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Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

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Related in: MedlinePlus

The 176 amino acid sequence of the AZP. Different Motifs were shown by distinct background colors.
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fig3: The 176 amino acid sequence of the AZP. Different Motifs were shown by distinct background colors.

Mentions: The sequence (4) 5′-ACT CGC GGA AAC AAA GGG-3′ was submitted to ZF tools 3.0 again to obtain the amino acid sequence of the corresponding AZP (Figure 3). As a result of “Sequence Feature Scan”, “InterproScan” [26] showed that the AZP consisted of six Cys2-His2 zinc finger motifs, which were located at the 8–30, 36–58, 64–86, 92–114, 120–142, and 148–170 amino acid residues of the AZP. “Secondary Structure & Disorder prediction” showed the assignment of domains in the AZP and indicated that there were few disordered regions in the AZP. Homology modeling showed the conformation of the six zinc finger motifs. The modeling was based on the template 2i13A (a stable AZP). The sequence identity was 79.221%, and the E-value was 1.26e − 44. To each amino acid residue of the AZP, the Anolea value was negative and the Verify 3D value ranged from 0 to +1. Taken together, these results suggested that the AZP was quite stable and justified further experiments.


Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

The 176 amino acid sequence of the AZP. Different Motifs were shown by distinct background colors.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2913762&req=5

fig3: The 176 amino acid sequence of the AZP. Different Motifs were shown by distinct background colors.
Mentions: The sequence (4) 5′-ACT CGC GGA AAC AAA GGG-3′ was submitted to ZF tools 3.0 again to obtain the amino acid sequence of the corresponding AZP (Figure 3). As a result of “Sequence Feature Scan”, “InterproScan” [26] showed that the AZP consisted of six Cys2-His2 zinc finger motifs, which were located at the 8–30, 36–58, 64–86, 92–114, 120–142, and 148–170 amino acid residues of the AZP. “Secondary Structure & Disorder prediction” showed the assignment of domains in the AZP and indicated that there were few disordered regions in the AZP. Homology modeling showed the conformation of the six zinc finger motifs. The modeling was based on the template 2i13A (a stable AZP). The sequence identity was 79.221%, and the E-value was 1.26e − 44. To each amino acid residue of the AZP, the Anolea value was negative and the Verify 3D value ranged from 0 to +1. Taken together, these results suggested that the AZP was quite stable and justified further experiments.

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

Show MeSH
Related in: MedlinePlus