Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.
Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.
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DOI：
10.1016/j.freeradbiomed.2010.06.020
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发表时间：
2010-09-01
影响因子：
7.4
通讯作者：
Srivenugopal, Kalkunte S.
中科院分区：
文献类型：
--
作者：
Yusuf, Mohd A.;Chuang, Trinette;Bhat, G. Jayarama;Srivenugopal, Kalkunte S.
关键词：
以前，我们报告说，人p53功能失活的S-谷胱甘肽在Cys-141氧化和DNA损伤治疗。在这里，我们描述了存在的巯基化p53和动态性质的这种修饰在人体组织中使用独特的和特定的多克隆抗体提出了对12个残基的p53肽轴承混合二硫化物在Cys-141。亲和纯化的抗体（p53）是序列特异性的，因为它们在ELISA中识别抗原肽，但不识别未巯基化的肽或乱序谷胱甘肽化的肽。在免疫印迹上，纯化的抗体不与天然p53或重组p53（rp 53）反应，但仅在非还原条件下容易地检测到谷胱甘肽化或半胱氨酸化或乙硫醇处理的rp 53。未经处理的HCT 116细胞显示出低水平的p53，其在H2 O2、联胺、顺铂和多柔比星处理后显著增加。将细胞传递至无氧化剂培养基后，Glut-p53水平急剧下降，表明有效的去硫醇作用。还识别了HT 29和T47 D人癌细胞中存在的突变型p53。体外实验表明，兔网织红细胞裂解物可迅速降解p53。人前列腺和前列腺癌组织显示在腔上皮中大量存在p53，这是众所周知的产生ROS的位点。黑色素瘤和结肠癌样品也对p53呈阳性。巯基化特异性抗体的可用性应该增强我们对在包括癌症在内的各种疾病中发现的氧化还原扰动状态下的p53调节的认识。
Previously, we reported that human p53 is functionally inactivated by S-glutathionylation at Cys-141 during oxidative and DNA-damaging treatments. Here, we describe the presence of thiolated p53 and dynamic nature of this modification in human tissues using unique and specific polyclonal antibodies raised against a 12-residue p53 peptide bearing a mixed disulfide at Cys-141. The affinity- purified antibodies (glut-p53) were sequence-specific in that they recognized the antigenic peptide but not the unthiolated peptide or a scrambled glutathionylated peptide in ELISAs. On immunoblots, the purified antibodies did not react with native p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or ethanethiol-treated rp53 only under non-reducing conditions. Untreated HCT116 cells showed low levels of glut-p53 which increased markedly after H2O2, diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after passing cells to oxidant-free media, suggesting efficient dethiolation. The mutant p53 present in HT29 and T47D human cancer cells was also recognized. In vitro, the glut-p53 was rapidly degraded by rabbit reticulocyte lysates. Human prostate and prostate cancer tissues showed abundant presence of glut-p53 in luminal epithelium, a site well-known to generate ROS. Melanoma and colon cancer samples were also positive for glut-p53. Availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.
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影响因子：
--
作者：
Johansson M;Lundberg M
通讯作者：
Lundberg M
影响因子：
11.2
作者：
Findlay, Victoria J.;Townsend, Danyelle M.;Tew, Kenneth D.
通讯作者：
Tew, Kenneth D.
DOI：
10.1073/pnas.81.1.90
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
HOUGH, R;RECHSTEINER, M
通讯作者：
RECHSTEINER, M
影响因子：
--
作者：
Chen, Lin;Annis, Ioana;Barany, George
通讯作者：
Barany, George
影响因子：
4.8
作者：
Bennett, RG;Hamel, FG;Duckworth, WC
通讯作者：
Duckworth, WC