PSMB8 ENSP00000406878

This Project

The purpose of this project is to annotate specific genes in the newly sequenced whale shark genome in order to contribute to the scientific study of the whale shark and the immune system.

Background Information
The PSMB8 gene (proteasome subunit beta type 8) is also used to cleave peptides in order to promote normal antigen processing in order to produce class I binding peptides (“PSMB8” 2015). This gene also has ATP-dependent proteolytic activity, which allows the gene to be used in the production of class I binding peptides (“Proteasome Subunit Beta Type-8” 2014). Furthermore, the PSMB8 gene is utilized to degrade excess or abnormal proteins that may be present in cells. This cellular degradation helps to regulate immune response against bacteria and viruses (“PSMB8 Gene” 2015). In a study conducted by Akiko Kitamura, it was concluded that a mutation in the PSMB8 gene could cause autoinflammation in humans. Autoinflammation causes irregular, recurrent fevers in humans, which disrupts proper cellular function (Kitamura 2015). The lack or a mutated PSMB gene can also affect the human body as it may lead to Nishimura syndrome, which causes a change in a single amino acid, which then leads to a reduction of proteins, causing the immune system to holistically malfunction (“PSMB8 Gene” 2015). The PSMB8 gene has various functions which essentially allows for the promotion of normal antigen processing and immune system function.
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Figure 1: The cellular effect of a PSMB8 mutation in a patient with Nishamura syndrome (Arima 2015)


Protein Domain


The first illustrated domain in figure 2 is the proteasome_beta_type_5 specific hit domain that is an enzyme quintessential in non-lysosomal protein degradation in the cytoplasm and nucleus of eukaryotic cells. The alpha and beta subunits of the enzyme are part of the N-terminal nucleophile (Ntn)-hydrolase superfamily; responsible for peptide bond hydrolysis. The proteasome subunits are responsible for protein degradation. All other superfamilies, multi-domains, non-specific hits, and specific hits are categorized as proteasome_beta_type_5 as subunits that assist in functions of the grander domain. All information on these domains was obtained from the NCBI BLAST site cited in the description below.



external image wkwg6inr60uHBZC5MSB6Hcx4HDDGybE6Xzo4zHiRzKZqci9Kf6qQdfXOKvU8F5ddGvGZTb2fqS3J-lS9c6d_jmP73k4TTFp_vO3eoeWY1-q_fsroD0jefwlfEdz4UmNDLh8Duus
(Figure 2: Protein Domain of IFI 30, illustrated as 1 GILT superfamily. Image obtained courtesy of http://blast.ncbi.nlm.nih.gov)
Methods
Whale Shark Protein Sequence
The PSMB8 protein sequence was found by searching for the sequence ID (ENSP00000406878) the Ensembl database and downloading the sequence from the top hit in the fasta format. From there the protein sequence was used as the query for a search of the NCBI BLAST database. The full protein sequence of the best match was then copied and used as the query for a BLAST against the predicted whale shark proteome on the galaxy server. The top predicted protein matches, determined by lowest E-value and highest alignment length, were then themselves used as queries for a BLAST against the NCBI human proteome database to check for reciprocity.
Orthologs
The same human PSMB8 sequence used previously was used at the query for BLASTs against various species’ NCBI and galaxy proteome databases to find orthologs to the human protein. The match with the lowest E-value of each BLAST was tabulated and itself used as a query for a BLAST against the NCBI human proteome database to determine reciprocity.
Phylogenetic Tree
The best matches obtained in the previous section were added to a clustalw multiple sequence alignment to create the phylogenetic tree as well as the four whale shark with the smallest E-values. Additionally, human PSMB5 was added to the tree as well as PSMB8 to further elucidate the divide in the tree. Default settings of the clustalw website were used.


Whale Shark Protein Sequence
The human protein sequence was used as the query of the predicted whale shark proteins database. The best four matches are tabulated in Table 1 below and ranked by descending E-value.
Sequence ID
E-Value
Alignment Length
Percentage of Positive Matches
Reciprocal Name
Reciprocal E-value
g21003.t1
1e-101
194
85.05
proteasome subunit beta type-5 isoform 1
1e-118
g28391.t1
6e-39
111
67.57
proteasome subunit beta type-8 isoform E1 proprotein
7e-34
g26495.t1
3e-36
68
91.18
proteasome subunit beta type-8 isoform E1 proprotein
4e-31
g36697.t1
4e-14
125
48.80
proteasome subunit beta type-7 proprotein
8e-68
Table 1. Predicted whale shark protein sequences that matched with human PSMB8. Percentage of positive matches refers to the percentage of amino acids that have similar function in sequence, and reciprocal name/E-value refers to the name/E-value of the best match (by smallest E-value) of the human protein returned by doing a BLAST of the NCBI human protein database with the whale shark predicted protein as the query. The top hit returned PSMB5 instead of PSMB8, indicating that it is a likely ortholog of human PSMB5. However, the next two hits did return human PSMB8 as their best matches, indicating that they are likely orthologs of human PSMB8, perhaps different isoforms.
Since the predicted protein g21003.t1 matched with human PSMB5 and not PSMB8 it cannot be considered a likely ortholog for human PSMB8, and in most likely instead an ortholog of human PSMB5. Both g28391.t1 and g26495.t1 returned human PSMB8 as their best matches and both have an extremely low E-value for their length. As a result, it seems that they are likely orthologs of the two isoforms of human PSMB8. The large increase in E-value in the PSMB5 ortholog could be a result of more information about and a larger portion of the PSMB5 protein in whale sharks being known whereas less is known about the PSMB8 orthologs, leading to a smaller portion of those proteins being included in the predicted whale shark proteome. Since subject length is a large factor in E-value, the PSMB8 orthologs with smaller subject lengths would have smaller E-values, everything else being equal.


Orthologs
The PSMB8 human protein was investigated to find orthologs in various other species and results are shown in Table 2. Every BLAST done either returned PSMB8 or PSMB5 as its reciprocal best match, and PSMB5 was only the best match when the species did not have a type-8 subunit listed at all in their database. This demonstrates the high degree of conservation of the proteasome subunit beta throughout eukaryotic life. Based on which species have direct PSMB8 orthologs, it seems that either PSMB8 evolved in a common ancestor of cartilaginous fishes and mammals and was lost in the evolution of elephant sharks, atlantic cod, and chicken from said ancestor, or PSMB8 evolved independently in mammals, the clawed frog, zebrafish and the whale shark.
Species
Name
ID
Length
E-Value
Reciprocity
Human
proteasome subunit beta type-8 isoform E2 proprotein
NP_683720.2
276
N/A
N/A
Mouse
proteasome subunit beta type-8 precursor
NP_034854.2
276
0.0
Yes
Chicken
proteasome subunit beta type-5
NP_001264655.1
254
2e-94
No
Clawed Frog
proteasome subunit beta type-8
NP_001103512.1
271
6e-135
Yes
Zebrafish
proteasome subunit beta type-8
NP_571467.3
271
2e-130
Yes
Atlantic Cod
N/A
gadMor1_genscan_HE568814.49_1
1061
4e-83
No
Elephant Shark
N/A
calMil1_genscan_KI636893.1_1
175
7e-68
No
Fruit Fly
proteasome beta5 subunit, isoform A
NP_652014.1
282
1e-92
No
Yeast
proteasome core particle subunit beta 5
NP_015428.1
287
4e-93
No
Rice
Os06g0153800
NP_001056842.1
277
3e-83
No
Table 2. The best match (by smallest E-value, query coverage, and percent identity) for each species when a query of human PSMB8 is BLASTed against its NCBI or galaxy database. Length refers to the amino acid length of the protein and reciprocity refers to whether or not an NCBI BLAST of the human proteome with the protein listed as the query returns either isoform of proteasome subunit beta type 8 as the best match.

Phylogeny and Phylogenetic Tree
The phylogenetic tree shown in figure 3 contains the protein sequences of the best matches in table 2 as well as the four best whale shark sequences in table 1 as well as human PSMB5 for comparison.The tree is mostly split into two large groups. The top group is that of the PSMB8 orthologs whereas the bottom group is that of the PSMB5 orthologs. The only result not in either group g36697.t1 is the least related of the whale shark predicted proteins and is likely an ortholog to a different proteasome subunit. The tree shows g21003.t1 to be more closely related to human PSMB5 than either are to any other protein, indicating it is an ortholog of human PSMB5 and not humanPSMB8. However, both g28391.t1 and g26495.t1 seem to be as related to human PSMB8 as the ortholog proteins in the clawed frog and zebrafish. This further demonstrates the likelihood of g28391.t1 and g26495.t1 to be different isoforms of the whale shark PSMB8.
external image FHzEdzSq8l_BfeWaErCuI06FnUdv_8EIJfSEfHsQinYQUfiMfrJNivRdbxGqbGpweai8RpJRSPqE2IJ5plgDus8vggF9Xauru8jD8cQsh9kmP15SFjDSQPyh8cruvLL5IQQ9xZE
Figure 3. The phylogenetic tree of PSMB8 best matches from Table 2 and the four best predicted whale shark proteins from Table 1 were aggregated using Clustalw where the point of divergence indicated the most recent common ancestor and length of the branches represents evolutionary time. Two large groups appear, one of PSMB8 orthologs one of PSMB5 orthologs of species without PSMB8. Human PSMB5 is added to this phylogenetic tree to demonstrate that the second group clusters around it, indicating PSMB5 orthologs.



Conclusion


The data obtained in the orthologs section of this page, and the relatively low E-values obtained in regards to genome similarity indicate that these genes are most likely orthologous. Additionally, the alignment of both g28391.t1 and g26495.t1 are from 180 to 269 and 247 respectively, indicating that both align within the proteasome beta type-5 domain. The phylogenetic tree shows common ancestry, and the data with E-values between closely related, and distantly related species indicate that the species have a related ortholog in this protein sequence. The more specific comparison is that of humans and Whale Sharks, which have promising data for the conclusion that these protein sequences are orthologous.



References


Arima, Kazuhiko. Proteasome Assembly Defect Due to a Proteasome Subunit Beta Type 8 (PSMB8) Mutation Causes the Autoinflammatory Disorder, Nakajo-Nishimura Syndrome. Digital image. PNAS, 2015. Web.

"Basic Local Alignment Search Tool." BLAST:. N.p., n.d. Web. 15 Apr. 2015.

Kitamura, Akiko. "JCI - A Mutation in the Immunoproteasome Subunit PSMB8 Causes Autoinflammation and Lipodystrophy in Humans." JCI - A Mutation in the Immunoproteasome Subunit PSMB8 Causes Autoinflammation and Lipodystrophy in Humans. Journal of Clinical Investigation, 27 July 2011. Web. 15 Apr. 2015.

"Proteasome Subunit Beta Type-8." PSMB8. Uniprot, 16 May 2014. Web. 15 Apr. 2015.

"PSMB8" GeneCards. Weizmann Institute of Science, 30 Mar. 2015. Web. 10 Apr. 2015.

"PSMB8 Gene." Genetics Home Reference. Genetics Home Reference, 13 Apr. 2015. Web. 15 Apr. 2015.