title: Peptidomics approach to elucidate the proteolytic regulation of bioactive peptides.
abstract: Peptide hormones and neuropeptides have important roles in physiology and therefore the regulation of these bioactive peptides is of great interest. In some cases proteolysis controls the concentrations and signaling of bioactive peptides, and the peptidases that mediate this biochemistry have proven to be extremely successful drug targets. Due to the lack of any general method to identify these peptidases, however, the role of proteolysis in the regulation of most neuropeptides and peptide hormones is unknown. This limitation prompted us to develop an advanced peptidomics-based strategy to identify the peptidases responsible for the proteolysis of significant bioactive peptides. The application of this approach to calcitonin gene-related peptide (CGRP), a neuropeptide associated with blood pressure and migraine, revealed the endogenous CGRP cleavage sites. This information was then used to biochemically purify the peptidase capable of proteolysis of CGRP at those cleavage sites, which led to the identification of insulin-degrading enzyme (IDE) as a candidate CGRP-degrading enzyme. CGRP had not been identified as an IDE substrate before and we tested the physiological relevance of this interaction by quantitative measurements of CGRP using IDE null (IDE(-/-)) mice. In the absence of IDE, full-length CGRP levels are elevated in vivo, confirming IDE as an endogenous CGRP-degrading enzyme. By linking CGRP and IDE, this strategy uncovers a previously unknown pathway for CGRP regulation and characterizes an additional role for IDE. More generally, this work suggests that this may be an effective general strategy for characterizing these pathways and peptidases moving forward.
authors.affiliation: Department of Chemical Engineering,  Soongsil University, Seoul 156-743,  South Korea.
authors.forename: Yun-Gon
authors.initials: YG
authors.lastname: Kim
authors.forename: Anna Mari
authors.initials: AM
authors.lastname: Lone
authors.forename: Whitney M
authors.initials: WM
authors.lastname: Nolte
authors.forename: Alan
authors.initials: A
authors.lastname: Saghatelian
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015740:Calcitonin Gene-Related Peptide / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D005260:Female; D007339:Insulysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013116:Spinal Cord / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D007339:Insulysin; D015740:Calcitonin Gene-Related Peptide