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Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations

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ABSTRACT

Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein’s nature, transduction efficiency and stability.

No MeSH data available.


In vitro anti-apoptotic effects of PTD-conjugated fusion proteins. a IC50 was measured with different conc. of NaAsO2. b Cell viability was measured to check anti-apoptotic activity by 5 µM PTD-Hsp27
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Fig6: In vitro anti-apoptotic effects of PTD-conjugated fusion proteins. a IC50 was measured with different conc. of NaAsO2. b Cell viability was measured to check anti-apoptotic activity by 5 µM PTD-Hsp27

Mentions: To evaluate the effects of PTD-Hspp27 fusion proteins in hypoxic condition, H9c2 cells were treated with 5 μM of PTD-Hsp27 fusion proteins for 24 h. Cell viability in T-Hsp27 treated cells increased about ~25 % while there was no significant effect in Hsp27 treated cells. Cell viability in 9R-Hsp27, Hsp27-9R and T-Hsp27-9R groups increased about ~20, 17 and 15 %, respectively (Fig. 6b). In the case of 9R-Hsp27, Hsp27-9R and T-Hsp27-9R, the protective effects were much higher than with just Hsp27, indicating that Hsp27 itself could not be transduced efficiently into cells. These results indicate that transduction of Hsp27 into cells is essential for the protection of cells against hypoxia and that Hsp27 is an anti-hypoxic protein.Fig. 6


Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations
In vitro anti-apoptotic effects of PTD-conjugated fusion proteins. a IC50 was measured with different conc. of NaAsO2. b Cell viability was measured to check anti-apoptotic activity by 5 µM PTD-Hsp27
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037156&req=5

Fig6: In vitro anti-apoptotic effects of PTD-conjugated fusion proteins. a IC50 was measured with different conc. of NaAsO2. b Cell viability was measured to check anti-apoptotic activity by 5 µM PTD-Hsp27
Mentions: To evaluate the effects of PTD-Hspp27 fusion proteins in hypoxic condition, H9c2 cells were treated with 5 μM of PTD-Hsp27 fusion proteins for 24 h. Cell viability in T-Hsp27 treated cells increased about ~25 % while there was no significant effect in Hsp27 treated cells. Cell viability in 9R-Hsp27, Hsp27-9R and T-Hsp27-9R groups increased about ~20, 17 and 15 %, respectively (Fig. 6b). In the case of 9R-Hsp27, Hsp27-9R and T-Hsp27-9R, the protective effects were much higher than with just Hsp27, indicating that Hsp27 itself could not be transduced efficiently into cells. These results indicate that transduction of Hsp27 into cells is essential for the protection of cells against hypoxia and that Hsp27 is an anti-hypoxic protein.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein’s nature, transduction efficiency and stability.

No MeSH data available.