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Identification of incompletely processed potential carboxypeptidase E substrates from CpEfat/CpEfat mice
E J Bures1, P L Courchesne, J Douglass
1Departments of Biochemistry and Genetics, Amgen, Thousand Oaks, CA, USA.
Abstract:
In an attempt to identify peptides that may be involved in the obese phenotype observed in CpEfat/CpEfat mice (deficient in Carboxypeptidase E, CpE) samples from fourteen neuroendocrine tissues in wild-type and CpEfat/CpEfat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that captures peptides with basic C-termini. Bound peptides were subjected to tryptic digestion and followed by liquid chromatography-mass spectrometry analysis. The relative levels of CpEfat/CpEfat versus wild-type peptides were determined by comparison of the ion intensities. Peptide ions elevated in the CpEfat/CpEfat samples were identified by targeted liquid chromatography-tandem mass spectrometry. From those ions, 27 peptides derived from known neuropeptides (including CpE substrates) were identified, together with another 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recently discovered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohormone processing. The approach demonstrated the feasibility of using an affinity-based method for identifying differences in specific classes of peptides between normal and mutant mice.
Insights
Researchers identified peptides linked to obesity in Carboxypeptidase E-deficient mice. This study used an affinity-based method to uncover differences in neuropeptide processing, revealing potential therapeutic targets for metabolic disorders.
Area of Science:
- Neuroendocrinology
- Molecular Biology
- Metabolomics
Background:
- Obesity is a complex metabolic disorder associated with dysregulation of neuropeptide processing.
- Carboxypeptidase E (CpE) plays a crucial role in the post-translational modification of prohormones and neuropeptides.
- CpE-deficient (CpEfat/CpEfat) mice exhibit an obese phenotype, suggesting CpE's involvement in weight regulation.
Purpose of the Study:
- To identify specific peptides affected by CpE deficiency in neuroendocrine tissues.
- To investigate the role of CpE in neuropeptide processing and its contribution to the obese phenotype.
- To develop and validate an affinity-based enrichment strategy for identifying CpE substrates.
Main Methods:
- Neuroendocrine tissue samples were collected from wild-type and CpEfat/CpEfat mice.
- Peptides with basic C-termini, potential CpE substrates, were enriched using an anhydrotrypsin affinity column.
- Enriched peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) and targeted LC-tandem mass spectrometry (LC-MS/MS).
Main Results:
- An affinity-based enrichment strategy successfully isolated peptides with basic C-termini.
- LC-MS/MS analysis identified 27 peptides derived from known neuropeptides and 25 peptides from novel protein sources.
- Elevated levels of specific neuropeptide-derived peptides were observed in CpEfat/CpEfat mice, including substrates of CpE such as proSAAS.
Conclusions:
- The study successfully identified peptides differentially regulated in CpE-deficient mice, implicating altered neuropeptide processing in obesity.
- The affinity-based enrichment method is a feasible approach for discovering CpE substrates and studying neuropeptide processing pathways.
- These findings provide insights into the molecular mechanisms underlying CpE-related obesity and suggest potential targets for therapeutic intervention.
