NOD1


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 a orthologous protein.


Background:
NOD1, nucleotide binding oligomerization domain containing 1, is a cytosolic protein that “contains an N-terminal caspase recruitment domain (CARD), a centrally located nucleotide-binding domain (NBD), and 10 tandem leucine-rich repeats (LRRs) in its C terminus. NOD1 is an innate immune receptor and intracellular pattern-recognition receptor (PRR) that initiates inflammation in response to a subset of bacteria through the detection of bacterial diaminopimelic acid (Irving et.al) . This protein “detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells (Irving et.al). So far studies have shown that NOD1 detects PG [peptidoglycan] within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection (Irving et.al) . Researchers are currently trying to develop small molecules capable of modulating NOD1 and NOD2 signaling in order to influence pathogen clearance and inflammation (Caruso et.al). NOD1 and NOD2 play a role in the clearance of invading pathogens and act redundantly and cooperatively with TLRs in the detection of bacteria and production of proinflammatory molecules (Caruso et.al) .
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Figure 1: NOD1, as an intracellular innate immune receptor detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells. Studies by Irving et.al 2014 have found that NOD1 detects PG within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection. (Irving et.al 2014).

Methods
Finding Whale Shark Orthologs:
The human protein sequence of NOD1 (ENSP00000222823) was used in a Blast search against the predicted whale shark protein genome to find the orthologs. First, the sequence for NOD1 was obtained in FASTA format. This was done through the Ensembl search.This database was then used as a query in a Blast against the predicted whale shark protein database. This was done using the Galaxy server which was accessed through www.whalesharkgeorgiaaquarium.org. The protein hits were all evaluated and the best five predicted hits, based on E- value and query length ( Table 1) were used as queries in protein Blasts against the NCBI human protein database.
Results:
Whale Shark ID
E value
Alignment length
Predicted protein length
% Identity
g22867.t1
7e-19
214
304
26.17 %
g36672.t1
5e-28
345
690
26.09 %
g46384.t1
4e-36
386
658
27.24 %
g37533.t1
2e-31
540
571
24.81 %
g31399.t1
3e-22
352
475
25.57 %
Table 1: Table of the best five predicted protein hits based on lowest E-value and longest query length produced by the Whale Shark genome.


Since none of the predicted proteins from the Blast of the whale shark proteins using the human NOD1 sequence as query returned NOD1 as the best hit against the human protein database, we are not confident in stating that any of these were homologues. We then repeated this process using the Rabbit predicted NOD1 protein as query against the whale shark predicted protein database to see if a more closely related species would return different best hits. There were several E-value hits, however the lowest was 0.0 and had one of the highest percent identity matches we had seen within any of our database hits with an identity of 82% and the mouse showed a homolog of 81%.



Other Orthologs:
NCBI BLASTs were used to conduct search for different species in order to search for orthologs between the Mouse, Yeast, Fruit Fly and Zebrafish against human protein database NOD1 (ENSP00000222823). These BLASTS were performed using single species protein databases. The best hits are shown below (Table 2).


Constructing a Phylogenetic tree:
The best hits with the lowest E-values of the whale shark and human protein BLAST and the NCBI best BLASR hits of Mouse, Yeast, and Zebrafish were used to construct a phylogenetic tree diagram using the ClustalW2 program (see Figure 3 below).


Protein Domains :
The whale shark gene NOD1 aligned with the homosapien protein family LRR_RI with an e-value of 5.6E-25. LRR_RI is short for Leucine-Rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily. The closest, specific hit was NACHT with an e-value of 1.86e-48. This NTPase domain is found in apoptosis proteins as well as those involved in MHC transcription activation and is demonstrated by Figure 2 below. This family is closely related to pfam00931.
Results:
BioLab.png
BioLab.png

Figure 2:General domains of whale shark NOD1 best hit predicted proteins. All of the four best-hit whale shark predicted proteins contain putative LRR_RI superfamily and homeo domains as predicted by NCBI BLAST server analyses.


Ortholog's Results:

The human NOD1 protein sequence [ENSP00000222823] was used as query in NCBI BLAST searches against individual species’ protein databases. The NOD1 protein sequence found in humans found near identical matches with orthologs in mice and rabbit, and along with the match found in fruit flies all belong in the LRR_RI protein superfamily. No such matches were to be found in yeast, however.
Species
ID
Alignment Length
E-value
Percent Identity
Homo sapiens
__NP_006083.1__
953
0.0
100%
Mouse
NP_766317.1
953
0.0
81%
Zebrafish
__XP_002665106.3__
940
0.0
51%
Fruit Fly
NP_001163077.1
1296
6e-06
23%
Yeast
__NP_012495.1__
1683
0.62
25%
Arabidopsis
__NP_563871.1__
605
2e24
34%
Clawed Frog
__XP_002937900.2__
945
0.0
53%
Rabbit
__XP_002713781.1__
953
0.0
82%
Table 2: Best hits with human NOD1 protein BLAST. The human NOD1 sequence was compared against sequences in individual species using protein BLASTs. ID, alignment length, E-value, and percent identity.


Phylogeny: ( Using the FASTA sequences) found while blasting the best hits of individual species. This was done through protein database searches using the NOD1 protein.
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WhaleShark:0.42978
Yeast:0.44022
Human:0.08833
Rabbit:0.09426
Mouse:0.10290
ClawedFrog:0.24435
ZebraFish:0.2690
Arabidopsis:0.42027
FruitFly:0.4469

Figure 3: Based on table one, and the data seen in the Phylogenetic Tree above it can be seen that humans and whale sharks share no homologs. Table one depicts that there were no high percent identities.



Conclusions:
Based on data from table 1 and table 2 the phylogenetic tree (Figure 3) depicts the homologs between different species. Whale sharks do not have homologs with humans for this protein. The closest species that have homologs are rabbits and mouse. This is reflected in current clinical practices because most drugs and devices are tested on mouse models before going into clinical trials. Unfortunately as the data from table 1, table 2 and figure 3 shows, for the NOD1 protein there are no homologs between humans and Whale Sharks. Since NOD1 doesn’t have a homolog between humans and whale sharks it was concluded that the innate immune receptors and intracellular pattern-recognition receptor (PRR) that initiates inflammation in response to a subset of bacteria through the detection of bacterial diaminopimelic acid in whale sharks will not be applicable for humans.This data means that since NOD1 is used as an innate immune receptor, future research can take a deeper look into the role that this protein plays in humans and similar proteins that mirror the NOD1 protein. Once researchers find similar proteins to NOD1 in terms of structure and function in the immune system, researchers can see if those similar proteins are homologs with the whaleshark. This will then help researchers better understand the innate mechanisms of the whale shark immune system.





References:

NOD1 nucleotide-binding oligomerization domain containing 1 [ Homo sapiens (human) ]. (2015, March 15). Retrieved March 29, 2015, from http://www.ncbi.nlm.nih.gov/gene/10392


Irving AT, Mimuro H, Kufer TA, Lo C, Wheeler R, Turner LJ, Thomas BJ, Malosse C, Gantier MP, Casillas LN, Votta BJ, Bertin J, Boneca IG, Sasakawa C, Philpott DJ, Ferrero RL, Kaparakis-Liaskos M. The immune receptor NOD1 and kinase RIP2 interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling. Cell Host Microbe. 2014 May 14;15(5):623-35. doi: 10.1016/j.chom.2014.04.001. Epub 2014 Apr 17. PubMed PMID: 24746552. http://www.ncbi.nlm.nih.gov/pubmed/24746552?report=abstract


Caruso, R., Warner, N., Inohara, N., & Nunez, G. (2014). NOD1 and NOD2: Signaling, Host Defense, and Inflammatory Disease. Immunity, 41(6), 898-908. Retrieved March 30, 2015, from Web of Science.


"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 30 Mar. 2015.


Strober, Warren, Peter J. Murray, Atsushi Kitani, and Tomohiro Watanabe. "Signalling Pathways and Molecular Interactions of NOD1 and NOD2."Nature Reviews Immunology 6.1 (2005): 9-20. Web.


Inohara, N., Koseki, T., del Peso, L., Hu, Y., Yee, C., Chen, S., Carrio, R., Merino, J., Liu, D., Ni, J., Nunez, G. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappa-B. J. Biol. Chem. 274: 14560-14567, 1999.


Strober, W., Murray, P., Kitani, A., & Watanabe, T. (2006). Signalling pathways and molecular interactions of NOD1 and NOD2. Retrieved April 14, 2015.


Activation of NOD1 by DAP Contributes to Myocardial Ischemia/reperfusion Injury via Multiple Signaling Pathways, 20(4), 512-522. (2015). Retrieved April 14, 2015, from Web of Science.