Conclusions
In the investigations of my specific aims I employed the use of many online computational programs that provided me with information on the SLC26A4 gene and the Pendrin protein.
Aim 1: To identify sites of amino acid changes between species
Why? Identifying specific sites of amino acid variability between species with different hearing mechanisms and hearing capabilities could elucidate specific amino acids that are important to the acute hearing mechanism and hearing capabilities.
Approach MEME alignments using the Pendrin sequences of species with different hearing mechanisms and species with acute hearing mechanisms and different hearing capabilities
Results I found that the amino acid variability was incredibly well conserved even between species with different hearing mechanisms, however I have identified and highlighted specific amino acid changes between the zebrafish which uses a vibrational hearing, the intermediate hearing mechanism seen in the frog and sea turtle, and the remaining species which have acute hearing. The amino acid changes that result in changes in phosphorylation sites as well as those with differences in polarity were also highlighted due to their potential to change protein function by changing the protein folding or protein polarity. Finally, the three amino acid changes seen between the echolocating Brandt's bat and the Flying Fox, which has poor hearing capabilities, were of particular interest and will be revisited in future directions.
Approach MEME alignments using the Pendrin sequences of species with different hearing mechanisms and species with acute hearing mechanisms and different hearing capabilities
Results I found that the amino acid variability was incredibly well conserved even between species with different hearing mechanisms, however I have identified and highlighted specific amino acid changes between the zebrafish which uses a vibrational hearing, the intermediate hearing mechanism seen in the frog and sea turtle, and the remaining species which have acute hearing. The amino acid changes that result in changes in phosphorylation sites as well as those with differences in polarity were also highlighted due to their potential to change protein function by changing the protein folding or protein polarity. Finally, the three amino acid changes seen between the echolocating Brandt's bat and the Flying Fox, which has poor hearing capabilities, were of particular interest and will be revisited in future directions.
Aim 2: Identify interaction partners in species with acute hearing
Why? Differences between the amino acid sequences seen in species with different hearing mechanisms are likely to cause changes in the interaction partners of Pendrin, thus reflecting the differences in the proteins function in species with different hearing mechanisms
Approach Yeast-2-Hybrid and Tap-tag assays
Results I expect that the species with acute hearing will all have very similar interaction networks but that Pendrin in the species with differing hearing mechanisms will have different interaction networks, reflecting both the amino acid changes in the Pendrin sequence as well as the differing function of Pendrin within the organisms
Approach Yeast-2-Hybrid and Tap-tag assays
Results I expect that the species with acute hearing will all have very similar interaction networks but that Pendrin in the species with differing hearing mechanisms will have different interaction networks, reflecting both the amino acid changes in the Pendrin sequence as well as the differing function of Pendrin within the organisms
Aim 3: Identify proteins regulated by Pendrin
Why? Elucidation of the role of Pendrin in the early development of the inner ear and the effect of Pendrin on other developmental genes and gene products could help further understanding of not only the hearing mechanism but also pinpoint the source of hearing loss in Pendred patients
Approach Microarray analysis using a mouse model, both wild-type and Pendrin mutant lines, and analyzing the expression levels of developmental genes throughout development, starting in the embryonic stage and continuing through the mouse equivalent of adolescence--due to the adolescent development of a goiter commonly seen in Pendred patients.
Results I hypothesize that knockdown of Pendrin will affect expression levels of other developmental genes specifically inner ear, thyroid, and kidney genes
Approach Microarray analysis using a mouse model, both wild-type and Pendrin mutant lines, and analyzing the expression levels of developmental genes throughout development, starting in the embryonic stage and continuing through the mouse equivalent of adolescence--due to the adolescent development of a goiter commonly seen in Pendred patients.
Results I hypothesize that knockdown of Pendrin will affect expression levels of other developmental genes specifically inner ear, thyroid, and kidney genes
Future Directions
Investigation of the amino acid changes between species with different hearing capabilities could provide further insight into which amino acids are particularly crucial to hearing in species with acute hearing mechanisms. For example, swapping the lysine amino acid found in the first motif at the 39 position in the intermediate and acute hearing species for the polar histidine amino acid found in zebrafish, which use a vibrational hearing mechanism, followed by a behavioral study of the organisms to quantify change in hearing capabilities using mice as the model organism to assess the importance of specific changes in the amino acid sequence. Identification of specific amino acid changes that cause significant hearing loss would then be candidate sites for further investigation and potentially candidates for gene therapies in diminishing hearing loss attributable to mutations in the SLC26A4 gene and the Pendrin protein.