Background Information:

IRAK-4 (interleukin-1 receptor-associated kinase) is a protein kinase used in signaling innate immune responses.
Toll-like receptors (TLRs), are receptors used to recognize pathogenic molecules and are found in the cytoplasmic membrane (Figure 1). TLRs have a pattern recognition receptor that is used to differentiate between the pathogen and the host molecules. TLRs and molecules like IRAK-4 are used collectively to initiate immune response. These molecules are found in the cytoplasm. IRAK4 is involved in a signal transduction pathway that triggers a phosphorylation cascade, relaying the signal from protein to protein until the response is eventually registered in the cell nucleus (Li, 2002).

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Figure 1. Cell signaling pathway.

The initiation of immune response is essential in defense against pathogens. Figure 2 shows this pathway. TLRs recognize the pathogenic molecules.

When TLRs are activated (when a microbe is detected), they “recruit adapter molecules within the cytoplasm of cells in order to propagate a signal."(Picard, 2010) These adapters then activate protein kinases within the cell, like IRAK4. In response, IRAK4 proteins initiate or suppress genes that cause inflammatory responses. IRAK4 functions in nonspecific immunity or "innate immunity" (NCBI, 2015). This means that IRAK4 functions in response to general foreign pathogens rather than specific bacteria or viral pathogens.


IRAK process.png


Figure 2. Process of initiating adaptive immune responses.


Two isoforms of this protein are known to exist through alternative RNA-splicing: one of which contains 124 codons less than the "canonical" RNA transcript. This second isoform is believed to be a source of IRAK4 deficiency (Uniprot, 2015). Studies show that those with IRAK-4 and MyD88 (adapter molecule) deficiencies are more susceptible to bacterial infection (Picard, 2010). Patients with homozygous mutations in IRAK-4 or MYD88 gene have this deficiency. Heterozygous carriers do not show any symptoms. In a study published by The Journal of Experimental Medicine, an individual exhibiting a heightened frequency of developing bacterial infections displayed a lesser response to lipopolysaccharide, a receptor molecule on the membrane of many gram-negative bacteria, and interleukin-1, a protein involved in signalling IRAK4 in the signal transduction pathway, exposure. If the individual had a functioning IRAK4 protein, then the individual would exhibit a direct response from interleukin-1. With a deficiency in IRAK4, the individual could not register a bacterial cell surface signal. By representing a case where the IRAK4 protein is compromised, this experiment demonstrates the importance of IRAK4 to register certain bacteria, as well as its importance in the phosphorylation cascade (Medvedev, 2003).
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Figure 3. Clinical manifestation of IRAK-4 and MyD88 deficiencies

Figure 3 shows the increased susceptibility to bacterial infections caused by IRAK-4 and MyD88 deficiencies. 60% of meningitis patients were IRAK or MyD88 deficient. This demonstrates the role IRAK in regulating the immune system.

Methods:
Whale Shark Predicted Orthologs
The FASTA format of the human protein sequence (ENSP00000390651) was obtained from Ensembl.org. This FASTA format sequence was blasted against the whale share predicted genes using whaleshark.georgiaaquarium.org. The top five hits of IRAK protein were obtained, based on E-values and percent identity.

Predicted Orthologs
The IRAK4 protein was identified in organisms other than whale shark using the NCBI Blast website. The FASTA format was used to blast against cow, mouse, elephant shark, zebra fish, fruit fly, and clawed fog.

Phylogenetic Tree
FASTA formats for the top five hits were obtained using the Galaxy website. The FASTA format for IRAK protein was blasted against other organisms. The best hit proteins for all the organisms were obtained and used to make the phylogenetic tree. The phylogenetic tree was made via clustalw.

Protein Domain
IRAK-4 is in the Protein Kinase C family. This family consists of protein kinases involved in regulating the function of other proteins by phosphorylation. Being a part of the DD superfamily (Death domain), IRAK4 can therefore bind to other proteins containing the same domain within their amino acid sequence. This is important for the formation of the Myddosome (shown in figure 1) because IRAK4 must bind with IRAK1 and MyD88.

Protien Domain.png


Finding the IRAK4 ortholog in Whale sharks
The human sequence was blasted against the whale shark predicted gene to determine matches. Table 1 shows the best five hits in the blast between human and whale shark. These hits were blasted against the human database using NCBI Blast. The resulting e-values were not low enough to be significantly conclusive. Therefore, we are unable to confidently identify any homologies. However, when we tried the same procedure using an elephant shark, we found that Interleukin-1 receptor-associated kinase 4 is, in fact, present in the human gene. Additionally, its e-value score was 8e-70, indicating an extremely significant match.

Table 1. Top five hits in blast between human and whale shark.
Whale shark ID
E-value
Alignment Length
Predicted Protein Length
% Identity
g14910.t1
2e-04
30
118
56.67
g16132.t1
3e-05
31
88
54.84
g20572.t1
2e-04
49
156
42.86
g36266.t1
3e-06
49
86
53.06
g46616.t1
2e-04
54
693
48.15

Orthologs
The human protein (IRAK-4) was found in all organisms expect fruit flies. Mouse and cow (highlighted) sequences had the lowest e value and were thus the best matches for the protein. This value shows that mice (2e-120) and cows (2e-125) have high homology with humans. Elephant shark and zebra fish also yielded low e-values. This indicates that they are orthologs of humans, though not as closely related as the mouse and cow. However, the clawed frog and fruit fly queries yielded insignificant e-values. Although clawed frog had the IRAK4 protein, the result was inconclusive because of its low e-value. Since fruit fly did not have the IRAK4 protein or a good e-value, we concluded that it is not an ortholog of human.

Table 2. Top matches with human IRAK4 protein BLAST – the human IRAK4 sequence was used as query against six species. The name, ID, length, and E-value of the top match from each species are reported here.
Species
Name
ID
Length
E-value
Mouse
interleukin-1 receptor-associated kinase 4
EDL04265.1
239
2e-120
Elephant Shark
interleukin-1 receptor-associated kinase 4
XP_007903160.1
771
8e-70
Zebra Fish
interleukin-1 receptor-associated kinase 4
NP_956457.1
382
8e-53
Clawed Frog
interleukin-1 receptor-associated kinase 4
NP_001079489.1
672
8e-04
Fruit fly
GH16651 [Drosophila grimshawi]
XP_001994379.1
489
7e-04
Cow
interleukin-1 receptor-associated kinase 4
NP_001069466.1
461
2e-125

Phylogenetic Tree
A phylogenetic tree was created to show homologous genes and relationships among the organisms we selected. Figure 4 shows the phylogenetic tree for the whale shark best hits and all the the orthologs.

Results
The result indicates that cows and humans share a common ancestor. However, neither organism shares a close ancestor with the whale shark predicted proteins. The clawed frog appears to be the whale shark's closest ancestor. This explains the human protein was not found in either the whale shark or the clawed frog. It is important to note that the elephant shark has a common ancestor with the cow, human and mouse. The phylogenetic tree supports the results from our ortholog table, which indicates orthologs in the mouse, cow and human. Additionally, fruit flies and clawed frogs have a distant ancestry with humans, which is also supported by the table.

Tree 2.png
Figure 4. Phylogenetic tree of whale shark genes and orthologs.

Conclusion:

This study attempted to locate the IRAK-4 protein in the whale shark genome. IRAK-4 is a kinase protein used in initiating immune responses. The five prospective genes from the whale shark are shown to be farther related to the mammal than fruit fly and clawed frog. Also, the high e-values and mediocre percent identities demonstrate poor matches to the IRAK-4 sequence. This implies that the closest matches found in a whale shark weren’t in fact homologs or orthologs to the human IRAK-4 protein. As a result, the elephant shark was used to blast against human. The result showed that IRAK-4 was present in elephant shark. Since the human IRAK-4 protein has an ortholog in the elephant shark and not the whale shark, it can be concluded that an ancestor IRAK-4 protein was present in a common ancestor of elephant sharks and homo sapiens. However, any common ancestor of elephant sharks and homo sapiens must also be an ancestor of whale sharks as well, since whale sharks and elephant sharks are closer related to each other than with humans. Therefore, it is concluded that whale sharks must have evolved in a way that eradicated use for that particular sequence, or adapted the sequence so greatly that it is now unrecognizable when compared to the human IRAK-4 sequence.


Reference:

  1. "Genes and Mapped Phenotypes." <i>National Center for Biotechnology Information</i>. U.S. National Library of Medicine, 22 Mar. 2015. Web. 1 Apr. 2015. &lt;http://www.ncbi.nlm.nih.gov/gene/51135>.
  2. "Interleukin-1 Receptor-associated Kinase 4." <i>IRAK4</i>. UniPort Consortium. Web. 1 Apr. 2015. &lt;http://www.uniprot.org/uniprot/Q9NWZ3>.
  3. Li, Shyun, Astrid Strelow, Elizabeth Fontana, and Holger Wesche. "IRAK-4: A Novel Member of the IRAK Family with the Properties of an IRAK-kinase." <i>Proceedings of the National Academy of Sciences</i> 99.8 (2002): 5567-572. <i>JSTOR</i>. Web. 1 Apr. 2015.
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