Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications
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- 作者：Heping Cao
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：November 2, 2004
- 年：2004
- 卷：43
- 期：43
- 页码：13724 - 13738
- 全文大小：760K
- 年卷期：v.43,no.43(November 2, 2004)
- ISSN：1520-4995
文摘
Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding toan AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. Thebiochemical properties of TTP have not been adequately characterized, due to the difficulties in proteinpurification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE bindingactivity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosisfactor 
mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion proteinexpressed in Escherichia coli. Cellular localization studies of the transfected cells indicated thatapproximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from bothlocations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact byN-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Resultsfrom size exclusion chromatography are consistent with the predominant form of active TTP being atetramer. TTP's ARE binding activity was increased by 10 
M Zn2+. The half-maximal binding of TTPfrom HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was abouttwice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoreticmobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not byPNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitrophosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest thatTTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainlyby phosphorylation but not by glycosylation, in mammalian cells. The results support a model in whicheach subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of theARE, resulting in a stable TTP-ARE complex.
mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion proteinexpressed in Escherichia coli. Cellular localization studies of the transfected cells indicated thatapproximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from bothlocations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact byN-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Resultsfrom size exclusion chromatography are consistent with the predominant form of active TTP being atetramer. TTP's ARE binding activity was increased by 10 M Zn2+. The half-maximal binding of TTPfrom HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was abouttwice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoreticmobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not byPNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitrophosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest thatTTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainlyby phosphorylation but not by glycosylation, in mammalian cells. The results support a model in whicheach subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of theARE, resulting in a stable TTP-ARE complex.