Transduced protein transduction domain linked HSP27 protected LECs against UVB radiation-induced damage

• Published in: Experimental eye research 2014-03, Vol.120, p.36-42
• Main Authors: Liu, Lian, Yu, Rongjie, Shi, Yuehuan, Dai, Yun, Zeng, Zhixing, Guo, Xiaoling, Ji, Qingshan, Wang, Guifang, Zhong, Jingxiang
• Format: Article
• Language: English
• Subjects: Animals; Anterior Chamber - metabolism; apoptosis; Apoptosis - drug effects; Apoptosis - radiation effects; cataract; Cell Line; Electrophoresis, Polyacrylamide Gel; Epithelial Cells - metabolism; Epithelial Cells - radiation effects; Flow Cytometry; Fluorescein-5-isothiocyanate - metabolism; Gene Expression - physiology; Genetic Therapy; HSP27; HSP27 Heat-Shock Proteins - genetics; human lens epithelial cell (HLEC); Humans; Lens, Crystalline - metabolism; Lens, Crystalline - radiation effects; Male; Membrane Potentials; Microscopy, Fluorescence; Mitochondria - metabolism; Peptide Fragments - genetics; Protein Structure, Tertiary; protein transduction domain (PTD); Rabbits; Radiation Injuries, Experimental - metabolism; Radiation Injuries, Experimental - pathology; Radiation Injuries, Experimental - prevention & control; Recombinant Fusion Proteins - genetics; tat Gene Products, Human Immunodeficiency Virus - genetics; transactivator of transcription (TAT); Transduction, Genetic; Ultraviolet Rays
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2013.12.016

PTD-fusion protein technology was used to transduce heat shock protein 27 (HSP27), an anti-apoptotic protein, into human lens epithelial cells (HLECs) (SRA01/04). The protein transduction domain (PTD) of the 11-amino acid YGRKKRRQRRR was tagged at the N-terminus of HSP27. The fusion protein was purified from bacteria transformed with a pKYB-PTD-HSP27 construct. The HLECs were incubated with PTD-HSP27-FITC and the fluorescence inside HLECs was found by fluorescence microscopic examination. To test the ability of PTD-HSP27 to pass through the corneas, PTD-HSP27-FITC was dropped onto the conjunctival sacs of rabbits; fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor. After being incubated with the PTD-HSP27 protein and irradiated with ultraviolet-B (UVB) light, HLECs was analyzed by flow cytometry, Hoechst 33258 staining and measurement of the potential of the mitochondrial transmembrane. HLECs incubated with PTD-HSP27 had a lower apoptotic rate and a higher mitochondrial membrane potential than the control cells. PTD-HSP27 appears to be sufficient to protect HLECs against UVB-induced apoptosis. •The PTD was first and successfully tagged at the N-terminus of HSP27.•PTD-HSP27 is able to get through the HELCs (SRA01/04) membrane.•PTD-HSP27 is able to pass through the rabbit's cornea.•PTD-HSP27 inhibits the HLECs (SRA01/04) apoptosis under UVB irradiating.