Group Members: Jennifer, Mandy, George, and Anita 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 The gene that encodes for STX11 is apart of the syntaxin family. Syntaxins are nervous system specific proteins involved in the docking of synaptic vesicles with the presynaptic plasma membrane (Bauer et al. 2015). This family is also involved in the integration of Q-SNARE proteins into membranes which participate in the process of exocytosis. Exocytosis is the process in which material from the cell is exported into the exterior through a sac or a vesicle. SNARE proteins mediate vesicle fusion in the process of exocytosis. STX11 is mainly responsible for regulating protein transport between late endosomes and trans Golgi network. It is also used to target and fuse intracellular transport vesicles. A defect in this gene can cause familial hemophagocytic lymophistocytosis(FHL), a life threatening disease that causes a disorder in the immune system which produces several activated immune cells known as T cells, B cells, and macrophages. More importantly, patients with FHL have an exceeding amount of natural killer (Nk) cells which rely on efficiency of STX11. However, it is unknown how STX11 is regulated and how it functions in NK cells. However, what is known about STX11 is that a mutation in STX11 (Syntaxin 11) can cause defects on cytokines, which are proteins that play a vital role in cell signaling, and also affect cytotoxic (substance toxic to cells) granules maturation and membrane transport. This mutation can make NK cells produce an exceeding amount cytotoxin and lead to the death of blood producing cells.
stx 11 pic.png
Figure 1: NK cell effector functions are triggered through activating receptors and/or cytokines. A cascade of intracellular events will induce the biogenesis of perforin and maturation of granzyme B that will be secreted via cytotoxic granules leading to apoptosis of target cells via DNA fragmentation. The mutations affecting maturation of the cytotoxic granules or their transport to the cell membrane are shown. (Moussa 2013)
Methods/ Approach: Finding whale shark predicted orthologs: In order to find whale shark predicted orthologs, the human protein sequence (ENSP00000356540) was obtained from Ensembl and then was used as query in a Blast against the predicted whale shark protein database using the Galaxy server: whaleshark.georgiaaquarium.org . From the data collected from Galaxy, the top 4 predicted protein hits were chosen according to the lowest and most significant e-values. The top 4 predicted hits were then used as queries (using the full predicted sequence) in protein BLASTs against the NCBI human protein database. Orthologs were found if the predicted protein returned STX 11 for homo sapiens as the top hit.
Predicted orthologs The NCBI Blast server was used to identify STX 11 predicted orthologs in other species other than whale sharks. To search for potential orthologues, protein BLASTs were performed using single species protein databases for the following species: mouse, zebrafish, cows, fruit flies, yeast, and dogs.The human STX11 protein (ENSP00000356540) was used as query sequence in these searches with default settings. Orthologues were found if the predicted protein returned STX11 as the top hit. To verify if the returned hit was a potential ortholog, the full protein sequence for the top hit was then blasted against the human protein database. If STX11 was returned as a top hit, then a potential ortholog was found.
Phylogenetic tree To construct the phylogenetic tree, the full protein sequence was obtained for each top hit with the lowest E-value for each non-whale shark species search (using the human protein as query). These were added onto a document along with the protein sequences from top 4 hits from the data collected in Galaxy for the whale shark genome. All of the protein sequences were in FASTA format and then the collection of the sequences was used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and the option “rooted phylogenetic tree” was used to create the tree.
Searching for STX11 for in the Whale Shark The human STX11 protein sequence was used to query the whale shark predicted protein database and results are shown in Table 2. There were 4 hits with the smallest and most significant being 2e-108 with the next smallest E-value being 2e-06. These 4 best hits were then Blasted against the human protein database using NCBI BLASTp. From all of the 4 genes, g29623.t1 returned STX11 protein as it’s best hit. This indicates that Gene ID 29623.t1 is a potential ortholog.
Table 1: Significant Predicted Protein Hits in the Whale Shark Genome
Gene ID
E-Value
Alignment Length
Predicted protein Length
Identity%
g29623.t1
2e-108
256
255
59.38
g27800.t1
2e-06
110
230
30
g39987.t1
2e-05
49
158
34.69
g27798.t1
4e-05
49
146
41.03
Table 1. This table indicates the best Human STX11 BLASTp hits against the whale shark predicted protein database. The Galaxy server was used to query the predicted whale shark protein database using the human STX11 protein sequence. The top 4 hits according to the lowest and most significant E-values are reported here with their database ID and amino acid length. These sequences were also used as queries against the NCBI human protein database. The top hit (highlighted in red) is a potential ortholog as the STX11 returned as the best hit when that protein sequence was blasted against the human protein database.
Protein Domain The protein domains were obtained by using the human STX11 protein (ENSP00000356540) as a query in Ensembl. From the results, it is evident that STX-11 like proteins all belong to the t-SNARE superfamily (Figure 2). The t-SNARE proteins are members of the SNARE family of proteins. Members of this family are essential to fusion events in living organisms and are expressed in eukaryotic species (Shiavo et al. 1997). This superfamily consists of more than 60 members in yeast and mammalian cells (Karp 2002). SNAREs are bound to distinct membrane compartments of the endocytic pathways and contribute to distinct intracellular membrane fusion processes. The SNARE family is divided into v-SNARES and t-SNARES. V-SNARES are apart of membranes of transport during cell budding and the proteins known as t-SNARES are believed to be involved in the fusion of transport vesicles to their target membranes (Weimbs et. al 1997). The assemblage of v-SNARE and t-SNARE proteins are essential to the process of docking transport vesicles to target membranes to enable fusion to occur (Paumet 2001). Two other smart domains were identified known as the target SNARE coiled-coil domain and the Syntaxin N-terminus domain (Figure 2). Proteins of the target SNARE coiled coil domain contains t-SNARE proteins with several regions with coiled coil propensity in their cystolic part (Weimbs et. al 1997). The Syntaxin N-terminus domain is a family of receptors for intracellular transport vesicles. The function of syntaxins is determined by their localization and they are also involved in neuronal exocytosis, ER Golgi transport, and Golgi-endosome transport; they are also involved in the interaction with other proteins as well as those apart of SNARE complexes (Teng et al. 2001).
Figure 2: Protein domains for ENSP00000356540.4.
Screen Shot 2015-04-11 at 6.02.02 PM.png
Figure 2: The following domains were identified for the STX 11 protein (ENSP00000356540): t-SNARE superfamily/domain, Syntaxin N-terminal domain, and Target SNARE coiled-coil domain.
The potential ortholog identified by blasting the entire protein sequence for the best hit from Galaxy against the human protein database had both of the conserved domains- Syntaxin N terminal Smart domain and the t-SNARE superfamily domain(Figure 3). This is supporting evidence for the fact that this hit is a potential ortholog as it shares putative conserved domains with humans for the STX 11 protein (ENSP00000356540).
Figure 3: Putative Conserved Domains in Whale Shark Ortholog of human STX 11 protein
Screen Shot 2015-04-11 at 5.49.32 PM.png
Figure 3. Putative conserved domains of whale shark STX11 best hit predicted protein. This is supporting evidence for this hit being an ortholog as it shares the SynN and t-SNARE domains with the STX11 proteins of humans.
Orthologues The human STX11 protein sequence (ENSP00000356540) was used as a query in NCBI BLAST searches against individual species' protein databases. The sequence was blasted against the following species: mice, zebrafish, cows, fruit flies, yeast, and dogs. Orthologues were found in mice, zebrafish, cows, and dogs as they each returned the STX 11 protein as their best hit. To verify these were orthologues, the best hit protein sequence was blasted against the human protein database to see if the best hit returned was STX11. By performing this method, it was determined that the mice, zebra fish, cows, and dogs are potential orthologues. The Syntaxin N terminal domain and the t-SNARE domain was also found in each hit, which also was supporting evidence for these species being potential orthologues .
Table 2: BLAST Search for STX 11 Human Protein Orthologues in Different Species
Animal name
Most significant E-value
Query Coverage
Protein Length
Accession Number
Mouse
0.0
99%
313
XP_011534515.1
Zebra Fish
6e-93
99%
294
NP_998075.1
Cow
6e-166
99%
287
NP_001192665.1
Fruit Fly
9e-34
83%
291
NP_524475.1
Yeast
3e-11
70%
290
NP 015092.1
Dog
7e-174
99%
287
XP_854544.1
Table 2. Best hits with human STX11 protein BLAST. The human STX11 sequence was used in protein BLASTs against individual species. Name, accession number, protein length, and the E-value of the best hit from each search is reported here. The mouse, zebrafish, cow, and dog entries (all highlighted in red) returned STX11 as the best hit when used as queries in a BLAST against the human protein database.
Phylogeny The best hits from protein database searches using the human STX11 protein as a query were used to create a phylogenetic tree. From this tree, it is evident that the potential ortholog that was identified in whale sharks for the human STX11 protein shares a high degree of similarity with dogs and some similarity with cows, mice, and humans (figure 4). This is not surprising as this protein is found in several mammals and a high degree of similarity between the sequences with few gaps were found between these particular species (whale shark, dog, cow, mouse, and humans). The mouse shared the highest degree of similarity for the STX 11 protein with humans. The other 3 genes from the whale shark genome that had significant e-values also share a high degree of similarity with each other.
Phylogenetic Tree- STX 11.png
Figure 4. Phylogenetic tree of STX 11 best hits. The best hit from the BLAST searches of protein databases (or the best 4 hits for whale sharks) were used in ClustalW2 program to create a phylogenetic tree. Branch length represents relative evolutionary time. Each bracket also forms a clade which is a group of organisms that is believed to have evolved from the same ancestor.
Conclusion: We were able to identify a predicted STX11 ortholog in the whale shark genome which suggests that this protein can potentially play a vital role in fusion events and regulating protein transport between late endosomes and the trans golgi network. This is implied by the fact that this protein is apart of the t-SNARE family whose primary functions deal with these processes. From the results of the phylogenetic tree, there is a high degree of similarity between several of the species and this implicates that this protein is found in several mammalian species in which the protein serves similar functions. As mentioned before, mutations in this gene have been linked to an immune disorder in humans known as hemophagocytic lymphohistiocytosis in which the immune system becomes overstimulated and activated. This suggests that further research should be conducted to see what potential effects and immune disorders can result from a mutation in the STX 11 protein in whale sharks, as this can lead to further insight on the whale shark immune system and potential diseases and disorders that whale sharks can contract.
References:
Cytotoxic Granules.” Journal of Cellular and Molecular Medicine 16.1 (2012): 129–141.PMC. Web. 12 Apr. 2015.
Dabrazhynetskaya, A., J. Ma, AO Guerreiro-Cacais, Z. Arany, E. Rudd, JI Henter, K. Karre, J. Levitskaya, and V. Levitsky. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 16 Jan. 2012. Web. 14 Apr. 2015. <http://www.ncbi.nlm.nih.gov/pubmed/21342435>.
Paumet, Fabienne, et al. "A T-SNARE Of The Endocytic Pathway Must Be Activated For Fusion." Journal Of Cell Biology155.6 (2001): 961. Academic Search Complete. Web. 13 Apr. 2015.
Schiavo G, Stenbeck G. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25.. Proceedings Of The National Academy Of Sciences Of The United States Of America [serial online]. February 4, 1997;94(3):997. Available from: Academic Search Complete, Ipswich, MA. Accessed April 13, 2015.
Teng, FY, Y. Wang, and BL Tang. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 24 Oct. 2001. Web. 13 Apr. 2015. <http://www.ncbi.nlm.nih.gov/pubmed/11737951>.
Weimbs T, Seng Hui L, Chapin S, Mostov K, Bucher P, Hofmann K. A conserved domain is present in different families of vesicular fusion proteins: A new superfamily. Proceedings Of The National Academy Of Sciences Of The United States Of America [serial online]. April 1997;94(7):3046. Available from: Academic Search Complete, Ipswich, MA. Accessed April 13, 2015.
Group Members: Jennifer, Mandy, George, and Anita
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
The gene that encodes for STX11 is apart of the syntaxin family. Syntaxins are nervous system specific proteins involved in the docking of synaptic vesicles with the presynaptic plasma membrane (Bauer et al. 2015). This family is also involved in the integration of Q-SNARE proteins into membranes which participate in the process of exocytosis. Exocytosis is the process in which material from the cell is exported into the exterior through a sac or a vesicle. SNARE proteins mediate vesicle fusion in the process of exocytosis.
STX11 is mainly responsible for regulating protein transport between late endosomes and trans Golgi network. It is also used to target and fuse intracellular transport vesicles. A defect in this gene can cause familial hemophagocytic lymophistocytosis(FHL), a life threatening disease that causes a disorder in the immune system which produces several activated immune cells known as T cells, B cells, and macrophages. More importantly, patients with FHL have an exceeding amount of natural killer (Nk) cells which rely on efficiency of STX11. However, it is unknown how STX11 is regulated and how it functions in NK cells. However, what is known about STX11 is that a mutation in STX11 (Syntaxin 11) can cause defects on cytokines, which are proteins that play a vital role in cell signaling, and also affect cytotoxic (substance toxic to cells) granules maturation and membrane transport. This mutation can make NK cells produce an exceeding amount cytotoxin and lead to the death of blood producing cells.
Figure 1: NK cell effector functions are triggered through activating receptors and/or cytokines. A cascade of intracellular events will induce the biogenesis of perforin and maturation of granzyme B that will be secreted via cytotoxic granules leading to apoptosis of target cells via DNA fragmentation. The mutations affecting maturation of the cytotoxic granules or their transport to the cell membrane are shown. (Moussa 2013)
Methods/ Approach:
Finding whale shark predicted orthologs:
In order to find whale shark predicted orthologs, the human protein sequence (ENSP00000356540) was obtained from Ensembl and then was used as query in a Blast against the predicted whale shark protein database using the Galaxy server: whaleshark.georgiaaquarium.org . From the data collected from Galaxy, the top 4 predicted protein hits were chosen according to the lowest and most significant e-values. The top 4 predicted hits were then used as queries (using the full predicted sequence) in protein BLASTs against the NCBI human protein database. Orthologs were found if the predicted protein returned STX 11 for homo sapiens as the top hit.
Predicted orthologs
The NCBI Blast server was used to identify STX 11 predicted orthologs in other species other than whale sharks. To search for potential orthologues, protein BLASTs were performed using single species protein databases for the following species: mouse, zebrafish, cows, fruit flies, yeast, and dogs.The human STX11 protein (ENSP00000356540) was used as query sequence in these searches with default settings. Orthologues were found if the predicted protein returned STX11 as the top hit. To verify if the returned hit was a potential ortholog, the full protein sequence for the top hit was then blasted against the human protein database. If STX11 was returned as a top hit, then a potential ortholog was found.
Phylogenetic tree
To construct the phylogenetic tree, the full protein sequence was obtained for each top hit with the lowest E-value for each non-whale shark species search (using the human protein as query). These were added onto a document along with the protein sequences from top 4 hits from the data collected in Galaxy for the whale shark genome. All of the protein sequences were in FASTA format and then the collection of the sequences was used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and the option “rooted phylogenetic tree” was used to create the tree.
Searching for STX11 for in the Whale Shark
The human STX11 protein sequence was used to query the whale shark predicted protein database and results are shown in Table 2. There were 4 hits with the smallest and most significant being 2e-108 with the next smallest E-value being 2e-06. These 4 best hits were then Blasted against the human protein database using NCBI BLASTp. From all of the 4 genes, g29623.t1 returned STX11 protein as it’s best hit. This indicates that Gene ID 29623.t1 is a potential ortholog.
Table 1: Significant Predicted Protein Hits in the Whale Shark Genome
Protein Domain
The protein domains were obtained by using the human STX11 protein (ENSP00000356540) as a query in Ensembl. From the results, it is evident that STX-11 like proteins all belong to the t-SNARE superfamily (Figure 2). The t-SNARE proteins are members of the SNARE family of proteins. Members of this family are essential to fusion events in living organisms and are expressed in eukaryotic species (Shiavo et al. 1997). This superfamily consists of more than 60 members in yeast and mammalian cells (Karp 2002). SNAREs are bound to distinct membrane compartments of the endocytic pathways and contribute to distinct intracellular membrane fusion processes. The SNARE family is divided into v-SNARES and t-SNARES. V-SNARES are apart of membranes of transport during cell budding and the proteins known as t-SNARES are believed to be involved in the fusion of transport vesicles to their target membranes (Weimbs et. al 1997). The assemblage of v-SNARE and t-SNARE proteins are essential to the process of docking transport vesicles to target membranes to enable fusion to occur (Paumet 2001).
Two other smart domains were identified known as the target SNARE coiled-coil domain
and the Syntaxin N-terminus domain (Figure 2). Proteins of the target SNARE coiled coil domain contains t-SNARE proteins with several regions with coiled coil propensity in their cystolic part (Weimbs et. al 1997). The Syntaxin N-terminus domain is a family of receptors for intracellular transport vesicles. The function of syntaxins is determined by their localization and they are also involved in neuronal exocytosis, ER Golgi transport, and Golgi-endosome transport; they are also involved in the interaction with other proteins as well as those apart of SNARE complexes (Teng et al. 2001).
Figure 2: Protein domains for ENSP00000356540.4.
The potential ortholog identified by blasting the entire protein sequence for the best hit from Galaxy against the human protein database had both of the conserved domains- Syntaxin N terminal Smart domain and the t-SNARE superfamily domain(Figure 3). This is supporting evidence for the fact that this hit is a potential ortholog as it shares putative conserved domains with humans for the STX 11 protein (ENSP00000356540).
Figure 3: Putative Conserved Domains in Whale Shark Ortholog of human STX 11 protein
Figure 3. Putative conserved domains of whale shark STX11 best hit predicted protein. This is supporting evidence for this hit being an ortholog as it shares the SynN and t-SNARE domains with the STX11 proteins of humans.
Orthologues
The human STX11 protein sequence (ENSP00000356540) was used as a query in NCBI BLAST searches against individual species' protein databases. The sequence was blasted against the following species: mice, zebrafish, cows, fruit flies, yeast, and dogs. Orthologues were found in mice, zebrafish, cows, and dogs as they each returned the STX 11 protein as their best hit. To verify these were orthologues, the best hit protein sequence was blasted against the human protein database to see if the best hit returned was STX11. By performing this method, it was determined that the mice, zebra fish, cows, and dogs are potential orthologues. The Syntaxin N terminal domain and the t-SNARE domain was also found in each hit, which also was supporting evidence for these species being potential orthologues .
Table 2: BLAST Search for STX 11 Human Protein Orthologues in Different Species
Phylogeny
The best hits from protein database searches using the human STX11 protein as a query were used to create a phylogenetic tree. From this tree, it is evident that the potential ortholog that was identified in whale sharks for the human STX11 protein shares a high degree of similarity with dogs and some similarity with cows, mice, and humans (figure 4). This is not surprising as this protein is found in several mammals and a high degree of similarity between the sequences with few gaps were found between these particular species (whale shark, dog, cow, mouse, and humans). The mouse shared the highest degree of similarity for the STX 11 protein with humans. The other 3 genes from the whale shark genome that had significant e-values also share a high degree of similarity with each other.
Figure 4. Phylogenetic tree of STX 11 best hits. The best hit from the BLAST searches of protein databases (or the best 4 hits for whale sharks) were used in ClustalW2 program to create a phylogenetic tree. Branch length represents relative evolutionary time. Each bracket also forms a clade which is a group of organisms that is believed to have evolved from the same ancestor.
Conclusion:
We were able to identify a predicted STX11 ortholog in the whale shark genome which suggests that this protein can potentially play a vital role in fusion events and regulating protein transport between late endosomes and the trans golgi network. This is implied by the fact that this protein is apart of the t-SNARE family whose primary functions deal with these processes. From the results of the phylogenetic tree, there is a high degree of similarity between several of the species and this implicates that this protein is found in several mammalian species in which the protein serves similar functions. As mentioned before, mutations in this gene have been linked to an immune disorder in humans known as hemophagocytic lymphohistiocytosis in which the immune system becomes overstimulated and activated. This suggests that further research should be conducted to see what potential effects and immune disorders can result from a mutation in the STX 11 protein in whale sharks, as this can lead to further insight on the whale shark immune system and potential diseases and disorders that whale sharks can contract.
References:
Cytotoxic Granules.” Journal of Cellular and Molecular Medicine 16.1 (2012): 129–141.PMC. Web. 12 Apr. 2015.Dabrazhynetskaya, A., J. Ma, AO Guerreiro-Cacais, Z. Arany, E. Rudd, JI Henter, K. Karre, J. Levitskaya, and V. Levitsky. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 16 Jan. 2012. Web. 14 Apr. 2015. <http://www.ncbi.nlm.nih.gov/pubmed/21342435>.
"Familial Hemophagocytic Lymphohistiocytosis." Genetics Home Reference. Genetics Home Reference, Nov. 2014. Web. 14 Apr. 2015. <http://ghr.nlm.nih.gov/condition/familial-hemophagocytic-lymphohistiocytosis>.
Gerald K (2002). "Cell and Molecular Biology (4th edition)". John Wiley & Sons, Inc.
"InterPro." T-SNARE (IPR010989). N.p., n.d. Web. 13 Apr. 2015.
<__http://www.ebi.ac.uk/interpro/entry/IPR010989__>.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43(Database issue):D222-6.
Paumet, Fabienne, et al. "A T-SNARE Of The Endocytic Pathway Must Be Activated For Fusion." Journal Of Cell Biology155.6 (2001): 961. Academic Search Complete. Web. 13 Apr. 2015.
Schiavo G, Stenbeck G. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25.. Proceedings Of The National Academy Of Sciences Of The United States Of America [serial online]. February 4, 1997;94(3):997. Available from: Academic Search Complete, Ipswich, MA. Accessed April 13, 2015.
Teng, FY, Y. Wang, and BL Tang. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 24 Oct. 2001. Web. 13 Apr. 2015. <http://www.ncbi.nlm.nih.gov/pubmed/11737951>.
Weimbs T, Seng Hui L, Chapin S, Mostov K, Bucher P, Hofmann K. A conserved domain is present in different families of vesicular fusion proteins: A new superfamily. Proceedings Of The National Academy Of Sciences Of The United States Of America [serial online]. April 1997;94(7):3046. Available from: Academic Search Complete, Ipswich, MA. Accessed April 13, 2015.