ACE1


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:
Angiotensin I is a converting enzyme (peptidyl-dipeptidase A) that converts angiotensin I to angiotensin II by release of the terminal His-Leu. ACE1 results in an increase of the vasoconstrictor activity of angiotensin and inactivates bradykinin, a potent vasodilator. ACE1 is also important "for glycosidase activity and releases GPI-anchored proteins from the membrane by cleaving the mannose linkage in the GPI moiety" (STRING interaction network). As seen in figure 1, ACE is also critical "in the regulation of blood pressure by converting the decapeptide angiotensin I into the potent vasoconstriction octapeptide angiotensin II" (Vermeirssen). This protein is likely to be found in the extracellular space and region of the vesicular exosome. As demonstrated by Figure 1, this protein is associated with hypertension, heart disease and sarcoidosis (The Human Protein Atlas). This enzyme has also shown "to be correlated with neurologic diseases such as Alzheimers" (Farrer). ACE1 also has "membranous expression in selected glandular cells, including small intestine, renal tubules, glandular cells in uterus and male genitalia" (ACE Localizations).


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Figure 1: The RAA pathway ensures that it will correct for all three of these triggers. It makes this a very important reflex because it controls your blood pressure and your two most important minerals in your body: sodium and potassium. When Renin is secreted, it stimulates a protein in your blood stream called angiotensinogen. The liver makes most of these plasma proteins (such as albumin). This angiotensinogen is always circulating in the blood stream. Renin activates it and turns it into angiotensin-1 (The Renin Angiotensin Aldosterone Reflex).


Methods
Finding Whale Shark Orthologs
The human protein sequence of ACE1 (ENSP00000290866) was used in a BLAST search against the predicted whale shark protein genome to find orthologs. The whole ACE1 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
g36554.t1
1e-88
162
163
74.69
g36554.t1
7e-80
158
163
67.72
g40202.t1
4e-56
114
114
71.05
g40202.t1
2e-55
114
114
68.42
g30771.t1
2e-19
85
85
43.53
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 the predicted protein g36554.t1 from the Blast of the whale shark proteins using the human ACE1 sequence as query returned ACE1 as the best hit against the human protein database, we were confident that it was an ACE1 ortholog.

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Figure 2: Through reciprocal blasting, we confirmed that g36554.t1 was an ACE1 ortholog


Other Ortholog:
BLAST searches were conducted to find different species in order to find othologs between the Mouse, Yeast and Zebra fish against human protein database ACE [ENSP00000290866]. These BLAST searches were condcted 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 of Mouse, Yeast, FruitFly, Arabidopsis, Clawed Frog, Rabbit, and Zebrafish were used to construct a phylogenetic tree diagram using the ClustalW2 program. (see Figure 3 below) .



Protein Domains:
As seen in figure 3, the whale shark gene ACE1 aligned with the homosapien protein family GluZincin superfamily with an e-value of 1.50E-04. Gluzincin family is short for (thermolysin-like proteinases, TLPs) in the oligoendopeptidase, M3 family. The M3 peptidases have two subfamilies: M3A, includes thimet oligopeptidase (TOP; endopeptidase 3.4.24.15), neurolysin (3.4.24.16), and the mitochondrial intermediate peptidase; M3B contains oligopeptidase F (Fernandez). M2 peptidase angiotensin converting enzyme (ACE, EC 3.4.15.1) catalyzes the conversion of decapeptide angiotensin I to the potent vasopressor octapeptide angiotensin II. ACE is a key part of the renin-angiotensin system that regulates blood pressure, thus ACE inhibitors are important for the treatment of hypertension. The next closest hit was Peptidase _M2 with an e-value of 634e-1228. Peptidase_M2 is a Angiotensin-converting enzyme. Members of the Peptidase_M2 family are dipeptidyl carboxydipeptidases (cleave carboxyl dipeptides) and have the main function of converting angiotensin I to angiotensin II (Riordan). Many members of this family contain a tandem duplication of the 600 amino acid peptidase domain, both of these are catalytically active. Most members are secreted membrane bound ectoenzymes.
Results:

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Figure 3: General domains of whale shark ACE1 best hit predicted proteins. All of the four best-hit whale shark predicted proteins contain the general GlyZincin and homeo domains as predicted by NCBI BLAST server analyses.



Orthologs:
The human ACE1 protein sequence [ENSP00000290866] was used as query in NCBI BLAST searches against individual species’ protein databases. As seen in Table 2, the ACE1 protein sequence found in humans found near identical matches with ortholog in mice and zebrafish, and along with the match found in fruit flies all belong in the Gluzincin protein superfamily. No such matches were to be found in yeast, however.
Results:
Species
Accession
Alignment Length
E- Value
Percent Identity
Homo Sapiens
NP_000780.1
1306
0.0
100%
Mouse
NP_997507.1
1312
0.0
83%
Zebrafish
XP_694336.5
1324
0.0
95%
Fruit Fly
NP_477046.1
615
2e-176
45%
Yeast
NP_0148879.1
211
.008
22%
Arabidopsis
NP_177267.2
557
1.5
29%
Clawed Frog
NP_001116882.1
1284
0.0
66%
Rabbit
NP_001075864.1
1310
0.0
88%
Table 2. Best hits with human ACE1 protein BLAST. The human ACE1 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. 5



Phylogeny:
The best hits of individual species acquired through protein database searches using the ACE1 protein were used to create a phylogenetic tree. From this tree, it is seen that the ACE1 protein deviated from the evolutionary tree from its mammalian cousins. Interestingly, the yeast homologue showed to be the most deviated from the human ACE1 protein, although the ACE1 protein is known to be present in fruit flies.

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ZebraFish:0.06212,
Rabbit:0.06345
Mouse:0.08018
FruitFly:0.28421
Yeast:0.42000
Arabidopsis:0.46152
ClawedFrog:0.14836
WhaleShark:0.04182

Figure 4: Phylogenetic tree of the best return hits for ACE1. 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.


Conclusions:
Results from table 2 and figure 3, show strong evidence for a whale shark ortholog for the ACE1 protein found in humans. The ortholog deviated from the evolutionary timeline before non-mammalian species, an E-value of 1e-88 coupled with a percent identity of 74.69% strongly displays the high correlation between the human and whale shark ACE1 proteins. The ACE1 protein is also found in Zebrafish in spite of the large evolutionary distance between the Zebrafish and human ACE1 proteins. This finding could be explained do to similar evolutionary patterns after deviating from the phylogenetic timeline. From the reciprocal blasts done, there was no difference between the whale shark and human indicating any differences between both. Indicating that the human and whale shark are indeed a homologue for this protein.

Future Research:
G36554.t1 was confirmed to be an ortholog for the ACE protein, and therefore, can be used to help understand how whale sharks regulate blood pressure. Further research can be done to understand how to use ACE to prevent diseases in whale sharks.


References:
ACE protein (Homo sapiens) - STRING interaction network. (n.d.). Retrieved from http://string90.embl.de/version_9_0/newstring_cgi/show_network_section.pl?identifier=9606.ENSP00000290866&all_channels_on=1

ACE Localizations. (n.d.). Retrieved from http://compartments.jensenlab.org/Entity?figures=subcell_cell_%%&knowledge=10&textmining=10&predictions=10&type1=9606&type2=-22&id1=ENSP00000290866

Tissue expression of ACE - Summary - The Human Protein Atlas. (n.d.). Retrieved from http://www.proteinatlas.org/ENSG00000159640-ACE/tissue

The Renin Angiotensin Aldosterone Reflex. (n.d.). Retrieved from http://antranik.org/the-renin-angiotensin-aldosterone-reflex/

Farrer LA, Sherbatich T, Keryanov SA, et al. Association Between Angiotensin-Converting Enzyme and Alzheimer Disease. Arch Neurol. 2000;57(2):210-214. doi:10.1001/archneur.57.2.210.

Vermeissen, V., Camp, J., & Verstraete, W. (2001, July 1). Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides. Retrieved from http://www.sciencedirect.com/science/article/pii/S0165022X02000064

Fernandez, M., Liu, X., Wouters, M., Heyberger, S., & Husain, A. (2000, November 6). Angiotensin I-converting Enzyme Transition State Stabilization by His1089 EVIDENCE FOR A CATALYTIC MECHANISM DISTINCT FROM OTHER GLUZINCIN METALLOPROTEINASES. Retrieved April 14, 2015, from http://www.jbc.org/content/276/7/4998.full

Riordan, J. (2003, July 25). Angiotensin-I-converting enzyme and its relatives. Retrieved April 14, 2015, from http://www.biomedcentral.com/content/pdf/gb-2003-4-8-225.pdf