This Project This page is the product of an assignment in Emory University’s 142 Biology lab course. The goal of this assignment was to conduct genomic analysis and aid in identifying possible ortholog proteins in the whale shark genome. This page is dedicated to the analysis of the PSMB5 protein.
Background PSMB5 has been identified as a proteasome, which is a structure made up of proteins within a cell that can digest a variety of proteins into short polypeptides and amino acids. Proteasomes are hollow and have openings on either end to allow target proteins to enter and be digested. In order to achieve their digestive functions, proteasomes require ATP (medicinenet.com). The primary function of PSMB5 is to work as an enzyme and cleave peptide bonds. PSMB5 also plays a role in protecting the cell against oxidative damage. Mutations to the PSMB5 are believed to play a role in cell resistance to apoptosis as seen in various types of cancer.
Methods Whale shark predicted orthologs The human protein sequence (ENSP00000355325) was used as query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. We then used the full sequences of the top predicted protein hits as queries in protein BLASTs against the NCBI human protein database. Predicted orthologs PSMB5 predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for the mouse, yeast, and zebrafish. We used the human PSMB5 protein (ENSP00000355325) as the query sequence in these searches with default settings. Phylogenetic Tree For each of the whale shark and non-whale shark species, we used the hit with the lowest e-value (using the full human protein sequence as query) to create a phylogenetic tree and multiple sequence alignment. To create the multiple sequence alignment and phylogenetic tree, each of the sequences were input in ClustalW2 with default setting.
Searching for PSMB5 in the Whaleshark The human PSMB5 protein sequence was used to query the whale shark predicted protein database (__http://whaleshark.georgiaaquarium.org/__) and results of the BLAST are shown below (Table 1). The top 5 hits had e-values below 1e-06, and the best hit had an e value of 7e -124. The top 5 hits were then used as queries against the human genome database (NCBI BLASTp).
Table 1: The best BLASTp human PSMB5 hits against the Whale shark predicted protein database. The Galaxy database was used to BLAST the human PSMB5 protein sequence against the predicted Whale shark protein database. The ID, e-value, Alignment Length, Predicted protein length, and percent (%) identity of the top 5 hits from that BLAST are displayed.
Two of the top 5 predicted proteins from the BLAST of the whale shark predicted proteins were used as queries against the human protein database. The BLAST of both proteins returned PSMB5 as the best hit. See Table 2 below.
Table 2. Top 2 predicted whale shark protein BLASTp hits against human protein database. The top results of each BLASTp are shown here with their e-values, name/descriptions, protein length, and percent (%) identity.
Because 2 of the BLASTs returned PSMB5 as a best hit while using the predicted whale shark proteins as queries, we believe that there is evidence that the 2 best predicted whale shark proteins and the human PSMB5 may be orthologs.
Protein Domains As shown in Figure 1, all 5 (human, mouse, yeast, elephant shark, and whale shark) proteins contain the Ntn_hydrolase super-family domain. This shows that the domain of the human PSMB5 protein is conserved in all of the top predicted whale shark proteins. The Ntn_hydrolase (N-terminal nucleophile) domain is constructed of several different enzymes that are activated autocatalytically. Studies suggest the discovery of eight entirely conserved secondary structures of the Ntn_hydrolase (Oinonen, 2000).
Human Mouse Yeast Elephant shark
Whales hark Figure 1. The figure displays the protein domains of the best mouse, yeast, elephant shark, whale shark predicted proteins and human PSMB5 protein hits. The proteins were obtained using the
Orthologs The human PSMB5 protein sequence (ENSP00000355325) was used as query in NCBI BLAST searches against individual species' protein databases. Ntn_hydrolase domain, proteasome beta type 5 orthologues were found using this method in mice, yeast, and elephant sharks. Based on the consistently low e-values, % identities, and consistently conserved domains, we have reason to believe that the PSMB5 ortholog may exists in each species included in the study.
Table 3. The table above displays the best hits with human PSMB5 protein BLAST. The human PSMB5 sequence was used in protein BLASTs against the individual species listed above. The Species, name, ID, protein length, and e-value are listed.
Phylogenetic Tree and Alignment Data
Figure 3. The figure above displays a multiple sequence alignment of individual species. The alignment of the species was generated by enter the top/best hit FASTA sequences for all of the species listed above, using the ClustalW site.
Using the alignment data from the figure above, a phylogenetic tree was generated. The tree displays the similarity between the human and mouse sequences by grouping them together on one branch. The whale shark and mouse are also group together, suggesting noteworthy similarity in their PSMB5 protein sequences. The whale shark predicted protein sequence is grouped very closely to the human, suggesting that there is a whale shark homolog to the human PSMB5 (Figure 4). Figure 4. The figure above displays a phylogenetic tree of PSMB5 best hits of each of the species listed. The best hits from BLASTp searches of the protein databases for individual species and the best whale shark predicted protein were used to create the phylogenetic tree. The ClustalW site was used to generate the tree/image.
Conclusion After running several BLASTs of the human PSMB5 against the whale shark predicted protein sequence, and reciprocal BLASTs, there is indication of a potential PSMB5 whale shark ortholog. To support this conclusion, several BLASTs were run using several different species more closely related to the whale shark (i.e Elephant shark) against the human PSMB5 protein. While all of them did not return direct indications of orthologs, each one displayed the conservation of the Ntn_hydrolase domain.
Ri, M., S. Iida, T. Nakashima, H. Miyazaki, F. Mori, A. Ito, A. Inagaki, S. Kusumoto, T. Ishida, H. Komatsu, Y. Shiotsu, and R. Ueda. "Bortezomib-resistant Myeloma Cell Lines: A Role for Mutated PSMB5 in Preventing the Accumulation of Unfolded Proteins and Fatal ER Stress." Leukemia 24.8 (2010): 1506-512. Web.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 31 Mar. 2015. <__http://www.ncbi.nlm.nih.gov/gene/5693__>.
Proteasome subunit beta type-5 (PSMB5)
This Project
This page is the product of an assignment in Emory University’s 142 Biology lab course. The goal of this assignment was to conduct genomic analysis and aid in identifying possible ortholog proteins in the whale shark genome. This page is dedicated to the analysis of the PSMB5 protein.
Background
PSMB5 has been identified as a proteasome, which is a structure made up of proteins within a cell that can digest a variety of proteins into short polypeptides and amino acids. Proteasomes are hollow and have openings on either end to allow target proteins to enter and be digested. In order to achieve their digestive functions, proteasomes require ATP (medicinenet.com). The primary function of PSMB5 is to work as an enzyme and cleave peptide bonds. PSMB5 also plays a role in protecting the cell against oxidative damage. Mutations to the PSMB5 are believed to play a role in cell resistance to apoptosis as seen in various types of cancer.
Methods
Whale shark predicted orthologs
The human protein sequence (ENSP00000355325) was used as query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. We then used the full sequences of the top predicted protein hits as queries in protein BLASTs against the NCBI human protein database.
Predicted orthologs
PSMB5 predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for the mouse, yeast, and zebrafish. We used the human PSMB5 protein (ENSP00000355325) as the query sequence in these searches with default settings.
Phylogenetic Tree
For each of the whale shark and non-whale shark species, we used the hit with the lowest e-value (using the full human protein sequence as query) to create a phylogenetic tree and multiple sequence alignment. To create the multiple sequence alignment and phylogenetic tree, each of the sequences were input in ClustalW2 with default setting.
Searching for PSMB5 in the Whaleshark
The human PSMB5 protein sequence was used to query the whale shark predicted protein database (__http://whaleshark.georgiaaquarium.org/__) and results of the BLAST are shown below (Table 1). The top 5 hits had e-values below 1e-06, and the best hit had an e value of 7e -124. The top 5 hits were then used as queries against the human genome database (NCBI BLASTp).
Table 1: The best BLASTp human PSMB5 hits against the Whale shark predicted protein database. The Galaxy database was used to BLAST the human PSMB5 protein sequence against the predicted Whale shark protein database. The ID, e-value, Alignment Length, Predicted protein length, and percent (%) identity of the top 5 hits from that BLAST are displayed.
Two of the top 5 predicted proteins from the BLAST of the whale shark predicted proteins were used as queries against the human protein database. The BLAST of both proteins returned PSMB5 as the best hit. See Table 2 below.
Table 2. Top 2 predicted whale shark protein BLASTp hits against human protein database. The top results of each BLASTp are shown here with their e-values, name/descriptions, protein length, and percent (%) identity.
Because 2 of the BLASTs returned PSMB5 as a best hit while using the predicted whale shark proteins as queries, we believe that there is evidence that the 2 best predicted whale shark proteins and the human PSMB5 may be orthologs.
Protein Domains
As shown in Figure 1, all 5 (human, mouse, yeast, elephant shark, and whale shark) proteins contain the Ntn_hydrolase super-family domain. This shows that the domain of the human PSMB5 protein is conserved in all of the top predicted whale shark proteins. The Ntn_hydrolase (N-terminal nucleophile) domain is constructed of several different enzymes that are activated autocatalytically. Studies suggest the discovery of eight entirely conserved secondary structures of the Ntn_hydrolase (Oinonen, 2000).
Human
Mouse
Yeast
Elephant shark
Whales hark
Figure 1. The figure displays the protein domains of the best mouse, yeast, elephant shark, whale shark predicted proteins and human PSMB5 protein hits. The proteins were obtained using the
Orthologs
The human PSMB5 protein sequence (ENSP00000355325) was used as query in NCBI BLAST searches against individual species' protein databases. Ntn_hydrolase domain, proteasome beta type 5 orthologues were found using this method in mice, yeast, and elephant sharks. Based on the consistently low e-values, % identities, and consistently conserved domains, we have reason to believe that the PSMB5 ortholog may exists in each species included in the study.
Table 3. The table above displays the best hits with human PSMB5 protein BLAST. The human PSMB5 sequence was used in protein BLASTs against the individual species listed above. The Species, name, ID, protein length, and e-value are listed.
Phylogenetic Tree and Alignment Data
Figure 3. The figure above displays a multiple sequence alignment of individual species. The alignment of the species was generated by enter the top/best hit FASTA sequences for all of the species listed above, using the ClustalW site.
Using the alignment data from the figure above, a phylogenetic tree was generated. The tree displays the similarity between the human and mouse sequences by grouping them together on one branch. The whale shark and mouse are also group together, suggesting noteworthy similarity in their PSMB5 protein sequences. The whale shark predicted protein sequence is grouped very closely to the human, suggesting that there is a whale shark homolog to the human PSMB5 (Figure 4).
Figure 4. The figure above displays a phylogenetic tree of PSMB5 best hits of each of the species listed. The best hits from BLASTp searches of the protein databases for individual species and the best whale shark predicted protein were used to create the phylogenetic tree. The ClustalW site was used to generate the tree/image.
Conclusion
After running several BLASTs of the human PSMB5 against the whale shark predicted protein sequence, and reciprocal BLASTs, there is indication of a potential PSMB5 whale shark ortholog. To support this conclusion, several BLASTs were run using several different species more closely related to the whale shark (i.e Elephant shark) against the human PSMB5 protein. While all of them did not return direct indications of orthologs, each one displayed the conservation of the Ntn_hydrolase domain.
References
“Proteasome Subunit Beta Type-5.” PSMB5. N.p., Web. 31 Mar. 2015.__http://www.uniprot.org/uniprot/P28074__
Ri, M., S. Iida, T. Nakashima, H. Miyazaki, F. Mori, A. Ito, A. Inagaki, S. Kusumoto, T. Ishida, H.
Komatsu, Y. Shiotsu, and R. Ueda. "Bortezomib-resistant Myeloma Cell Lines: A Role for Mutated
PSMB5 in Preventing the Accumulation of Unfolded Proteins and Fatal ER Stress." Leukemia 24.8 (2010): 1506-512. Web.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 31 Mar. 2015. <__http://www.ncbi.nlm.nih.gov/gene/5693__>.
"PSMB5 (human)." PSMB5 (human). Cell Signaling Technology, Inc., n.d. Web. 11 Apr. 2015. <__http://www.phosphosite.org/proteinAction.do?id=8939&showAllSites=true__>.
Oinonen, Carita, and Juha Rouvinen. "Structural Comparison of Ntn-hydrolases." Protein Science 9.12 (2000): 2329-337. Web. <__http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2144523/pdf/11206054.pdf__>.
"Multiple Sequence Alignment - CLUSTALW." Multiple Sequence Alignment - CLUSTALW. N.p., n.d. Web. 14 Apr. 2015. <__http://www.genome.jp/tools/clustalw/__>.
"Basic Local Alignment Search Tool." BLAST:. N.p., n.d. Web. 14 Apr. 2015. <__http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome__>.
"Galaxy / Whale Shark." Galaxy / Whale Shark. N.p., n.d. Web. 14 Apr. 2015. <__http://whaleshark.georgiaaquarium.org/__>.