Dataset Information

Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath.

ABSTRACT:

Background

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.

Findings

In order to be able to fully characterize CNPase structurally and functionally, we have set up expression systems for different domains of CNPase, using a total of 18 different expression constructs. CNPase was expressed in E. coli with a TEV-cleavable His-tag. Enzymatic activity assays indicated that the purified proteins were active and correctly folded. The folding of both the full-length protein, as well as the N- and C-terminal domains, was also studied by synchrotron CD spectroscopy. A thermal shift assay was used to optimize buffer compositions to be used during purification and storage. The assay also indicated that CNPase was most stable at a pH of 5.5, and could be significantly stabilized by high salt concentrations.

Conclusions

We have been able to express and purify recombinantly several different domains of CNPase, including the isolated N-terminal domain, which is folded mainly into a beta-sheet structure. The expression system can be used as an efficient tool to elucidate the role of CNPase in the myelin sheath.

SUBMITTER: Myllykoski M

PROVIDER: S-EPMC2843729 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Publications

Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath.

Myllykoski Matti M Kursula Petri P

BMC research notes 20100121

<h4>Background</h4>2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.<h4>Findings</h4>In order to be able to fully characterize CNPase structurally and functionally, we have set up expressi ...[more]

PMID: 20180985

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Project description:Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.

Dataset Information

Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath.

Background

Findings

Conclusions

Publications

Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath.

Similar Datasets

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