Core C will provide essential functions for Projects 1-5 of this Program Project. These include the synthesis of peptides that are required by the Projects for biological and biochemical investigations. In addition, the Core will characterize synthetic, natural or modified peptides and proteins by mass spectrometry and Edman-based chemical sequencing. Core C will synthesize and characterize peptides, and develop new HPLC- and CZE-based analytical systems to improve resolution and efficiency. Additionally, this Core will perform circular dichroism experiments as requested. Each year, using Merrifield's automated SPPS, Core C will synthesize a total of approximately 10-15 peptides (20 to 40 residues in length;linear, cyclic, phosphorylated). Using the latest techniques for the preparative purification of peptides/proteins, we will provide 10 to 100 mg of highly purified peptides to support investigations carried out in most Projects. All peptides will be extensively characterized by the techniques available to the Core, including HPLC, CZE, MS, and Edman degradation. The protein chemical characterization of peptides and proteins involved in neuroendocrine regulation is an essential part of this Program. The Core provides both state-of-the-art instrumentation and highly trained investigators and support personnel to carry out these characterizations. The Core will characterize peptides, proteins and their posttranslational modifications using highly resolving mass spectrometric methods. In particular, the disulfide arrangement of soluble forms of CRFRs, both recombinant and naturally occurring, will be determined. In addition, we will confirm the covalent structure of recombinant proteins that the Projects generate for use in biological studies. The processing and posttranslational modifications of urocortins will be established by isolating the peptides from tissues or cell lines and determining their primary structure by mass spectrometry and Edman degradation.

Public Health Relevance

This Core fulfills important support functions for all of the projects of this Program by providing highly purified peptides and by characterizing ligands and binding proteins of the CRF/Urocortin system. This system is the major modulator of the mammalian stress response. In addition, it plays an important role in diseases such as diabetes and those of the cardiovascular system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK026741-35
Application #
8662241
Study Section
Special Emphasis Panel (ZDK1)
Project Start
Project End
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
35
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Erchegyi, Judit; Wang, Lixin; Gulyas, Jozsef et al. (2016) Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins). J Med Chem 59:854-66
Zhang, Cheng; Kuo, Ching-Chang; Moghadam, Setareh H et al. (2016) Corticotropin-releasing factor receptor-1 antagonism mitigates beta amyloid pathology and cognitive and synaptic deficits in a mouse model of Alzheimer's disease. Alzheimers Dement 12:527-37
Cui, Changhai; Noronha, Antonio; Warren, Kenneth R et al. (2015) Brain pathways to recovery from alcohol dependence. Alcohol 49:435-52
Perrin, Marilyn H; Tan, Laura A; Vaughan, Joan M et al. (2015) Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies. J Pharmacol Exp Ther 353:307-17
van der Meulen, Talitha; Donaldson, Cynthia J; Cáceres, Elena et al. (2015) Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion. Nat Med 21:769-76
Radley, Jason J; Sawchenko, Paul E (2015) Evidence for involvement of a limbic paraventricular hypothalamic inhibitory network in hypothalamic-pituitary-adrenal axis adaptations to repeated stress. J Comp Neurol 523:2769-87
Mulak, Agata; Larauche, Muriel; Biraud, Mandy et al. (2015) Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice. Peptides 63:71-80
van der Meulen, Talitha; Huising, Mark O (2015) Role of transcription factors in the transdifferentiation of pancreatic islet cells. J Mol Endocrinol 54:R103-17
Jappelli, Roberto; Perrin, Marilyn H; Lewis, Kathy A et al. (2014) Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli. PLoS One 9:e84013

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