Figure 1. Computer model of STIM1. [1]
Figure 1. Computer model of STIM1. [1]

This Project

This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.

Background Information

STIM1 stands for the name of the protein called Stromal Interaction Molecule 1. This protein is an endoplasmic reticulum Ca2+ sensor that becomes activated when there are depleted amounts of Ca2+ in the ER. Calcium is important for a variety of cell functions like gene transcription, contraction,
and, cell growth (Takahashi et al, 2007). The mechanism of how the protein transduces signals from the endoplasmic reticulum lumen to the plasma membrane is still unknown. (Liou et al, 2007). It also activates Ca2+ channels in the plasma membrane. The changes in the Ca2+ concentrations are linked to cytoplasmic
Ca2+ inflow through the calcium release-activated channels (Stathopulos, 2006). STIM1 is modified by N-linked glycosylation from asparagine residues, providing evidence that it is located across the membrane (Williams et al., 2002). STIM1 is also a tumor suppressor gene and codes for a transmembrane phosphoprotein. Studies show that STIM1 gene sequence is highly conserved, which indicates that it arose from a single common ancestor (William et al, 2001).


Methods

Identifying Human Protein Sequence

Given the Ensembl protein ID, ENSP00000300737, a search for the full STIM1 amino acid sequence was performed through the Ensembl website [2]. The resulting sequence was recorded in FASTA format.

Identifying Orthologs

Using the human amino acid sequence found, it was then blasted against the Whale shark database on the Whale Shark Genome Galaxy website [3]. The top hit protein match’s FASTA sequence was then recorded and underwent a reciprocal blast in the NCBI human genome database [4]. Top hits were identified using the lowest e-value, highest query cover, and highest percent identity. Other orthologs were then detected in a similar manner by blasting the human STIM1 sequence on the NCBI website against the databases of the zebra fish, elephant shark, mouse, and yeast. The whale shark protein sequence was also blasted against the same organism. The resulting best hit orthologous sequences were recorded.

Generating Phylogenetic Tree

With the orthologous protein sequence of the human, whale shark, zebra fish, elephant shark, mouse, and yeast in FASTA format, they were entered into ClustalW [5] along with the whale shark and human protein sequences to compare the protein sequences and deduce phylogeny and speciation. A phylogenetic tree was generated from the protein comparison.


Results & Discussion

Protein Domains

The BLAST searches performed using the human STIM1 protein against the proteome database of zebra fish, elephant shark, mouse, and yeast yielded protein domain in Figure 3. The results show that there were conserved protein domain in the SAM superfamily, Mod r superfamily, BAR superfamily, PRK13461 domain, and the SOAR superfamily. The reciprocal blast of the whale shark sequence g37094.t1 against the human database as well as all the other species mentioned showed only the conservation of the SOAR superfamily. The SOAR chromosomal region is responsible for activating the STIM1 protein (Marchler-Bauer et al., 2015). The conservation of such a protein domain means there should be a form of the protein existing in the whale shark that is orthologous to the other species'.
Screen Shot 2015-04-07 at 4.42.41 PM.png
Figure 2. A reciprocal blast of the whale shark protein g37094.t1 was performed against the human genome. The resulting diagram above shows the conserved protein domains of the protein alignment between the human database and whale sharks query.



Screen Shot 2015-04-07 at 5.07.25 PM.png
Figure 3. A NCBI blast of the human STIM1 protein was performed against the genomes of organisms zebra fish, mouse, elephant shark, and yeast. The resulting diagram above shows all of the conserved protein domains of the protein alignment between the human query and the organisms' databases listed.

Orthologs

BLAST searches of the human STIM1 protein against the whale shark, elephant shark, zebra fish, yeast, and mouse were performed and the reciprocal BLAST searches of the whale shark g37094.t1 against the same organisms' proteomes were also performed. The results listed in Table 1 show that the initial blast of between human and whale shark have a higher e-value but a low query cover and high percent identity. Compared to the alignments between the human STIM1 against other organisms' database, we found that the the human protein is most similar to that of the mouse (100% query cover, 0.0 e-value, and 97% identity). The reciprocal was done to see consistency within the results and it was found that the whale shark protein more closely matched with the STIM1 isoform 3 of the human database. This alignment had a query cover of 87% and percent identity of 71%. Similar results were found in blasts of the whale shark sequence with other species' databases (with the exception of yeast; Table 1) in which different isoforms matched best. This suggests that the whale shark could likely have the STIM1 protein but just a different form that has differentiated through evolution.

Query
Database
Description
Query Cover
E Value
Identity
Human
Whale Shark
g37094.t1
30%
1e-96
71%
Human
Elephant Shark
Stromal interaction molecule 1 (XP_007883685.1)
52%
0.0
80%
Human
Zebra Fish
Stromal interaction molecule 1 precursor (NP_001038264.1)
93%
0.0
67%
Human
Yeast
Ste50p (NP_009898.1)
5%
1.3
27%
Human
Mouse
Stromal interaction molecule 1 precursor (NP_033313.2)
100%
0.0
97%
Whale Shark
Human
Stromal interaction molecule 1 isoform 3 precursor (NP_001264891)
87%
1e-92
71%
Whale Shark
Zebra Fish
Stromal interaction molecule 1 isoform X4 (XP_009290147.1)
87%
6e-98
73%
Whale Shark
Mouse
Stromal interaction molecule 1 isoform X5 (XP_006507600.1)
87%
3e-93
72%
Whale Shark
Elephant Shark
Stromal interaction molecule 1
87%
4e-107
77%
Whale Shark
Yeast
Pet100p (NP_010364.1)
20%
0.38
28%
Table 1. Running BLAST searches of the human STIM1 protein against the whale shark, elephant shark, zebra fish, yeast, and mouse and running the reciprocal BLAST searches of the whale shark g37094.t1 against the same organisms' proteome gave the above results. The top hits that are recorded above were chosen based on the highest query cover and percent identification and the lowest e-value in each search.

Phylogeny

The phylogenetic tree generated from with the comparison of the amino acid sequences of the orthologs listed in Table 1 (Figure 4) confirm the previous data. The human and mouse protein are most closely related, followed by the elephant shark. The zebra fish and whale shark are most closely related and yeast is least similar to all the other organisms as it diverged first on the tree (split between unicellular and multicellular eukaryotes). Following the tree, it would be likely that the whale shark has a protein similar to the STIM1 isoform X4 found in zebra fish and also the STIM1 found in elephant sharks.
Screen Shot 2015-04-07 at 4.54.34 PM.png
Figure 4. Using the orthologous proteins of the human STIM1 protein found in mouse, elephant shark, zebra fish, whale shark, and yeast (in Table 1), a phylogenetic tree was generated from the comparisons of each organism's protein sequences in respect to each other. The phylogenetic tree traces the divergence in the original ancestral protein sequence through speciation.



Conclusions

Based on the data collected, we conclude that the whale shark has some form of the STIM1 protein. The initial BLAST between the human protein against the whale shark protein database came up with a short query cover of the human protein sequence but with a low e-value and a 71% identity. Analyzing different protein domains, there is a main protein domain that matched up between the human and whale shark protein sequences. This protein domain was the SOAR (STIM1 Orai1-activating region) superfamily. This is the chromosomal region that is responsible activating the STIM1 protein (Marchler-Bauer et al., 2015). The reciprocal BLAST between the whale shark and humans showed a match with isoform 3 of the protein. Similar BLASTs with other organisms (mouse, elephant shark, yeast, and zebrafish) point to other isoforms of the same STIM1 protein (except yeast). The query coverage are for all but the yeast are 87% and have low e-values and around 70-80% identity. This leads us to believe that the whale shark does indeed have the STIM1 protein but a different isoform of it that is partially different than all of the other organisms. The divergence of the STIM1 protein can be traced with the phylogenetic tree produced from the comparison between amino acid sequences of each organism's protein of best hit. Though the tree, we can see the evolution of the protein diverged between multicellular and unicellular eukaryotes and then once again between zebrafish and whale sharks and humans, mice, and elephant sharks. Using this data, the whale shark protein should be most similar to the STIM1 isoform X4 found in zebrafish and the STIM1 found in elephant shark. Further research should be conducted to find the exact function of the STIM1 ortholog found in whale sharks; possibly as a protein used for Ca2+ transport like in other organisms. The functionality of the orthologous protein found in whale sharks are important to learning more about evolutionary divergence of the protein over time and its function changed over time.


Works Cited

Liou, Jen, Marc Fivaz, Takanari Inoue, and Tobias Meyer. "Live-cell Imaging Reveals Sequential Oligomerization and Local Plasma Membrane Targeting of Stromal Interaction Molecule 1 after Ca2+ Store Depletion." PNAS, 29 Mar. 2007.

Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43(Database issue):D222-6.

Stathopulos, Peter B., Guang-Yao Li, Michael J. Plevin, James B. Ames, and Mitsuhiko Ikura. "Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region." The Journal of Biological Chemistry. The American Society for Biochemistry and Molecular Biology, 29 Aug. 2006.

Takahashi, Y., Watanabe, H., Murakami, M., Ono, K., Munehisa, Y., Koyama, T., Nobori, K. Iijima, T., Ito, H (2007). Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells. ScienceDirect 361: 934-940.
re 1. Image from the RCSB PDB (www.rcsb.org)of PDB ID 1MAJ Stathopulos, P.B., Schindl, R.,Fahrner, M., Zheng, L., Gasmi-Seabrook, G.M.,Muik, M., Romanin, C., Ikura, M. (2013)STIM1/Orai1 coiled-coil interplay in the regulationof store-operated calcium entry Nat Commun 4: 2963-29

Williams, Richard T., Paul V. Senior, Leonie Van Stekelenburg, Judith E. Layton, Peter J. Smith, and Marie A. Dziadek. "Stromal Interaction Molecule 1 (STIM1), a Transmembrane Protein with Growth Suppressor Activity, Contains an Extracellular SAM Domain Modified by N-linked Glycosylation." ScienceDirect. Elsevier, 14 Feb. 2002.

Williams, R.T., Manji, S.S., Parker, N.J., Hancock, M.S., Van, Stekelenburg L., Eid, J.P., Senior, P.V., Kazenwadel, J.S., Shandala, T., Saint, R., Smith, P.J., Dziadek, M.A (2001). Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins. Biochem Journal 357: 673-675.


References

1. ^ Stathopulos, P.B., Schindl, R.,Fahrner, M., Zheng, L., Gasmi Seabrook, G.M.,Muik, M., Romanin, C., Ikura, M. (2013) STIM1/Orai1 coiled-coil interplay in the regulationof store-operated calcium entry. RCSB PDB (www.rcsb.org). PDB ID 1MAJ. Nat Commun 4: 2963-29.

2. ^ http://useast.ensembl.org/index.html

3. ^ http://whaleshark.georgiaaquarium.org/

4. ^ http://blast.ncbi.nlm.nih.gov/Blast.cgi

5. ^http://www.genome.jp/tools/clustalw/