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Analysis in silico of the single nucleotide polymorphism G–152A in the promoter of the angiotensinogen gene of Indonesian patients with essential hypertension

Akhiyan Hadi Susanto
Akhiyan Hadi Susanto
, 
Widodo
Widodo
, 
Mohammad Saifur Rohman
Mohammad Saifur Rohman
, 
Didik Huswo Utomo
Didik Huswo Utomo
 e 
Mifetika Lukitasari
Mifetika Lukitasari
   | 31 dic 2018
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Article Category: Original article
Pubblicato online: 31 dic 2018
Pagine: 15 - 25
DOI: https://doi.org/10.1515/abm-2018-0027
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© 2018 Akhiyan Hadi Susanto et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Figure 1

3D structural models of DNA (3D-DART *.pdb files) from sequences of the AGT promoter hypoxia-response element (HRE) region: G allele (top), 5′–GCGTG–3′ (olive green) and A allele (bottom), 5′–GCATG–3′ (olive green) with the mutation region (G→A) (white). Molecular graphics were created using the Chimera package (version 1.11.2), developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from a U.S. National Institutes of Health grant P41-GM103311 [20].
3D structural models of DNA (3D-DART *.pdb files) from sequences of the AGT promoter hypoxia-response element (HRE) region: G allele (top), 5′–GCGTG–3′ (olive green) and A allele (bottom), 5′–GCATG–3′ (olive green) with the mutation region (G→A) (white). Molecular graphics were created using the Chimera package (version 1.11.2), developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from a U.S. National Institutes of Health grant P41-GM103311 [20].

Figure 2

3D representation of the complex between hypoxia-inducible factor 1 (HIF-1) with its aryl receptor nuclear translocator (ARNT) subunit (dark blue) and HIF-1a subunit (sky blue) and DNA (gold) with hypoxia-response element (HRE) (chain A 5′–ACGTG–3′) (olive green). Molecular graphics were created from Protein Data Base entry 1D7G.pdb [21] with the Chimera package (version 1.11.2) [20].
3D representation of the complex between hypoxia-inducible factor 1 (HIF-1) with its aryl receptor nuclear translocator (ARNT) subunit (dark blue) and HIF-1a subunit (sky blue) and DNA (gold) with hypoxia-response element (HRE) (chain A 5′–ACGTG–3′) (olive green). Molecular graphics were created from Protein Data Base entry 1D7G.pdb [21] with the Chimera package (version 1.11.2) [20].

Figure 3

Visualization of isolated hypoxia-inducible factor 1 (HIF-1) protein with its aryl receptor nuclear translocator (ARNT) subunit (dark blue) and HIF-1a subunit (sky blue). We removed the 3D DNA structure and optimized the isolated HIF-1 protein (HIF-1a and ARNT complex) using VEGA ZZ software (release 3.1.1.42) [22] by removing water molecules and adding hydrogen atoms before creating the molecular graphics with the Chimera package (version 1.11.2) [20].
Visualization of isolated hypoxia-inducible factor 1 (HIF-1) protein with its aryl receptor nuclear translocator (ARNT) subunit (dark blue) and HIF-1a subunit (sky blue). We removed the 3D DNA structure and optimized the isolated HIF-1 protein (HIF-1a and ARNT complex) using VEGA ZZ software (release 3.1.1.42) [22] by removing water molecules and adding hydrogen atoms before creating the molecular graphics with the Chimera package (version 1.11.2) [20].

Figure 4

Polymerase chain reaction (PCR) products. Agarose gel (1.5%) electrophoresis showing a 593 bp band for 8 PCR products after amplification of AGT; presented is AGT in the promoter area; M: Invitrogen TrackIt 100 bp DNA Ladder (Thermo Fisher Scientific).
Polymerase chain reaction (PCR) products. Agarose gel (1.5%) electrophoresis showing a 593 bp band for 8 PCR products after amplification of AGT; presented is AGT in the promoter area; M: Invitrogen TrackIt 100 bp DNA Ladder (Thermo Fisher Scientific).

Figure 5

The single nucleotide polymorphism (SNP) G–152A of AGT (rs11568020) by direct sequencing. Electropherograms indicate the polymorphic site of GG (G allele) (black arrow, top) and AG (A allele) (green arrow, bottom) genotypes. No patients with AA genotype were found. C, cytosine blue; A, adenine green; T, thymine magenta; G, guanine black.
The single nucleotide polymorphism (SNP) G–152A of AGT (rs11568020) by direct sequencing. Electropherograms indicate the polymorphic site of GG (G allele) (black arrow, top) and AG (A allele) (green arrow, bottom) genotypes. No patients with AA genotype were found. C, cytosine blue; A, adenine green; T, thymine magenta; G, guanine black.

Figure 6

Differences in the binding pattern between G allele– hypoxia-inducible factor 1 (HIF-1) (left) and A allele–HIF-1 (right) interactions; molecular graphics were created using the Chimera package (version 1.11.2) [20] from the present docking results using High Ambiguity Driven protein-protein DOCKing (HADDOCK) [24] between the structures shown in Figures 1 and 3. HIF-1 protein (blue) with DNA (gold); hypoxia-response element (HRE) recognized (olive green).
Differences in the binding pattern between G allele– hypoxia-inducible factor 1 (HIF-1) (left) and A allele–HIF-1 (right) interactions; molecular graphics were created using the Chimera package (version 1.11.2) [20] from the present docking results using High Ambiguity Driven protein-protein DOCKing (HADDOCK) [24] between the structures shown in Figures 1 and 3. HIF-1 protein (blue) with DNA (gold); hypoxia-response element (HRE) recognized (olive green).

Figure 7

2D schematic representation of DNA–protein contacts observed in the hypoxia-inducible factor 1 (HIF-1) DNA-binding domain using NUCPLOT (version 1.0) [26]. The aryl receptor nuclear translocator (ARNT) subunit corresponds to amino acid residues 1–59, and HIF-1a subunit corresponds to residues 60–116 (or 1–57, arbitrary numbering). Left, G allele–HIF-1 contacts; right, A allele–HIF-1 contacts. C, cytosine brown; A, adenine magenta; T, thymine blue; G, guanine green. (B) indicates the amino acid acting as a ligand, and * indicates strong bonding between amino acids and nucleotides (either favorable or unfavorable contact).
2D schematic representation of DNA–protein contacts observed in the hypoxia-inducible factor 1 (HIF-1) DNA-binding domain using NUCPLOT (version 1.0) [26]. The aryl receptor nuclear translocator (ARNT) subunit corresponds to amino acid residues 1–59, and HIF-1a subunit corresponds to residues 60–116 (or 1–57, arbitrary numbering). Left, G allele–HIF-1 contacts; right, A allele–HIF-1 contacts. C, cytosine brown; A, adenine magenta; T, thymine blue; G, guanine green. (B) indicates the amino acid acting as a ligand, and * indicates strong bonding between amino acids and nucleotides (either favorable or unfavorable contact).

Figure 8

Visualization of hypoxia-inducible factor 1 (HIF-1)–hypoxia-response element DNA at the 8th nucleotide, guanine in G allele (A) and adenine in A allele (B) using LIGPLOT [27] with LigPlot+ (version 2.1) [28]. Here, the G allele (guanine) contacts arginine (Arg) 75 favorably. By contrast, the A allele (adenine) makes less favorable contact with Arg 75. Color key to atoms: carbon, black; nitrogen, blue; oxygen, magenta; phosphorous, purple.
Visualization of hypoxia-inducible factor 1 (HIF-1)–hypoxia-response element DNA at the 8th nucleotide, guanine in G allele (A) and adenine in A allele (B) using LIGPLOT [27] with LigPlot+ (version 2.1) [28]. Here, the G allele (guanine) contacts arginine (Arg) 75 favorably. By contrast, the A allele (adenine) makes less favorable contact with Arg 75. Color key to atoms: carbon, black; nitrogen, blue; oxygen, magenta; phosphorous, purple.

Protein–DNA contacts (hydrogen bonds) in the hypoxia-inducible factor 1 (HIF-1) DNA-binding domain complex observed in LIGPLOT [27] and NUCPLOT [26].

DonorAcceptorDistance (Å)
Hydrogen bonds – G allele
Arg 63 NH1A5 O2P2.82
Arg 63 NH2A5 O2P2.82
Ser 67 OGG6 O2P2.70
Lys 42 NZC7 O1P2.86
Arg 74 NH2C7 O5′2.65
Arg 74 NH2G8 O1P2.65
Arg 75 NH2G8 O2P2.68
Arg 75 NH1G8 O1P2.98
Arg 16 NH1T9 O5′2.94
His 8 NE2G10 O2P2.88
Arg 16 NH2G10 O2P2.73
Arg 5 NH1A11 O2P2.74
Arg 2 NH1G12 O2P2.77
Arg 5 NH2G12 N72.87
Glu 1NA16 O2P2.79
Glu 1NA16 O1P2.72
Arg 68 NH2T20 O5′3.00
Arg 68 NEC21 O2P2.68
Lys 64 NZC21 O5′2.80
Lys 39 NZG27 O5′2.83
Hydrogen bonds – A allele
Arg 68 NH2G6 O2P2.94
Ser 67 OGC7 O1P2.87
Arg 72 NH1C7 O2P2.75
Arg 72 NH2A8 O2P2.64
Arg 75 NH2A8 O1P2.66
Arg 75 NH1A8 O2P2.72
His 8 NE2G10 O1P2.77
Arg 15 NH2G10 O2P2.72
Tyr 83 OHC19 O3′2.86
Arg 2 NH2C25 O2P2.75
Arg 2 NH1C25 O5′2.67
Arg 5 NH2T26 O3′2.86
Lys 64 NZG28 O1P2.64
Ser 60 OGG29 O5′2.84
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