino acid positions 183, 292, 453, and 564, the sites are shown within the protein domains as labeled by the red lines. As I mentioned, I chose to focus on the BTB and BACK domains and since there is a ubiquitination site only in BACK, this is my focus primary focus.
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Hypothesis 1: Ubiquitination of the BACK domain is important for Iron metabolism
To better summarize this hypothesis and explain the relationship between ubiquitination, the BACK domain, and iron metabolism, I will refer to the image on the left, taken from the paper Sleep fragmentation and motor restlessness in Drosophila model of restless leg syndrome by Freeman, A, et al. [1]. As mentioned, Cullin3 has been shown to bind BTBD9, BTBD9 is involved in ubiquitination, the BACK domain gets ubiquitinated, RLS has been linked to low iron levels. In the left part of the image you can see the normal condition in which Cullin3 and BTBD9 bind, thus inhibiting IRP2 and allowing Ferritin expression, which is important for normal iron storage and a lack of restless legs. However, if Cullin3 and BTBD9 do not bind (as I suggested in my hypothesis that the BACK domain gets ubiquitinated and the protein would thus be targeted for protein degradation and not present to bind Cullin 3) IRP2 is expressed and subsequently inhibits Ferritin resulting in low iron storage and restless legs phenotype.
Question 2: Are the BTBD9 ubiquitination sites conserved?
Experiment 2: I took the protein FASTA sequences for homologous BTBD9 proteins in numerous animals and aligned them using ClustalW2. I then found each ubquitination site at the human position. I first looked at position 183 (in the BACK domain) as it is my primary focus. My results are in the image at the left. The red box on the far left shows position 183. As you can see, this site is very conserved with K (for the amino acid Lysine) at this position for most animals. I then looked at conservation within the other sites and noticed C.elegans had a mutation at each of the four ubiquitination sites and therefore these sites are not ubiquitinated.
Question 3: Does fly BTBD9 still function without the ubiquitination site in the BACK domain?
Experiment 3: Re-place the the worm amino acid into the fly gene and analyze for BTBD9 protein presence and function. I would do a western blot or 2D gel-electrophoresis and Maldi-TOF to detect the presence of BTBD9 protein in the fly. To identify function I would transgenically tag the BTBD9 and Cullin3 proteins with different colored immunofluorescence and observe for co-localization to suggest BTBD9 protein still functions to localize with Cullin3. I would also check the iron levels of the mutated fly and compare them to a control, normal fly. I would expect the BTBD9 protein to still be present and function as I believe this site is only necessary for ubiquitination and protein degradation, without it, protein degradation would not occur and we would have a functional protein.
Future study for HoxA9
Why is restlessness only in the legs? This seemed like an obvious question to ask. In an attempt to find the answer, I looked at the protein interaction network of BTBD9 and found a Hox gene, HoxA9 specifically (shown in the image on the left). Hox genes are important for developing the layout of the body plan in developing animals and thus may provide the positional factor to restless leg syndrome.
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Where is HoxA9 localized?
I looked this up and found that HoxA9 is located in the lower legs, just where the uncomfortable sensations associated with restless leg syndrome occur! |
Hypothesis: HoxA9 localizes BTBD9 to the lower legs
Experiment: I would knockout the HoxA9 gene in mice and tag BTBD9 with immunofluorescence to see where it localizes, I would also tag BTBD9 in a control mouse to see where BTBD9 normally localizes. I would use a DNA microarray to measure expression levels of BTBD9 in the mutant and control mouse. Lastly I would test for restlessness in the mouse by checking for increased muscle sensitivity by placing a light over various muscles in the mouse to see if the mouse moves quicker than the control mouse, suggesting uncomfortable sensations in these muscles. I would expect BTBD9 to be widely dispersed instead of just in the lower legs, since HoxA9 would not be present to localize it (my hypothesis). Since BTBD9 would be widely distributed, I expect there to be widespread restlessness, not just in the calves and since BTBD9 would not be as highly concentrated as in the lower legs, I expect restlessness to be less severe.
Other future studies
The gene ontology for BTBD9 is very basic and needs to be further developed in the future, as does the protein interaction network. This would greatly aid in understanding the importance of BTBD9 in Restless Leg Syndrome.
Identifying the exact functions of the BTBD9 domains is important as well since the current knowledge is based on the domain functions in other proteins. Again this would be helpful in understanding the function of BTBD9 and it's relationship to Restless Leg Syndrome.
The ubiquitination sites should be further analyzed to identify their potential significance in causing iron deficiency and Restless Leg Syndrome.
Tourette Syndrome has also been associated with BTBD9, it might be beneficial to look into the relationship of restless leg syndrome, tourettes, and BTBD9.
Identifying the exact functions of the BTBD9 domains is important as well since the current knowledge is based on the domain functions in other proteins. Again this would be helpful in understanding the function of BTBD9 and it's relationship to Restless Leg Syndrome.
The ubiquitination sites should be further analyzed to identify their potential significance in causing iron deficiency and Restless Leg Syndrome.
Tourette Syndrome has also been associated with BTBD9, it might be beneficial to look into the relationship of restless leg syndrome, tourettes, and BTBD9.
Download the file below to view my final presentation in PowerPoint format.
rls.btbd9final.pptx | |
File Size: | 1108 kb |
File Type: | pptx |
References
[1] Freeman, A., et al. (2012). Sleep fragmentation and motor restlessness in Drosophila model of restless leg syndrome. Current Biology 22, 1142-1148. DOI 10.1016/j.cub.2012.04.027
[2] Molineaux, S.M., (2011). Molecular Pathways: Targeting Proteasomal Protein Degradation in Cancer. Clin Cancer Res; 18(1); 15-20.doi: 10.1158/1078-0432.CCR-11-0853.
[2] Molineaux, S.M., (2011). Molecular Pathways: Targeting Proteasomal Protein Degradation in Cancer. Clin Cancer Res; 18(1); 15-20.doi: 10.1158/1078-0432.CCR-11-0853.