Flores S C, Marecki J C, Harper K P, Bose S K, Nelson S K, McCord J M
Webb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, Denver 80262.
Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7632-6. doi: 10.1073/pnas.90.16.7632.
Using a HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1, we have found that the expression of the regulatory Tat protein suppresses the expression of cellular Mn-containing superoxide dismutase (Mn-SOD). This enzyme is one of the cell's primary defenses against oxygen-derived free radicals and is vital for maintaining a healthy balance between oxidants and antioxidants. The parental HeLa cells expressed nearly equivalent amounts of Cu,Zn- and Mn-SOD isozymes. Those cells expressing the Tat protein, however, contained 52% less Mn-SOD activity than parental cells, whereas that of the Cu,Zn enzyme was essentially unchanged. The steady-state levels of Mn-SOD-specific RNAs were also lower in the HeLa-tat cell line than in the parental line. No difference was seen in the steady-state levels of Cu,Zn-SOD-specific RNAs. In addition to the decreased Mn-SOD-activity, HeLa-tat cell showed evidence of increased oxidative stress. Carbonyl proteins were markedly higher, and total cellular sulfhydryl content decreased in cell extracts at a faster rate, probably reflecting ongoing lipid peroxidation. HeLa and HeLa-tat extracts were incubated with radiolabeled Mn-SOD transcripts, and the reaction products were subjected to UV crosslinking, digestion with ribonuclease A, and electrophoretic analysis. The results suggest a direct interaction between Tat protein and Mn-SOD gene transcripts.
利用稳定转染了1型人类免疫缺陷病毒tat基因的HeLa细胞系，我们发现调节性Tat蛋白的表达会抑制细胞内含锰超氧化物歧化酶（Mn-SOD）的表达。这种酶是细胞抵御氧衍生自由基的主要防御机制之一，对于维持氧化剂和抗氧化剂之间的健康平衡至关重要。亲本HeLa细胞表达几乎等量的铜锌和锰超氧化物歧化酶同工酶。然而，那些表达Tat蛋白的细胞，其Mn-SOD活性比亲本细胞低52%，而铜锌酶的活性基本未变。HeLa-tat细胞系中Mn-SOD特异性RNA的稳态水平也低于亲本细胞系。铜锌超氧化物歧化酶特异性RNA的稳态水平未见差异。除了Mn-SOD活性降低外，HeLa-tat细胞还表现出氧化应激增加的迹象。羰基蛋白明显更高，细胞提取物中的总细胞巯基含量下降速度更快，这可能反映了正在进行的脂质过氧化。将HeLa和HeLa-tat提取物与放射性标记的Mn-SOD转录本一起孵育，反应产物进行紫外线交联、核糖核酸酶A消化和电泳分析。结果表明Tat蛋白与Mn-SOD基因转录本之间存在直接相互作用。
