This project emerged in Emory University laboratory course in Biology 142 during the spring semester of 2015. Newly acquired whale shark proteins of interest were assigned to various student groups in hopes of better understanding the origins of these different proteins. Students were tasked to BLAST the assigned protein sequences to better understand the homology of the whale shark proteins as well as determine whether there was an orthologous protein within the whale shark genome.
Background Information
TBK1 is a serine/threonine kinase of the IKK family that plays a key role in controlling inflammatory responses to foreign agents (Fitzgerald). Following the activation of the receptors by bacterial or viral components, pro-inflammatory and antiviral genes are transcriptionally activated as well. TBK 1 steps in to create such an antiviral front by forming several different complexes whose composition varies in different types of cells and cellular stimuli ("Serine/threonine-protein"). Then, several scaffolding molecules including FADD, TANK, and TBKBP1/SINTBAD are recruited to theTBK1-containing-complexes within the cytoplasm ("Genes").
Figure1. Schematic model illustrating the signaling pathway through which TBK1 inhibits TNF induced apoptosis and the parallel pathway involving the activation of pro-inflammatory genes. (Delhase, Mireille)
Methods
Whaleshark Predicted Orthologs The human protein sequence (ENSP00000329967) was used as the query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. The top predicted protein hits were then used as queries, using the full predicted sequence, in protein BLASTs against the NCBI human protein database.
Predicted Orthologs TBK1 predicted orthologs were identified in species other than whale sharks using the NCBI Blast serves. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, yeast, whale shark, guinea pig, and fruit fly protein databases. The human TBK1 protein (ENSP00000329967) was used as the query sequence in these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each non-whale shark species search (using the human protein as query) along with the top 5 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and tree.
Searching forTBK1in the whale shark
Whale Shark ID
e- value
Alignment Length
Predicted Protein Length
% Identity
g24028.t1
2e-23
211
314
30.33
g43793.t1
2e-19
139
167
35.25
g43123.t1
7e-17
217
468
31.34
g25542.t1
6e-17
126
135
32.54
g44454.t1
4e-17
222
300
26.58
Table 1. Predicted proteins in the whale shark protein database using human TBK1 query.
Protein domains
The orthologs for the TBK1 protein found within the mouse, zebra fish, and the guinea pig all had the TBK1 protein. Even with the organisms that did not have a zero E-value, the best hits (lowest E-values) were for proteins that were from the PKc_like superfamily. The PK superfamily includes protein tyrosine kinase (PTK) , serine/thereonine kinase (STK) and dual-specificity protein kinases that phosphorylate both of the PTK and STK proteins (Marchler-Bauer et al. 2015). The PKc superfamily (Protein Kinase C) plays a essential role in cellular processes that include a variety of aspects including controlling autonomous activities and organismal functions, such as memory (Mellor and Parker 1998).
Figure 2. Results of the reciprocal cross after the shark protein sequence was BLAST searched against the human genome. PKc superfamily was the common family across all of the organisms tested.
Orthologs
The TBK1 human protein sequence (ENSP00000329967)was used as a query in the BLAST searches against other individual species's protein databases. The organisms that were tested included mouse, zebra fish, yeast, whale shark, guinea pig, and fruit fly. The mouse, zebra fish, and guinea pig had the best hits with an E-value of 0. The hit within these organisms was that of the TBK1 kinase protein. The other orthologs with significant hits were within the fruit fly (IKappaB Kinase- like 2 protein) and the yeast (RAD53 protein). The whale shark ortholog had higher E-values compared to the other organisms (2e-23).
IkappaB kinase-like 2, isoform B [Drosophila melanogaster]
NP_724278.1
1e-156
720
Table 2. Protein matches for human TBK1 across species using protein BLAST. The best entries return with the TBK1 protein (mouse, zebra fish and guinea pig). The data table displays the species, protein name, ID, protein length and the E-values.
Phylogeny
The protein sequences with the lowest E-values using the TBK1 protein query were used to create a phylogenetic tree (Figure 3). As seen below, the human, mouse, guinea pig, and zebra fish are the most similar when it pertains to the TBK1 protein. Surprisingly, the organisms stated above had a higher degree of similarity than the whale shark proteins had with each other. The yeast protein sequence had a higher degree of similarity with the whale shark proteins than the other organisms. This might suggest that the whale shark might not have the TBK1 protein but rather a distant family kinase protein that is most similar to RAD53.
Figure 3. Phylogenetic tree of TBK1 protein best hits. The best hits in the BLAST searches on the NCBI page were used in the clustalW2 program to produce the phylogenetic diagram. The five best hits for the whale shark proteins came from galaxy and were included in the creation of the phylogenetic diagram.
Conclusions
We searched the proteins of whale shark, zebra fish, mouse, guinea pig, fruit fly and human and our group found matches in the TBK1 protein sequence within all of these animals. The mouse, guinea pig, and zebra fish are most likely orthologs since their E-values are all zero. In this case, the whale shark turned out to be the least related to the human version of the protein sequence. We believe our results to be fairly accurate because the organisms stated above not only had the lowest E-values but also matched when we did a reciprocal test.
References
Delhase, Mireille. "TANK-binding Kinase 1 (TBK1) Controls Cell Survival through PAI-2/serpinB2 and Transglutaminase 2." TANK-binding Kinase 1 (TBK1) Controls Cell Survival through PAI-2/serpinB2 and Transglutaminase 2. N.p., n.d. Web. 14 Apr. 2015.
Fitzgerald, Katherine A., Sarah M. McWhirter, Kerrie L. Faia, Daniel C. Rowe, Eicke Latz, Douglas T. Golenbock, Anthony J. Coyle, Sha-Mei Liao, and Tom Maniatis. "IKKepsilon and TBK1 Are Essential Components of the IRF3 Signaling Pathway." Nature.com. Nature Immunology, 14 Apr. 2003. Web. 17 Apr. 2015.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 14 Apr. 2015.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43(Database issue):D222-6.
Mellor, H., & Parker, P. (1998). The extended protein kinase C superfamily.Biochem. j, 332, 281-292
This Project
This project emerged in Emory University laboratory course in Biology 142 during the spring semester of 2015. Newly acquired whale shark proteins of interest were assigned to various student groups in hopes of better understanding the origins of these different proteins. Students were tasked to BLAST the assigned protein sequences to better understand the homology of the whale shark proteins as well as determine whether there was an orthologous protein within the whale shark genome.
Background Information
TBK1 is a serine/threonine kinase of the IKK family that plays a key role in controlling inflammatory responses to foreign agents (Fitzgerald).
Following the activation of the receptors by bacterial or viral components, pro-inflammatory and antiviral genes are transcriptionally activated as well. TBK 1 steps in to create such an antiviral front by forming several different complexes whose composition varies in different types of cells and cellular stimuli ("Serine/threonine-protein"). Then, several scaffolding molecules including FADD, TANK, and TBKBP1/SINTBAD are recruited to theTBK1-containing-complexes within the cytoplasm ("Genes").
Figure1. Schematic model illustrating the signaling pathway through which TBK1 inhibits TNF induced apoptosis and the parallel pathway involving the activation of pro-inflammatory genes. (Delhase, Mireille)
Methods
Whaleshark Predicted Orthologs
The human protein sequence (ENSP00000329967) was used as the query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. The top predicted protein hits were then used as queries, using the full predicted sequence, in protein BLASTs against the NCBI human protein database.
Predicted Orthologs
TBK1 predicted orthologs were identified in species other than whale sharks using the NCBI Blast serves. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, yeast, whale shark, guinea pig, and fruit fly protein databases. The human TBK1 protein (ENSP00000329967) was used as the query sequence in these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each non-whale shark species search (using the human protein as query) along with the top 5 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and tree.
Searching for TBK1 in the whale shark
Protein domains
The orthologs for the TBK1 protein found within the mouse, zebra fish, and the guinea pig all had the TBK1 protein. Even with the organisms that did not have a zero E-value, the best hits (lowest E-values) were for proteins that were from the PKc_like superfamily. The PK superfamily includes protein tyrosine kinase (PTK) , serine/thereonine kinase (STK) and dual-specificity protein kinases that phosphorylate both of the PTK and STK proteins (Marchler-Bauer et al. 2015). The PKc superfamily (Protein Kinase C) plays a essential role in cellular processes that include a variety of aspects including controlling autonomous activities and organismal functions, such as memory (Mellor and Parker 1998).
Figure 2. Results of the reciprocal cross after the shark protein sequence was BLAST searched against the human genome. PKc superfamily was the common family across all of the organisms tested.
Orthologs
The TBK1 human protein sequence (ENSP00000329967)was used as a query in the BLAST searches against other individual species's protein databases. The organisms that were tested included mouse, zebra fish, yeast, whale shark, guinea pig, and fruit fly. The mouse, zebra fish, and guinea pig had the best hits with an E-value of 0. The hit within these organisms was that of the TBK1 kinase protein. The other orthologs with significant hits were within the fruit fly (IKappaB Kinase- like 2 protein) and the yeast (RAD53 protein). The whale shark ortholog had higher E-values compared to the other organisms (2e-23).
Phylogeny
The protein sequences with the lowest E-values using the TBK1 protein query were used to create a phylogenetic tree (Figure 3). As seen below, the human, mouse, guinea pig, and zebra fish are the most similar when it pertains to the TBK1 protein. Surprisingly, the organisms stated above had a higher degree of similarity than the whale shark proteins had with each other. The yeast protein sequence had a higher degree of similarity with the whale shark proteins than the other organisms. This might suggest that the whale shark might not have the TBK1 protein but rather a distant family kinase protein that is most similar to RAD53.
Figure 3. Phylogenetic tree of TBK1 protein best hits. The best hits in the BLAST searches on the NCBI page were used in the clustalW2 program to produce the phylogenetic diagram. The five best hits for the whale shark proteins came from galaxy and were included in the creation of the phylogenetic diagram.
Conclusions
We searched the proteins of whale shark, zebra fish, mouse, guinea pig, fruit fly and human and our group found matches in the TBK1 protein sequence within all of these animals. The mouse, guinea pig, and zebra fish are most likely orthologs since their E-values are all zero. In this case, the whale shark turned out to be the least related to the human version of the protein sequence. We believe our results to be fairly accurate because the organisms stated above not only had the lowest E-values but also matched when we did a reciprocal test.
References
Delhase, Mireille. "TANK-binding Kinase 1 (TBK1) Controls Cell Survival through PAI-2/serpinB2 and Transglutaminase 2." TANK-binding Kinase 1 (TBK1) Controls Cell Survival through PAI-2/serpinB2 and Transglutaminase 2. N.p., n.d. Web. 14 Apr. 2015.
Fitzgerald, Katherine A., Sarah M. McWhirter, Kerrie L. Faia, Daniel C. Rowe, Eicke Latz, Douglas T. Golenbock, Anthony J. Coyle, Sha-Mei Liao, and Tom Maniatis. "IKKepsilon and TBK1 Are Essential Components of the IRF3 Signaling Pathway." Nature.com. Nature Immunology, 14 Apr. 2003. Web. 17 Apr. 2015.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 14 Apr. 2015.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43(Database issue):D222-6.
Mellor, H., & Parker, P. (1998). The extended protein kinase C superfamily.Biochem. j, 332, 281-292
"Serine/threonine-protein Kinase TBK1." TBK1. UniProt Consortium, n.d. Web. 14 Apr. 2015.