Transduction of human catalase mediated by an HIV-1 Tat protein basic domain and arginine-rich peptides into mammalian cells
Transduction of human catalase mediated by an HIV-1 Tat protein basic domain and arginine-rich peptides into mammalian cells
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DOI：
10.1016/s0891-5849(01)00734-1
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发表时间：
2001-12-01
影响因子：
7.4
通讯作者：
Choi, SY
中科院分区：
文献类型：
--
作者：
Jin, LH;Bahn, JH;Choi, SY
抗氧化酶如超氧化物歧化酶（SOD）和过氧化氢酶（CAT）已被认为对由活性氧（ROS）介导的各种疾病具有有益作用。虽然已经产生了各种修饰的重组抗氧化酶来保护免受氧化应激，但缺乏它们向细胞中的转导能力导致了对细胞内ROS解毒的能力有限。为了使过氧化氢酶在细胞外添加时能够解毒细胞内ROS，产生了细胞可渗透的重组过氧化氢酶蛋白。克隆了人肝脏过氧化氢酶基因，并在细菌表达载体中与编码HIV-1达特蛋白转导结构域（RKKRRQRRR）和富含精氨酸肽（RRRRRRRRRR）的基因片段融合，分别产生遗传符合读框的Tat-CAT和9 Arg-CAT融合蛋白。表达纯化的融合蛋白可在体外以时间和剂量依赖性的方式转导至哺乳动物细胞（HeLa和PC 12细胞）中，且在60 h内具有稳定的酶活性。当暴露于H2 O2时，用Tat-CAT或9Arg-CAT融合蛋白转导的HeLa细胞的存活率显著增加。在与转导的SOD，转导的过氧化氢酶也导致在细胞活力的合作增加时，细胞用百草枯，细胞内的抗氧化阴离子发生器处理。然后，我们评估了过氧化氢酶融合蛋白渗入动物皮肤的能力。该分析表明，Tat-CAT和9Arg-CAT融合蛋白有效地渗透表皮以及皮下层的真皮，当喷雾在动物皮肤上，如通过免疫组织化学和特异性酶活性判断。这些结果表明，Tat-CAT和9Arg-CAT融合蛋白可用于与这种抗氧化酶相关的各种疾病的蛋白质治疗。(C)2001 Elsevier Science Inc.
Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme 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 (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H2O2, the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal 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 enzyme. (C) 2001 Elsevier Science Inc.