Transduction of human catalase mediated by TCTP protein transduction domain into HaCaT human keratinocytes

Abstract

For organisms living in an aerobic environment, exposure to reactive oxygen species (ROS) is continuous and unavoidable. ROS encompass a variety of partially reduced metabolites such as O₂-, H₂O₂,·OH, etc., which are known to be involved in the pathogenesis of many diseases. Cellular defense against ROS utilizes antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. Catalase is localized in peroxisomes, wherein many H₂O₂-producing enzymes reside. It is a tetrameric heme containing enzyme that is found in all aerobic organisms. Catalase is one of the most active catalysts produced by nature. It decomposes hydrogen peroxide at an extremely rapid rate. Catalase is unique among H₂O₂ degrading enzymes in that it degrades H₂O₂ without consuming cellular reducing equivalents. Hence, Catalase provides the cell with a very energy efficient mechanism to remove hydrogen peroxide. Although antioxidant enzymes including catalase have been considered to be potential therapeutic agents for ROS-mediated disease, the difficulties in transduction of antioxidant enzymes into cells was the limitation in their use for therapeutic purposes. Protein transduction domains (PTDs) have been developed to allow the delivery of therapeutic proteins into living cells. Recently, "protein transduction technology" has been successfully applied to a wide range of proteins to study their intracellular functions. We identified a novel cell-penetrating PTD from Translationally Controlled Tumor Protein (TCTP). TCTP was originally identified as a growth-related tumor protein whose synthesis is controlled at the translational as well as transcriptional level. We made mutants of TCTP PTDs to improve transduction efficiency. In this study, I manufactured TCTP PTD WT-CAT and TCTP PTD #16-CAT fusion constructs. The resulting TCTP PTD-CAT fusion proteins were analyzed for transduction efficiency into HaCaT human keratinocytes and for protective effect against oxidative stress. TCTP PTD-CAT fusion proteins were compared with the putative TAT-CAT fusion protein. TCTP PTD WT-CAT fusion protein was more effective than TAT-CAT fusion protein on cell viability under oxidative stress condition. The experimental data I report here suggest that this fusion protein would be useful therapeutic agent for various disorders including skin disease related to the function of catalase.;유산소환경에서 살아가는 유기체는 피할 수 없이 계속해서 과산화수소와 같은 활성산소종에 노출되게 된다. 활성산소종은 산소분자에 비해서 높은 반응성을 가지고 여러 질병을 유발하는 병태생리학적인 문제를 야기한다. 활성산소종에 대항하는 세포의 저항 기전으로 superoxide dismutase (SOD), catalase, blutathione peroxidase 같은 항산화효소들이 존재한다. 그 중 catalase는 peroxisome에 대부분 존재하고 있으며 모든 호기성 유기체에서 발견되는 효소로 자연에서 생산되는 가장 활성이 높은 catalyst로서 굉장히 빠른 속도로 과산화수소를 물분자와 산소분자로 분해시킨다. Catalase를 비롯한 많은 항산화효소들이 ROS와 관련될 질병의 치료제로서 사용될 수 있지만 세포안으로 들어가는 것에 제한이 있어서 쉽게 사용되지 못했다. Protein transduction domain (PTD) 는 여러 가지 cargo들을 살아있는 cell 안으로 가지고 들어갈 수 있는 짧은 peptide로 연구되어 왔다. 본 실험실에서는 이미 많은 기능을 가지고 있는 것으로 알려져 있는 Translationally Controlled Tumor Protein (TCTP) 에서 새로운 PTD를 발견했다. 세포투과능력을 높이기 위해 TCTP PTD의 여러 mutant들을 peptide level에서 만들어서 실험하였다. 그 중 본 논문에서는 TCTP PTD WT과 TCTP PTD #16을 이용하여 catalase와 fusion protein을 만들어 HaCaT cell을 이용하여 TAT fusion protein과 비교 실험을 하였다. 그 결과 TCTP PTD #16은 기대에 비해 효과가 좋지 않았지만, TCTP PTD WT의 경우 TAT보다 세포의 생존율을 더 놓게 유지시켰으며, 활성도 12 시간 이상 지속되었다. 따라서 본 연구결과는 새로운 PTD인 TCTP PTD WT와 융합된 catalase 단백질이 활성산소종에 의한 세포사멸에 대항하는 단백질 치료제로서 가능성을 보여주고 있다.