期刊
MOLECULAR AND CELLULAR BIOLOGY
卷 24, 期 1, 页码 84-95出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.24.1.84-95.2004
关键词
-
资金
- FIC NIH HHS [F05 TW005461, TW05461] Funding Source: Medline
- NIGMS NIH HHS [R01 GM030308, GM66355, R01 GM065872, GM30308, GM65872, R01 GM066355] Funding Source: Medline
- FOGARTY INTERNATIONAL CENTER [F05TW005461] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM066355, R01GM065872, R01GM030308] Funding Source: NIH RePORTER
Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据