Transduction of human catalase mediated by an HIV-1 Tat protein basic domain and arginine-rich peptides into mammalian cells

Citation
Lh. Jin et al., Transduction of human catalase mediated by an HIV-1 Tat protein basic domain and arginine-rich peptides into mammalian cells, FREE RAD B, 31(11), 2001, pp. 1509-1519
Citations number
41
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
FREE RADICAL BIOLOGY AND MEDICINE
ISSN journal
0891-5849 → ACNP
Volume
31
Issue
11
Year of publication
2001
Pages
1509 - 1519
Database
ISI
SICI code
0891-5849(200112)31:11<1509:TOHCMB>2.0.ZU;2-R
Abstract
Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) h ave been considered to have a beneficial effect against various diseases me diated by reactive oxygen species (ROS). Although a variety of modified rec ombinant antioxidant enzymes have been generated to protect against the oxi dative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase e nzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRR RRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CA T and 9Arg-CAT fusion proteins, respectively. The expressed and purified fu sion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medi um, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H2O2, the viability of HeLa cells transduced with Ta t-CAT or 9Arg-CAT fusion proteins was significantly increased. In combinati on with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an in tracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis show ed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epi dermis as well as the dermis of the subcutaneous layer when sprayed on anim al skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzym e. (C) 2001 Elsevier Science Inc.