NLRC5


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 an orthologous protein.

Background:
NLRC5, short for NOD-like receptor, is “an intracellular protein which plays an important part in homeostatic control of innate immunity and antiviral defense mechanisms”(Hartz 2010).In humans it is coded for by the NLRC5 gene and is identified as a negative regulator. NLRC5 (alternatively named NOD27 or CLR16.1) is an interesting exception in the NLR family. It has a typical NLR architecture but contains an effector domain. It is “a key regulator of MHC class I-dependent immune responses, this is important because its expression plays a role in the initiation of adaptive immune responses” (Kobayashi 2012).

.
external image q6OKiVd9zi1X5F2zQz6kYHdaF-YApDqppeJdbwTJ64A-WwBjHTVt_8XO4PD-ObNDzISI1ZqosGAF9dxWN3S0m0k7CSwv98LLgSvjN6QONdnHTWzkzTowseGlOcQ6JtVr4O3pNo8

Figure 1: In this image we see how NLRC5 is capable of inhibiting the RIG-I/MDA5 function and the IKK complex. By interacting with IKKα and IKKβ and blocking their phosphorylation NLRC5 inhibits NF-κB-dependent responses. We can see that NLRC5-specific siRNA knockdown has enhanced the activation of NF-κB and the and the type 1 interferon signaling (Cui, 2010).

Methods:
Finding Whale Shark Orthologs
The human protein sequence of NLRC5 (ENSP0000022510) was used in a BLAST search against the predicted whale shark protein genome to find orthologs. The whole NLRC5 sequence in FASTA form was produced from the Ensembl search. This database was used as a query in a BLAST search against the predicted whale shark protein database using the Galaxy server (whalesharkgeorgiaaquariumm.org). The top best five predicted protein hits based (determined by low E-value and query length (Table 1)) were then used as querries in protein BLASTs against the NCBI human protein database.
Results:
Whale Shark ID
E-value
Alignment length
Predicted protein length
% Identity
g46108.t1
7e-54
510
642
31.76%
g46616.t1
4e-29
570
693
24.74%
g44600.t1
2e-32
495
583
27.27%
g36672.t1
6e31
489
581
26.18%
g31399.t1
7e-27
339
468
28.02%
Table 1: Table of top five best 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 NLRC5 sequence as query returned NLRC5 as the best hit against the human protein database, we were not confident that any of these five were NLRC5 orthologs.


Other Orthologs:
BLAST searches were conducted to find different species in order to find orthologs between the Mouse, Yeast, zebrafish, fruit fly, clawed frog, arabidopsis, and rabbit against human protein database NLRC5 (ENSP0000022510). These BLAST searches were conducted using single species protein databases. The best hits are shown below (see Table 2 below).


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, as shown in Table 1, of Mouse, Yeast, Zebrafish, fruit fly, clawed frog, arabidopsis, and rabbit were used to construct a phylogenetic tree diagram using the ClustalW2 program. (see Figure 3 below).


Protein Domains:
The whale shark gene NLRC5 aligned with the homosapien protein family LRR_RI superfamily with an e value of 4.13e-12. Leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily. LRRs are 20-29 residue sequence motifs present in many proteins that participate in interactions, which are protein-protein interactions, these have varying functions a locations within the cell. LRRs correspond to structural units consisting of a beta strand and an alpha helix. This alignment contains 12 strands corresponding to 11 whole repeats, consistent with the extent observed in the subfamily acting as Ran GTPase Activating Proteins (RanGAP1).
Results:

external image 3LF1yWhshhSiLPU6G9oy-y0UErFrvfoVZuy9pE7G1hvaW3gfhnWgwIaXpskZqvanAL6ilBzC17pqQbxczFUVtJ5SQKfDRTD6YJfoBEzQyMIguXCYnsdn5EIs3n1h_BLa5gfFqco
external image JzeeDi9Nm3iloii6tCZomyN-EZ3-92w1HGOqQLyHTRV_u4amN1pwz0lFKquk_0lj4CTwaIPMzMpiu7DApTL1avrqjF25r3O2t5drkSqk39p-5gRf5VYzM9-78eY7RmzBBQFS6s8

Figure 2: General domains of whale shark NLRC5 best hit predicted proteins. All of the four best-hit whale shark predicted proteins contain the general LRR_RI and homeo domains as predicted by NCBI BLAST server analyses.


Orthologs:
The human NLRC5 protein sequence (ENSP0000022510) was used as query in NCBI BLAST searches against individual species’ protein databases. The protein NLRC5 sequence found in humans found near identical matches with orthologs in mice and rabbits, as shown in Table 2.
Results:
Species
Accession
Alignment Length
E- value
Percent Identity
Homo Sapiens
NP_115582.4
3749
0.0
100%
Mouse
XP_006531227.1
2194
0.0
63%
Zebrafish
XP_003200494.2
941
2e-130
27%
Fruit Fly
NP_001260579.1
469
1e-09
26%
Yeast
NP_010922.1
114
3e-04
30%
Rabbit
XP_008255544.1
2446
0.0
70%
Arabidopsis
NP_563871.1
366
4e-23
28%
Clawed frog
XP_004914291.1
359
7e-107
34%
Table 2. Best hits with human NLRC5 protein BLAST. The human NLRC5 sequence was compared against sequences in individual species using protein BLAST searches. The ID (Acession), alignment length, E-value, and percent identity from each best hit are given.


Phylogeny:
The best hits of individual species acquired through protein database searches using the NLRC5 protein were used to create a phylogenetic tree. From this tree, it is seen that the NLRC5 protein deviated from the evolutionary tree from its mammalian cousins. Interestingly, the yeast homologue showed to be the most deviated from the human NLRC5 protein.



external image Vp1OjNiEtk-2ZYr8DQ4IvF5XWwAVQdNazsgU7HZtVozS5y-a5rjPTCN51rV0A4bPCLr4ezFEpjfCrLinRsyvYEuPTn1syq3Gleie-S1s8TjtDS_8looAd4AFCDVDrVuimy7TbmM


Whaleshark:0.43301
Human:0.15930
Rabbit:0.16171
Mouse:0.19449
Clawed:0.33672,
Zebrafish:0.37510
Yeast:0.45223
Fruit:0.42436
Arabidopsis:0.42236

Figure 2: Phylogenetic tree of the best return hits for NLRC5. The best hits from BLAST searches of protein databases of individual species were used in the ClustalW2 program to create a phylogenetic tree. Branch lengths represent relative evolutionary time.


Conclusion:
The ortholog deviated from the evolutionary timeline before non-mammalian species. My results do not provide enough information to show whether or not the whale shark is a homolog. As shown in table 1, we are provided with a low e value of 7e-54, but a low very low percent identity of 31.76%. The NLRC5 protein is also found in Rabbits in spite of the large evolutionary distance between the Rabbit and human NLRC5 proteins. This finding could be explained due to similar evolutionary patterns after deviating from the phylogenetic timeline. As seen in table 2 and the phylogeny tree, mice also contain the protein. This is reflected in current clinical practices because most drugs and devices are tested on mouse models before going into clinical trials. In the future, proteins similar in function and structure to the NLRC5, should be sought after in the whale shark in order to learn more about its immune system.



References:
Lamkanfi, M., & Kanneganti, T. (n.d.). Regulation of immune pathways by the NOD-like receptor NLRC5. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225722/


Cui, J. (n.d.). NLRC5 Negatively Regulates the NF-κB and Type I Interferon Signaling Pathways. Retrieved from http%3A%2F%2F__www.cell.com%2Fabstract%2FS0092-8674(10)00359-4__


Neerincx, A., Castro, W., Guarda, G., & Kufer, T. A. (n.d.). NLRC5, at the Heart of Antigen Presentation. Retrieved from __http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837245/__


Du, L. (2010). The role of NLRC5 in monocytes upon bacterial infections.


Li, A. (n.d.). Download PDFs. Retrieved from __http://www.sciencedirect.com/science/article/pii/S128645791100311X__


Kobayashi, K. (2012, December). Retrieved from __http://www.nature.com/nri/journal/v12/n12/full/nri3339.html__


Biswas, A., Meissner, T. B., Kawai, T., Kobayashi, K. S. Cutting edge: impaired MHC class I expression in mice deficient for Nlrc5/class I transactivator. J Immun. 189: 516-520, 2012. [PubMed: 22711889, related citations] [Full Text: HighWire Press]


Hartz, P. A. Personal Communication. Baltimore, Md. 8/20/2010.