Pai Liu
Biology 142 Lab
Wiki-page
April. 5th 2015
Project Introduction
This project is originally an assignment in Emory University’s Biology 142 lab course. Our group
was assigned the protein IRAK1-ENSP00000358997 and to examine if the newly sequenced
whale shark genome had evidence of an orthologous protein.
Background information
IRAK1-ENSP00000358997, named interleukin-1 receptor-associated kinase 1, had 732 amino
acids in the sequence. It plays an important role in initiating innate immune response against
foreign pathogens. Also, it plays a role in toll-like receptor (TLR) and IL-1R signaling pathways.
IRAK1 is able to work with MYD88 so IRAK1 could be able to be phosphorylated by IRAK4,
and thus, the subsequent auto-phosphorylation and kinase activation can occur. Also, IRAK1
phosphorylates E3 ubiquitin ligases Pellino proteins (PELI1, PELI2, and PELI3). The
polyubiquitinated IRAK1 can bind with the ubiquitin-binding domain of IRBKG/NEMO and
become the IRAK1-MAP3K7/TAK1-TRAF6 complex and the NEMO-IKKA-IKKB complex.
The MAP3K7/TAK1 complex can activate IKKS, which later causes NP-kappa-B nuclear
translocation and activation. IRAK1 phosphorylates TIRAP to promote its ubiquitination,
subsequent degradation, and interferon regulatory factor 7 (IRF7) to induce its activation and
translocation to the nucleus, producing transcriptional activation of type I IFN genes and making
the cell into an antiviral state.
Here, we are able to identify a strong potential IRAK1 ortholog from the newly sequenced whale
shark genome.
Methods
Whale shark predicted orthologs
The human protein sequence (ENS0000047512) was used as query in the Blast against the
predicted whale shark protein database by using the website whaleshark.georgiaaquarium.org
Galaxy server. Top predicted protein hit were then used as querries (using the full predicted
sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein
database.
Predicted Orthologs
IRAK1 predicted Orthologs were identified in species other than whale sharks by using the NCBI
Blast Server. Protein BLASTs were performed using single species protein database for mouse,
zebra fish, clawed fogs, fruit flies, yeast, and pig databases. The human IRAK1 protein
(ENS0000047512) was used as query sequencein these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each search using human protein as query and the top 4 whale
shark BLAST hits were used to create a sequence alignment and phylogenetic tree. ClustalW2
with default settings was used to create the alignment and tree.
Searching for IRAK4 in the whale shark
The human IRAK4 protein sequence was the query sequence that in the BLAST against the
whale shark predicted protein database and results are shown in Table 1. Four hits with E-values
below 9e-30 with the second smallest E-value being 4e-11. There four E-values will later be
blasted using NCBI BLASTp.
ID
g14811.t1
g13678.t1
Alignment Length
70
129
Top hit E-value
9e-30
4e-11
Identity
68.57%
29.46%
Pai Liu
Biology 142 Lab
Wiki-page
April. 5th 2015
g35543.t1
98
1e-08
28.57%
g33456.t1
30
2e-07
53.33%
Table 1: Human IRAK1 best 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
IRAK1 protein sequence. The top 4 hits according to E-value are reported here, and their
database ID, alignment length, and identity are also recorded. These sequence were used as
querries against the NCBI human protein database. The top E-value are reported here according
to the lowest E-value.
All the predicted proteins from the Blast of the whale shark proteins using the human IRAK4
sequence as query returned IRAK1 as the best hit against the human protein database. The Evalues were all lower than 1e-06.
Protein domains
Protein IRAK1 in the whale shark (from BLAST results) contain both a Death_IRAK1 and a
STKc_IRAK1. The Death Domain (DD) superfamily is consisted of the DD, Pyrin, CARD
(Caspase activation and recruitment domain) and DED (Death Effector Domain) families. The
STKc_IRAK1 contains a PKc_like superfamily. The PK superfamily contains the large family of
typical PKs that includes serine/threonine kinases (STKs), protein tyrosine kinases (PTKs), and
dual-specificity PKs.
Figure 1: Putative domains of whale shark IRAK1 best hit predicted proteins. All of the four besthit whale shark predicted proteins contain putative Death-IRAK as predicted by NCBI BLAST
server analyses.
Orthologs
The human IRAK1 protein sequence (ENS0000047512) as used as query in NCBI BLAST
searches against individual species’ protein database. Death Domain Superfamily is found using
this method in mice, zebra fish, and etc.
Species
Name
ID
Length
E-value
Homo sapiens
Death-IRAK1
NP_0015602
712
N/A
Mouse
Death-IRAK1
NP_001171446.1 711
N/A
Zebra fish
Death-IRAK1
XP_005166760.1 686
2E-137
Clawed dog
Death-IRAK1
NP_001006713.1 721
5E-134
fruitfly
Pelle, isoform A
NP_476971.1
501
1E-42
Yeast
Saccharomyces
NP_012440.1
1478
2E-17
cerevisiae S288C
Arabidopisis
Protein kinase 2A NP_172889.1
426
3E-48
Elephant Shark
Callorhinchus
Xp_007888675.1 287
1E-101
milli
Pai Liu
Biology 142 Lab
Wiki-page
April. 5th 2015
Table 2: Best hits with human IRAK1 protein BLAST. The human IRAK1 sequence was used in
protein BLASTs against individual species. Name, ID, length and E-Value of the best hit from
each search are reported here.
Phylogeny
The best hits from protein database searches using the human IRAK1 protein as query were used
to create a phylogenetic tree.
(More-detailed Figure 2 needed)
Figure 2:
Conclusions
We are able to identify the predicted IRAK1 ortholog in whale sharks. More information will be
added after all the species were examined and blasted. The predicted IRAK1 ortholog in
the elephant shark does suggest that an IRAK1 ortholog may exist in the whale
shark. (more information will be added)
References
Jensen, L. E., and Alexander S. Whitehead. "IRAK1b, a Novel Alternative Splice Variant of
Interleukin-1 Receptor-associated Kinase (IRAK), Mediates Interleukin-1 Signaling and Has
Prolonged Stability." Journal of Biological Chemistry (2001): 29037-9044. Print.
"National Center for Biotechnology Information." National Center for Biotechnology Information. U.S.
National Library of Medicine. Web. 5 Apr. 2015. <http://www.ncbi.nlm.nih.gov/>.
"Basic Local Alignment Search Tool." BLAST:. Web. 5 Apr. 2015.
<http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome>.
"Galaxy / Whale Shark." Galaxy / Whale Shark. Web. 5 Apr. 2015.
<http://whaleshark.georgiaaquarium.org/>.