TMEM173/STING

Background Information
The Transmembrane Protein 173 is a human transmembrane protein involved in innate immune response to viral and bacterial infections. Its structure is depicted in figure 1. It senses the cytosolic DNA of viruses and bacteria, signaling creation of type I interferon. The process is started when the protein is triggered by messengers produced by bacteria and viral DNA by binding these messengers and activating two transcription pathways: NF-kappa-B and IRF3. These activate expression of type I expression. Ureterocele and Japanese Encephalitis are two diseases correlated to the disease. According to a 2014 Cell study, this STING pathway is involved in “innate immune” detection of cancer.



500px-STING_4EMU.jpg
500px-STING_4EMU.jpg

Figure 1: (__http://en.wikipedia.org/wiki/Stimulator_of_interferon_genes___) STING Protein - Crystal Structure. The protein’s crystal structure demonstrates how STING senses cyclic diguanylate monophosphate (c-di-GMP). When c-di-GMP binds, the two surface loops dissociate (Shang and Guijun, 2012)



nsmb.2332-F2.jpg
nsmb.2332-F2.jpg



Figure 2: (__http://www.nature.com/nsmb/journal/v19/n7/fig_tab/nsmb.2332_F2.html__)



840px-STING_signaling_20130422.png
840px-STING_signaling_20130422.png

Figure 3: STING Signaling: STING activates phosphorylation of IRF3 and STAT6 by activating TBK1. IRF3 and STAT6 then dimerize and enter the nucleus, which activates Type I interferon immune responses. AT6 then dimerize and enter the nucleus, which activates Type I interferon immune responses.
(__http://en.wikipedia.org/wiki/Stimulator_of_interferon_genes__)


Methods
Whale shark predicted orthologs:
On the whaleshark.georgiaaquarium.org Galaxy server, the protein sequence was used as the query in a BLAST search against the whale shark protein sequence. The best five protein hits were used as queries in BLASTs against the NCBI human protein sequence. The whale shark predicted protein database was searched as well, with the TMEM173 sequence as the query.


Predicted orthologs:
The TMEM173 protein was detected in species other than whale sharks with NCBI Blast. The FASTA format was used to blast against the mouse, cow, elephant shark, fruit fly, clawed frog, and zebrafish.


Phylogenetic tree:
A phylogenetic tree was created with ClustalW2 by using the non-whale shark species with the lowest E-values and the top five whale shark BLAST hits.
Searching for TMEM173/STING in the Whale Shark
The human TMEM173 protein sequence was used as a query to search the whale shark predicted protein database. The results of this query are shown in Table 1. None of the results had e-values below 1E-04 and all % identities were relatively low (under 50%) - thus we were unable to conclude that any of the hits were strong ortholog candidates.


Whale Shark ID
E-value
Alignment length
Predicted protein length
% identity
g28183.t1
1E-04
21
109
47.62%
g33175.t1
5E-04
28
100
50.00%
g13069.t1
7E-04
50
151
28.00%
g25371.t1
7E-04
48
156
35.42%
g23072.t1
8E-04
23
84
47.83%
Table 1. Top 5 hits in human/whale shark blast.


Because there were no strong matches when the human TMEM173 protein was used as query against the whale shark database, this process was repeated using the protein predicted for the Dolphin database as query against the whale shark predicted protein database. The results still yielded low e-values (the lowest here being 7E-06), so again there were no strong matches for possible orthologs.


Whale Shark ID
E-value
Alignment length
Predicted protein length
% identity
g46626.t1
7E-06
65
153
27.69%
g32883.t1
1E-04
21
98
42.86%
g23638.t1
2E-04
36
88
38.89%
g26983.t1
2E-04
55
135
30.91%
Table 2. Protein predicted for the dolphin database as query used against whale shark predicted protein database


Protein domains
Possible proteins similar to TMEM173 all include a STING_C superfamily. Located in the ER and composed of an N-terminal transmembrane region and a C-terminal c-di-GMP binding domain, STING is the master regulator that controls production of cytokine when there is a microbial invasion. It senses messengers like cyclic dinucleotide bis-(3’-5’)-cyclic dimeric GMP and activates the IFN regulatory factor 3 through the TANK-binding kinase 1 stimulation. STING is also a signaling adaptor in the IFN response to the cytosolic DNA. This quick detection of foreign substances is necessary for efficient immune responses.


external image 5G8ID2OCo6aAjYtl_pwYTgpvhRt2UzBdrrcCYun4ZjDptgFfU6U2jmqOHf7OOaumTFoCsMcy6iU8835ZYYQmIpQvc3JE8mV_4P4RWVfVUAsLBcPzUNAk_FUOAyoUy_qxooR8tAY
Figure 3. Putative conserved domains of TMEM173 best hit proteins. All five of the best hits have the STING_C superfamily.


The human TMEM173 protein sequence was used as query in NCBI BLAST searches against the following species’ protein databases: mouse, elephant shark, zebra fish, clawed frog, fruit fly, and cow.
Species
Name
ID
Length
E-Value
Mouse
stimulator of interferon genes protein isoform 3
NP_001276521.1
337
3e-166
Elephant Shark
stimulator of interferon genes protein
XP_007904451.1
378
2e-73
Zebrafish
stimulator of interferon genes protein isoform X1
XP_005157178.1
398
4e-52
Clawed Frog
stimulator of interferon genes protein
NP_001106445.2
355
2e-78
Fruit Fly
CG1667, isoform B
NP_001286256.1
343
9e-05
Cow
stimulator of interferon genes protein
NP_001039822.1
378
0.0
Table 3. The top matches with TMEM173 protein - the human TMEM173 sequence was used as a query against six species.


Phylogeny
The top hit from the protein database searches of each species (using the human TMEM173 protein as query) were entered into the Clustal2w program to create the following phylogenetic tree (Figure 4). The top 4 whale shark hits were used. As shown, the whale shark proteins appear to be highly similar as they are grouped towards the bottom of the tree. These proteins are most similar to that of the fruit fly, following by the zebrafish, elephant shark, clawed frog, mouse, cow, and human in subsequent order. These results are surprising consider the whale shark’s close relations to the elephant shark.


tree_upgma.png
tree_upgma.png

Figure 4. Phylogenetic tree showing gene ancestry between the individual species. Top hits from the BLAST searches were used in the Clustal2W program to create the phylogenetic tree.




Conclusion





We attempted to locate a strong predicted TMEM173 ortholog in the whale shark genome. TMEM173 is involved in innate immune signaling responses, thus identifying it in whale sharks would have provided insight into the human immune system, as well as the immune responses in related species. However, our search failed to identify a strong ortholog prospect in whale sharks. We then searched for the protein in several additional species, and our results yielded strong ortholog prospects in the mouse, cow, clawed frog, zebrafish, and elephant shark. These results are surprising, in light of the whale shark’s close genetic relationship to the elephant shark. The protein was also found in the other species, which are farther removed from the elephant shark than the whale shark. Thus, the TMEM173 protein must have been present in a common ancestor of whale sharks and elephant sharks, but was lost in the whale shark as the species diverged. It would be interesting to search for related genes in the whale shark, to determine what components of the immune system the lack of this protein affects. These results could be used to suggest paths of divergence between the elephant shark and whale shark and draw contrasts between the human and whale shark immune systems.








References
  1. "Stimulator of Interferon Genes Protein." TMEM173. N.p., n.d. Web. 13 Apr. 2015. <http://www.uniprot.org/uniprot/Q86WV6>.
  2. "TMEM173 Gene." - GeneCards. N.p., n.d. Web. 13 Apr. 2015. <http://www.genecards.org/cgi-bin/carddisp.pl?gene=TMEM173>.
  3. Ouyang, Songying, et al. "Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding." Immunity 36.6 (2012): 1073-1086.
  4. "Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 01 Apr. 2015. <http://www.ncbi.nlm.nih.gov/gene/340061>.
  5. "Stimulator of Interferon Genes Protein." TMEM173. N.p., n.d. Web. 01 Apr. 2015. <http://www.uniprot.org/uniprot/Q86WV6>.
  6. Zhiqing, Huang, et al. "Molecular Cloning And Functional Characterization Of Tibetan Porcine STING." International Journal Of Molecular Sciences 13.1 (2012): 506-515. Academic Search Complete. Web. 1 Apr. 2015.
  7. Shang, Guijun, et al. "Crystal structures of STING protein reveal basis for recognition of cyclic di-GMP." Nature structural & molecular biology 19.7 (2012): 725-727.