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 Interferon-inducible proteinAIM2 (also known as absent in melanoma 2) is a human protein which belongs to the interferon-inducible HIN-200 family (Ludlow, L. E. et al., 2005). AIM2 is made up of 343 protein residues and contains a N-terminal Pyrin domain and a C-terminus HIN-200 (Fernandes Alnemri et al., 2009).
The expression of the AIM2 protein plays a key role in the function of the inflammasome, an essential component of the innate immune system of multicellular organisms which is responsible for triggering an inflammatory immune pathway in response to the detection of foreign viral or bacterial DNA (Schroder & Tschopp, 2010). The expression of AIM2 activates the caspase-1 enzyme which is part of the innflamasome structure (Fernandes Alnemri et al., 2009). The function of caspase-1 within the inflammasome as a protein cleaver is vital to the activation of an inflammatory response and in inducing necrosis or pyroptosis within cells (Yu, J. W. et al., 2007). The activation of caspase-1 has been shown to be dependent on an intact Pyrin domain within the AIM2 protein (Fernandes Alnemri et al., 2009).
Recent studies have also implicated AIM2 in the process of tumorigenic reversion and in the proliferation of cancerous cells (Dihlmann et al., 2014). Furthermore, AIM2 expression has been identified as a veritable biomarker in the prognosis prediction for patients suffering from various types of cancer and has become the topic of many cancer studies (Dihlmann et al., 2014).
Methods Searching for an AIM2 ortholog in the whale shark genome
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used to BLAST against the predicted whale shark protein database on the whaleshark.georgiaaquarium.org Galaxy server in order to identify possible orthologs within the whale shark predicted protein sequence. The top hits were then used in a reverse BLAST against the human genome in order too verify if it was a possible ortholog. A bootstrapping technique, where the best African Ghostshark predicted AIM2 ortholog sequence was used as query in the BLAST of the whale shark predicted protein databse, was then attempted when our initial BLASTp returned insignificant e-values. Orthologs
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used as query to search the mouse, Rhesus monkey, and African Ghostshark genomes in addition to the whale shark genome for possible orthologs. Phylogenetic tree
The best hits from our general ortholog search of the mouse, Rhesus monkey, African Ghostshark and whaleshark genome (listed in Table 4) were used to run a multiple sequence alignment in ClustalW2 and to make a phylogenetic tree.
Searching for an AIM2 Ortholog in the Whale Shark Genome The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used to BLAST against the predicted whale shark protein database stored on the whales shark Galaxy server (see Table 1).
Table 1: Top hits for possible AIM2 ortholog in whale shark genome. The human AIM2 protein FASTA sequences was used as query to search for a possible ortholog within the whale shark predicted protein database. The following hits were returned.
None of these e-values nor alignments provided enough evidence for a possible homologue,
furthermore, in a reverse BLAST using the top hits none returned the human AIM2 protein when used as a query in a reverse BLAST against the human genome. A bootstrapping BLAST technique was then used to search for better hits by first attempting to identify the closest
African Ghostshark AIM2 ortholog and then using the African Ghostshark sequence to BLAST the whale shark genome. This method returned equally insignificant results as we were unable to identify a possible AIM2 ortholog within theAfrican Ghostshark genome and other related genomes.
From this search we were unable to identify a strong potential AIM2 ortholog within the whale shark genome.
Domains:
Pyrin Domain The Pyrin Domain, also known as the death fold, is a member of the death domain-fold superfamily of proteins believed to have evolved from an older, ancestral protein domain (Fairbrother et al., 2001). Members of the death domain superfamily are known mediators of many protein-protein interactions which program apoptosis and inflammation pathways (Fairbrother et al., 2001). Other members of the death fold superfamily include caspase recruitment domains (CARDs), which interact with caspase proteins in different multicellular organisms (Fairbrother et al., 2001). This is consistent with the results of recent studies which suggest that the AIM2 function of activating caspase-1 is dependent on it containing a terminal Pyrin domain; the Pyrin domain on the N-terminus of the AIM2 protein is able to interact with other Pyrin domains and activate caspase-1 to create functional inflamasomes (Fernandes Alnemri et al., 2009). The AIM2 protein shares approximately 18% sequence identity with the Pyrin domain (see Table 2).
In the reverse blast between the top whale shark hits from our original BLAST (listed in Table 1 above) and the human genome there were multiple hits which contained the Pyrin or PYD domain: Pyrin domain-containing protein 1 [Homo sapiens].These results indicated to us that the whale shark genome may contain fragments of the conserved Pyrin domain.
A follow-up BLAST between the known Pyrin domain sequence (NP_000234.1) and the whale shark genome returned hits with e-values as low as 2e -62 with a 50.57 % identity which would suggest that the whale shark genome contains fragments of the conserved Pyrin domain/ death domain superfamily sequences (see Table 3).
Table 3: Top hits in whale shark genome using Pyrin domain as query. The known Pyrin domain sequence (NP_000234.1) was used as query to BLASTp the predicted whale shark protein database. The following hits were returned.
Orthologs
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used as a BLAST query in order to identify possible orthologs within the mouse, rhesus monkey, and Australian Ghostshark genomes in addition to the whale shark genome. The best hits from each database are displayed in Table 4.
Table 4: Top hits for possible AIM2 ortholog within each respective genome.
As you can see in Table 4, the Mouse and Rhesus monkey genomes both contain similar AIM2 orthologs where the Rhesus monkey AIM2 was found to be identical to the human protein. The e-value of the best hit from the whale shark genome suggests the whale shark genome shares a possible homeodomain (which we identified as Pyrin domain/death domain superfamily) with the human AIM2 sequence which corroborates our findings in the Domains section of this page. It is unlikely that the Australian Ghostshark contains an AIM2 ortholog or shares a homeodomain with the human sequence, the e-value is too high.
Phylogenetic Tree
The FASTA sequences from the top hits of the multi species ortholog search displayed in in Table 4 were used to create a phylogentic tree. As expected, the species containing AIM2 orthologs are grouped closely together suggesting the conserved AIM2 protein sequence diverged similarly and more recently within these mammalian species than in the shark species. The early divergence between the mammalian protein and the shark protein also supports our findings in the Domains section of this page which suggest that the whale shark genome shares ancestral Pyrin domain/death domain superfamily sequences with the human AIM2 protein.
Figure1: Phylogenetic tree of best hit orthologs. The FASTA sequences of the best hits from the ortholog search (displayed in Table 4) were used to create a phylogenetic tree to map protein divergence and homogeny.
Conclusions
Although we were unable to identify an AIM2 ortholog in the whale shark genome our findings suggest that the genome might possibly contain conserved pyrin protein domains or an older ancestral member of the larger death domain superfamily. Our results from the protein BLAST of the Pyrin domain against the whale shark genome offer the strongest evidence for a possible homeodomain. The phylogenetic tree which indicates divergence between the mammalian and shark sequences further corroborates this finding and paints a picture of how an ancestral death domain protein might have diverged and eventually become a key domain within the AIM2 protein (Pyrin domain). Further research into the conservation and variation of these domain sequences within the whale shark and subsequent organisms could identify a more specific divergence of the Pyrin domain as well as the mammalian AIM2 protein and could provide valuable insight into the development of the innate immune system which these proteins pertain to.
Next Step Testing:
The expression of AIM2 affects inflammasome activation (Fernandes Alnemri et al., 2009). In order to test the function of the [possibly] conserved death-domain protein sequences for AIM2 like functioning we could [theoretically] knock-out the death-domain fragment sequences within a whale shark cell and test its immunity by injecting foreign bacterial DNA into the cytoplasm of that cell. If these Pyrin-esque (or Pyrin-ancestral) domains serve the same function as the Pyrin domain within the AIM2 protein we would expect that the knock out inhibit the activation of the innate immune response, i.e. the inability to activate important components of the inflammasome complex. Likely possible model organisms suitable for this research might include Drosophilia melanogaster (fruit fly) and Caenorhabditis elegans (flat worm) because these organisms are known to have well developed
innate immunity (Janeway et al., 2001).
References
Dihlmann, Susanne, et al. "Lack of Absent in Melanoma 2 (AIM2) expression in tumor cells is closely associated with poor survival in colorectal cancer patients." International journal of cancer 135.10 (2014):2387-2396.
Fairbrother, W. J., Gordon, N. C., Humke, E. W., O’Rourke, K. M., Starovasnik, M. A., Yin, J.-P., & Dixit, V. M. (2001). The PYRIN domain: A member of the death domain-fold superfamily. Protein Science : A Publication of the Protein Society, 10(9), 1911–1918.
Fernandes Alnemri, Teresa, et al. "AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA." Nature 458.7237 (2009):509-13.
Janeway CA Jr., Travers P., Walport M., et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. Evolution of the innate immune system.
Ludlow, L. E., Johnstone, R. W. & Clarke, C. J. The HIN-200 family: more than interferon-inducible genes?Exp. Cell Res.308, 1–17 (2005)
Schroder, Kate, and JurgTschopp. "The inflammasomes." Cell 140.6 (2010):821-32. (Yu, J. W. et al., 2007)
Yu, J. W. et al.Pyrin activates the ASC pyroptosome in response to engagement by autoinflammatory PSTPIP1 mutants. Mol. Cell28, 214–227 (2007)
This ProjectThis 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
Interferon-inducible proteinAIM2 (also known as absent in melanoma 2) is a human protein which belongs to the interferon-inducible HIN-200 family (Ludlow, L. E. et al., 2005). AIM2 is made up of 343 protein residues and contains a N-terminal Pyrin domain and a C-terminus HIN-200 (Fernandes Alnemri et al., 2009).
The expression of the AIM2 protein plays a key role in the function of the inflammasome, an essential component of the innate immune system of multicellular organisms which is responsible for triggering an inflammatory immune pathway in response to the detection of foreign viral or bacterial DNA (Schroder & Tschopp, 2010). The expression of AIM2 activates the caspase-1 enzyme which is part of the innflamasome structure (Fernandes Alnemri et al., 2009). The function of caspase-1 within the inflammasome as a protein cleaver is vital to the activation of an inflammatory response and in inducing necrosis or pyroptosis within cells (Yu, J. W. et al., 2007). The activation of caspase-1 has been shown to be dependent on an intact Pyrin domain within the AIM2 protein (Fernandes Alnemri et al., 2009).
Recent studies have also implicated AIM2 in the process of tumorigenic reversion and in the proliferation of cancerous cells (Dihlmann et al., 2014). Furthermore, AIM2 expression has been identified as a veritable biomarker in the prognosis prediction for patients suffering from various types of cancer and has become the topic of many cancer studies (Dihlmann et al., 2014).
Methods
Searching for an AIM2 ortholog in the whale shark genome
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used to BLAST against the predicted whale shark protein database on the whaleshark.georgiaaquarium.org Galaxy server in order to identify possible orthologs within the whale shark predicted protein sequence. The top hits were then used in a reverse BLAST against the human genome in order too verify if it was a possible ortholog. A bootstrapping technique, where the best African Ghostshark predicted AIM2 ortholog sequence was used as query in the BLAST of the whale shark predicted protein databse, was then attempted when our initial BLASTp returned insignificant e-values.
Orthologs
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used as query to search the mouse, Rhesus monkey, and African Ghostshark genomes in addition to the whale shark genome for possible orthologs.
Phylogenetic tree
The best hits from our general ortholog search of the mouse, Rhesus monkey, African Ghostshark and whaleshark genome (listed in Table 4) were used to run a multiple sequence alignment in ClustalW2 and to make a phylogenetic tree.
Searching for an AIM2 Ortholog in the Whale Shark Genome
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used to BLAST against the predicted whale shark protein database stored on the whales shark Galaxy server (see Table 1).
Table 1: Top hits for possible AIM2 ortholog in whale shark genome. The human AIM2 protein FASTA sequences was used as query to search for a possible ortholog within the whale shark predicted protein database. The following hits were returned.
None of these e-values nor alignments provided enough evidence for a possible homologue,
furthermore, in a reverse BLAST using the top hits none returned the human AIM2 protein when used as a query in a reverse BLAST against the human genome. A bootstrapping BLAST technique was then used to search for better hits by first attempting to identify the closest
African Ghostshark AIM2 ortholog and then using the African Ghostshark sequence to BLAST the whale shark genome. This method returned equally insignificant results as we were unable to identify a possible AIM2 ortholog within theAfrican Ghostshark genome and other related genomes.
From this search we were unable to identify a strong potential AIM2 ortholog within the whale shark genome.
Domains:
Pyrin Domain
The Pyrin Domain, also known as the death fold, is a member of the death domain-fold superfamily of proteins believed to have evolved from an older, ancestral protein domain (Fairbrother et al., 2001). Members of the death domain superfamily are known mediators of many protein-protein interactions which program apoptosis and inflammation pathways (Fairbrother et al., 2001). Other members of the death fold superfamily include caspase recruitment domains (CARDs), which interact with caspase proteins in different multicellular organisms (Fairbrother et al., 2001). This is consistent with the results of recent studies which suggest that the AIM2 function of activating caspase-1 is dependent on it containing a terminal Pyrin domain; the Pyrin domain on the N-terminus of the AIM2 protein is able to interact with other Pyrin domains and activate caspase-1 to create functional inflamasomes (Fernandes Alnemri et al., 2009). The AIM2 protein shares approximately 18% sequence identity with the Pyrin domain (see Table 2).
Table 2: Percentage identity and similarity between PYRIN domain sequences
source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2253208/table/t1/
In the reverse blast between the top whale shark hits from our original BLAST (listed in Table 1 above) and the human genome there were multiple hits which contained the Pyrin or PYD domain: Pyrin domain-containing protein 1 [Homo sapiens].These results indicated to us that the whale shark genome may contain fragments of the conserved Pyrin domain.
A follow-up BLAST between the known Pyrin domain sequence (NP_000234.1) and the whale shark genome returned hits with e-values as low as
2e -62 with a 50.57 % identity which would suggest that the whale shark genome contains fragments of the conserved Pyrin domain/ death domain superfamily sequences (see Table 3).
Table 3: Top hits in whale shark genome using Pyrin domain as query. The known Pyrin domain sequence (NP_000234.1) was used as query to BLASTp the predicted whale shark protein database. The following hits were returned.
Orthologs
The human AIM2 protein sequence (ENSP00000357112, NP_004824.1) was used as a BLAST query in order to identify possible orthologs within the mouse, rhesus monkey, and Australian Ghostshark genomes in addition to the whale shark genome. The best hits from each database are displayed in Table 4.
Table 4: Top hits for possible AIM2 ortholog within each respective genome.
As you can see in Table 4, the Mouse and Rhesus monkey genomes both contain similar AIM2 orthologs where the Rhesus monkey AIM2 was found to be identical to the human protein. The e-value of the best hit from the whale shark genome suggests the whale shark genome shares a possible homeodomain (which we identified as Pyrin domain/death domain superfamily) with the human AIM2 sequence which corroborates our findings in the Domains section of this page. It is unlikely that the Australian Ghostshark contains an AIM2 ortholog or shares a homeodomain with the human sequence, the e-value is too high.
Phylogenetic Tree
The FASTA sequences from the top hits of the multi species ortholog search displayed in in Table 4 were used to create a phylogentic tree. As expected, the species containing AIM2 orthologs are grouped closely together suggesting the conserved AIM2 protein sequence diverged similarly and more recently within these mammalian species than in the shark species. The early divergence between the mammalian protein and the shark protein also supports our findings in the Domains section of this page which suggest that the whale shark genome shares ancestral Pyrin domain/death domain superfamily sequences with the human AIM2 protein.
Figure1: Phylogenetic tree of best hit orthologs. The FASTA sequences of the best hits from the ortholog search (displayed in Table 4) were used to create a phylogenetic tree to map protein divergence and homogeny.
Conclusions
Although we were unable to identify an AIM2 ortholog in the whale shark genome our findings suggest that the genome might possibly contain conserved pyrin protein domains or an older ancestral member of the larger death domain superfamily. Our results from the protein BLAST of the Pyrin domain against the whale shark genome offer the strongest evidence for a possible homeodomain. The phylogenetic tree which indicates divergence between the mammalian and shark sequences further corroborates this finding and paints a picture of how an ancestral death domain protein might have diverged and eventually become a key domain within the AIM2 protein (Pyrin domain). Further research into the conservation and variation of these domain sequences within the whale shark and subsequent organisms could identify a more specific divergence of the Pyrin domain as well as the mammalian AIM2 protein and could provide valuable insight into the development of the innate immune system which these proteins pertain to.
Next Step Testing:
The expression of AIM2 affects inflammasome activation (Fernandes Alnemri et al., 2009). In order to test the function of the [possibly] conserved death-domain protein sequences for AIM2 like functioning we could [theoretically] knock-out the death-domain fragment sequences within a whale shark cell and test its immunity by injecting foreign bacterial DNA into the cytoplasm of that cell. If these Pyrin-esque (or Pyrin-ancestral) domains serve the same function as the Pyrin domain within the AIM2 protein we would expect that the knock out inhibit the activation of the innate immune response, i.e. the inability to activate important components of the inflammasome complex. Likely possible model organisms suitable for this research might include Drosophilia melanogaster (fruit fly) and Caenorhabditis elegans (flat worm) because these organisms are known to have well developed
innate immunity (Janeway et al., 2001).
References
Dihlmann, Susanne, et al. "Lack of Absent in Melanoma 2 (AIM2) expression in tumor cells is closely associated with poor survival in colorectal cancer patients." International journal of cancer 135.10 (2014):2387-2396.
Fairbrother, W. J., Gordon, N. C., Humke, E. W., O’Rourke, K. M., Starovasnik, M. A., Yin, J.-P., & Dixit, V. M. (2001). The PYRIN domain: A member of the death domain-fold superfamily. Protein Science : A Publication of the Protein Society, 10(9), 1911–1918.
Fernandes Alnemri, Teresa, et al. "AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA." Nature 458.7237 (2009):509-13.
Janeway CA Jr., Travers P., Walport M., et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. Evolution of the innate immune system.
Ludlow, L. E., Johnstone, R. W. & Clarke, C. J. The HIN-200 family: more than interferon-inducible genes? Exp. Cell Res. 308, 1–17 (2005)
Schroder, Kate, and JurgTschopp. "The inflammasomes." Cell 140.6 (2010):821-32. (Yu, J. W. et al., 2007)
Yu, J. W. et al. Pyrin activates the ASC pyroptosome in response to engagement by autoinflammatory PSTPIP1 mutants. Mol. Cell 28, 214–227 (2007)