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Characterization of single-stranded cAMP response element binding protein (ssCRE-BP) from mouse cerebellum
Abstract:
It has been speculated that opiate tolerance and dependence may occur at the level of gene expression. Our previous studies have shown that the binding activity of a nuclear factor (ssCRE-BP) to single-stranded CRE of somatostatin gene is altered by long-term treatment with morphine in the mouse cerebellum. ssCRE-BP was purified from the mouse cerebellum by a combination of chromatography on DNA affinity agarose and Mono Q HR. The native protein exhibited a molecular size of 110-150 kDa by gel filtration, and two polypeptides of about 35-40 kDa were observed on SDS-PAGE. The cloning and sequencing of a cDNA encoding ssCRE-BP showed that the protein possesses a glycine-rich domain and a glutamine-rich domain in the amino terminus and the carboxyl terminus, respectively. To investigate the function of ssCRE-BP in the brain, recombinant glutathione-S-transferase (GST) fusion proteins containing ssCRE-BP were expressed in bacterial systems. Rabbit anti-ssCRE-BP antibodies were raised against a GST-ssCRE-BP fusion protein. Using the antibodies in western blot analysis, a polypeptide of approximately 66 kDa was detected in the brain. These findings indicate that ssCRE-BP is involved in opiate tolerance and dependence.
Insights
Opiate tolerance and dependence may involve gene expression changes. A nuclear factor, single-stranded CRE-binding protein (ssCRE-BP), was identified and characterized, indicating its role in opiate-induced changes in the brain.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Opiate tolerance and dependence are complex phenomena potentially linked to alterations in gene expression.
- Previous research indicated that long-term morphine treatment affects the binding activity of a nuclear factor (ssCRE-BP) to the somatostatin gene in the mouse cerebellum.
Purpose of the Study:
- To purify and characterize the nuclear factor ssCRE-BP involved in opiate tolerance and dependence.
- To investigate the molecular structure and function of ssCRE-BP in the brain.
Main Methods:
- Purification of ssCRE-BP from mouse cerebellum using DNA affinity agarose and Mono Q HR chromatography.
- Protein characterization via gel filtration and SDS-PAGE.
- Cloning and sequencing of ssCRE-BP cDNA.
- Expression of recombinant glutathione-S-transferase (GST) fusion proteins.
- Antibody generation and Western blot analysis.
Main Results:
- ssCRE-BP was purified, showing native molecular size of 110-150 kDa and subunits of 35-40 kDa on SDS-PAGE.
- The cDNA sequence revealed glycine-rich and glutamine-rich domains.
- Western blot analysis using anti-ssCRE-BP antibodies detected a ~66 kDa polypeptide in brain tissue.
Conclusions:
- ssCRE-BP is a distinct nuclear factor implicated in the molecular mechanisms underlying opiate tolerance and dependence.
- The characterized protein structure and brain detection support its functional role in opiate-related neurobiological processes.