PROTOCOLS FOR THE DETECTION OF S-GLUTATHIONYLATED AND S-NITROSYLATED PROTEINS IN SITU

被引:31
作者
Aesif, Scott W. [1 ]
Janssen-Heininger, Yvonne M. W. [1 ]
Reynaert, Niki L. [2 ]
机构
[1] Univ Vermont, Coll Med, Dept Pathol, Burlington, VT 05405 USA
[2] Maastricht Univ, Dept Resp Med, Maastricht, Netherlands
来源
METHODS IN ENZYMOLOGY, VOL 474: THIOL REDOX TRANSITIONS IN CELL SIGNALING, PT B: CELLULAR LOCALIZATION AND SIGNALING | 2010年 / 474卷
关键词
SIGNAL-TRANSDUCTION; ESCHERICHIA-COLI; GLUTAREDOXIN; THIOREDOXIN; MECHANISMS; OXIDATION; SELENIUM; SULFUR;
D O I
10.1016/S0076-6879(10)74017-9
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The oxidation of protein cysteine residues represents significant posttranslational modifications that impact a wide variety of signal transduction cascades and diverse biological processes. Oxidation of cysteines occurs through reactions with reactive oxygen as well as nitrogen species. These oxidative events can lead to irreversible modifications, such as the formation of sulfonic acids, or manifest as reversible modifications such as the conjugation of glutathione with the cysteine moiety, a process termed S-glutathionylation (also referred to as S-glutathiolation, or protein mixed disulfides). Similarly, S-nitrosothiols can also react with the thiol group in a process known as S-nitrosylation (or S-nitrosation). It is the latter two events that have recently come to the forefront of cellular biology through their ability to reversibly impact numerous cellular processes. Herein we describe two protocols for the detection of S-glutathionylated or S-nitrosylated proteins in situ. The protocol for the detection of S-glutathionylated proteins relies on the catalytic specificity of glutaredoxin-1 for the reduction of S-glutathionylated proteins. The protocol for the detection of S-nitrosylated proteins represents a modification of the previously described biotin switch protocol, which relies on ascorbate in the presence of chelators to decompose S-nitrosylated proteins. These techniques can be applied in situ to elucidate which compartments in tissues are affected in diseased states whose underlying pathologies are thought to represent a redox imbalance.
引用
收藏
页码:289 / 296
页数:8
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