TANK


Background Information

TRAF family member-associated NF-kappa-B activator, or TANK, functions in a form of innate immunity that protects against viral infections (NCBI, 2015) and is also involved in acute phase and inflammatory responses (Nomura, 2000). Its structure is depicted in figure 1. TANK is an activator protein that stimulates the function of all known TRAF (Tumor receptor associated factors) proteins. TRAF proteins are known to activate NF-kappa-B (NFKB) which, in turn, initiates an immune response (Chin, 1999), (Rothe, 1996). TANK-binding kinase 1 activates NF-kappa-B and goes down its transcriptional pathway (Lee, 2014). By functioning in innate immunity, TANK therefore works with nonspecific immune responses, however TANK activation has been widely seen in the presence of viral pathogens. TANK is located within the cytoplasm of the cell.

Figure 1. TRAF family member-associated NF-kappa-B activator protein structure.

PDB 1kzz EBI.jpg
PDB 1kzz EBI.jpg

TANK structure from <http://en.wikipedia.org/wiki/TANK_%28gene%29>


In a study done by Dr. Song and team, those with one of two single nucleotide polymorphisms (SNPs) (rs2395309, rs9277535) in the TANK gene have been shown to have an increased sensitivity to Hepatitis B. This study was conducted using a random sample of Han Chinese participants and supports the conclusion that the TANK regulatory protein my have a role in resisting development of this viral liver disease (Song, 2012).


Methods:


Whale Shark Predicted Orthologs


The FASTA format of the human protein sequence (ENSP00000376505) was obtained from Ensembl.org. This FASTA format sequence was blasted against the whale share predicted genes using whaleshark.georgiaaquarium.org. The top five hits of TANK protein were obtained, based on E-values and percent identity.

Predicted Orthologs


The TANK protein was identified in organisms other than whale shark using the NCBI Blast website. The FASTA format was used to blast against cow, mouse, elephant shark, zebra fish, fruit fly, and clawed fog.

Phylogenetic Tree


FASTA formats for the top five hits were obtained using the Galaxy website. The FASTA format for TANK protein was blasted against other organisms. The best hit proteins for all the organisms were obtained and used to make the phylogenetic tree. The phylogenetic tree was made via clustalw.


Protein Domain


The TBD domain (Tbk1/Ikki binding domain) is a portion of the polypeptide chain that forms a helical structure designed for binding to protein kinases. Along with TANK, this domain is also found in NAP1 and SINTBAD. This particular domain makes sense when the function of TANK is considered. Since TANK is an activator molecule, it's function depends on its ability to bind to TRAF proteins.
tbd superfamily.png

Figure 2. Protein domain for the TANK protein.


Finding the TANK ortholog in Whale sharks

Table 1. Top five hits in blast between human and whale shark.
The human sequence was blasted against the whale shark predicted gene to determine matches. Table 1 shows the best five hits in the blast between human and whale shark. These hits were blasted against the human database using NCBI Blast. The resulting e-values were not low enough to be significantly conclusive. However, further homologies between the human sequence and other organisms were found.

|| Whale Shark ID

E-value
Alignment Length
Predicted Protein Length
% Identity
g38449.t1
6e-06
40
101
37.50
g15269.t1
5e-05
60
86
28.33
g26587.t1
5e-05
45
106
26.92
g45125.t1
6e-05
52
169
30.77
g30944.t1
8e-05
71
303
28.17



Orthologs

The protein (TANK) was found in all species listed below except the fruit fly. The mouse and the cow both have e-values of 0, making them an exact match and homologous to humans. The clawed frog had the second lowest e-value, the elephant shark the third, and the zebra fish the fourth. However, these e-values show that the clawed frog and elephant shark are orthologs of humans but the e-values aren't low enough to indicate that they are homologous to humans. The fruit fly, with an e-value of 1.4, can be disregarded.
Table 2. Top matches with human TANK protein BLAST -- the human TANK sequence was used as query against the six species. The species name, ID, length, and e-value are listed.
Species
Name
ID
Length
E-value
Mouse
TRAF family member-associated NF-kappa-B activator isoform 2
NP_001157544.1
413
0.0
Elephant Shark
TRAF family member-associated NF-kappa-B activator
XP_007888043.1
471
2e-24
Zebra Fish
TRAF family member-associated NF-kappa-B activator
NP_001070068.1
348
7e-19
Clawed Frog
TRAF family member-associated NF-kappa-B activator isoform
XP_004917754.1
486
1e-40
Fruit fly
GG10629 [Drosophila erecta]
XP_001970440.1
417
1.4
Cow
TRAF family member-associated NF-kappa-B activator
NP_001179193.1
422
0.0


Phylogenetic Tree


The top hits yielded from searching the species' protein databases using the human TANK protein as query were used to generate the following phylogenetic tree, using the Clustalw algorithm. As shown, the human protein was most closely linked to the cow, followed by the mouse, clawed frog, zebra fish, elephant shark, whale shark, and then fruit fly, in subsequent order. The results yielded from the whale shark appear to be divided into two groups, with two grouped near the top and sharing a distant common ancestor with the cow, mouse, human, elephant shark, clawed frog, and zebra fish, and two near the bottom most closely linked to the fruit fly. We established that the whale shark hits were not strong ortholog possibilities - thus, it appears the closest matches in the whale shark are found in two different groups of proteins, neither orthologous to TANK.

external image tree_upgma.png
Figure 3. Phylogenetic tree generated from top hits of protein database searches for TANK protein. The top hit from the human, elephant shark, mouse, zebra fish, fruit fly, clawed frog, and cow searches and the top 5 hits from the whale shark search were entered into the Clustalw algorithm to generate the tree.





Conclusion

We attempted to identify the human TANK protein in the whale shark genome. The TANK protein is involved in the innate immune system, specifically inflammatory and acute phase responses. Thus, finding a strongly predicted ortholog in humans would indicate similarity in immune responses. However, our searches did not yield a strong prospect for an ortholog – neither the percent identities nor the e-values were significant enough to find a strong possibility. The phylogenetic tree indicates that, of the strongest predicted orthologs (which were still not sufficiently significant), two main groups exist: the two proteins grouped at the top and the three towards the bottom, most closely related to the fruit fly. We did, however, find strong ortholog possibilities in the mouse, elephant shark, zebra fish, clawed frog, and cow. Because the elephant shark and whale shark are closely related species, this indicates that a common ancestor possessed the protein, but the whale shark lost the gene as it diverged from the elephant shark. Further study could test for similar proteins in the whale shark, to determine the extent to which the whale shark’s innate immune system diverged from that of the elephant shark, human, and other species.



References


  1. Chin, Arnold I-Dah et al. “TANK Potentiates Tumor Necrosis Factor Receptor-Associated Factor-Mediated c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase Activation through the Germinal Center Kinase Pathway.” Molecular and Cellular Biology 19.10 (1999): 6665–6672. Print.
  2. "Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Apr. 2015. Web. 14 Apr. 2015. <http://www.ncbi.nlm.nih.gov/gene/10010>.
  3. Nomura, Fumiko, et al. "NF-Κb Activation Through IKK-I-Dependent I-TRAF/TANK Phosphorylation." Genes To Cells 5.3 (2000): 191. Academic Search Complete. Web. 14 Apr. 2015.
  4. Rothe, M et al. “I-TRAF Is a Novel TRAF-Interacting Protein That Regulates TRAF-Mediated Signal Transduction.” Proceedings of the National Academy of Sciences of the United States of America 93.16 (1996): 8241–8246. Print.
  5. Seung Hoon, Lee, et al. "Angiogenin Reduces Immune Inflammation Via Inhibition Of TANK-Binding Kinase 1 Expression In Human Corneal Fibroblast Cells."Mediators Of Inflammation (2014): 1-12. Academic Search Complete. Web. 14 Apr. 2015.
  6. Song, Qi-Long, Xing-Xing He, Hong Yang, Jin Li, Man Chen, Meng-Yi Wang, Qing Liu, Jin-Ling Yu, Jin-Jian Yao, Li-Feng Liu, Shu-Zhen Sun, and Ju-Sheng Lin. "Association of a TANK Gene Polymorphism with Outcomes of Hepatitis B Virus Infection in a Chinese Han Population." Viral Immunology (2012): 120106121209007. Print.
  7. "TANK (gene)." Wikipedia. Wikimedia Foundation, n.d. Web. 14 Apr. 2015. <http://en.wikipedia.org/wiki/TANK_%28gene%29>.