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Review
. 2013 Feb;17(1):83-9.
doi: 10.1016/j.cbpa.2012.10.038. Epub 2013 Jan 15.

Peptidomics methods for the identification of peptidase-substrate interactions

Anna Mari Lone  1 , Yun-Gon Kim, Alan Saghatelian
Affiliations
Review

Peptidomics methods for the identification of peptidase-substrate interactions

Anna Mari Lone et al. Curr Opin Chem Biol. 2013 Feb.

Abstract

Peptidases have important roles in controlling physiological signaling through their regulation of bioactive peptides. Understanding and controlling bioactive peptide regulation is of great biomedical interest and approaches that elucidate the interplay between peptidases and their substrates are vital for achieving this goal. Here, we highlight the utility of recent peptidomics approaches in identifying endogenous substrates of peptidases. These approaches reveal bioactive substrates and help characterize the biochemical functions of the enzyme. Most recently, peptidomics approaches have been applied to address the challenging question of identifying the peptidases responsible for regulating specific bioactive peptides. Since peptidases are of great biomedical interest, these approaches will begin to impact our ability to identify new drug targets that regulate important bioactive peptides.

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Figures

Figure 1
Figure 1
Peptides control a wide range of important biological processes. A) Bioactive peptides are found in many organs and control diverse physiological processes. B) DPP4 regulates GLP-1 levels. GLP-1(7–36) amide is released from the gut in response to food intake, and stimulates biosynthesis and secretion of insulin. DPP4 inactivates this species by removing the N-terminal dipeptide, resulting in the inactive species GLP-1(9–36) amide. By inhibiting DPP4, recently developed diabetes drugs increase levels of GLP-1 and insulin, thus affording better control of blood glucose levels.
Figure 2
Figure 2
Peptide profiling reveals endogenous substrates of peptidases. A) In the peptide profiling technique, mass spectrometry is used for a global comparison of peptide levels in samples with and without enzyme activity. In the sample without enzyme activity, there should be no substrate turnover, resulting in substrate accumulation and lower levels of the peptidase product, relative to a sample with normal enzyme activity. Either chemical inhibitors or genetic knockouts can be used to achieve the removal of enzyme activity. B) Applying this approach to DPP4, a number of previously unknown endogenous substrates of this enzyme were identified. For some of these substrates, the corresponding product could also be seen elevated in the sample containing enzyme activity. C) Inhibiting POP using the specific chemical inhibitor S17092 and application of the peptide profiling approach resulted in the identification of several new substrates of this enzyme, including some bioactive peptides (substance P). The profiling further revealed that not all internal prolines are processed by POP, as previously believed and that POP has a length preference, as the CGRP sequence identified as a substrate was no longer cleaved efficiently when part of the complete CGRP sequence.
Figure 3
Figure 3
A new peptidomics technique reveals the proteolytic processing pathway and enzyme responsible for inactivating CGRP. A) The technique involved three main steps. Initially, peptidomics is used to identify the biologically relevant fragments of a given bioactive peptide. In step 2, this crucial information about the proteolytic processing pathway is used to design an assay used for purifying the enzyme responsible for this cleaving activity. Proteomics is used to identify candidate enzymes in the most active purified fractions, and these are then recombinantly expressed to identify the enzyme(s) with the desired, biologically relevant, cleaving activity(B). In the final step, the identified enzyme is tested for its ability to regulate levels of the bioactive peptide in vivo(C). Here, the enzyme is either chemically inhibited or genetically knocked out, and levels of the intact bioactive peptide species compared between samples, the expectation being that in the sample without enzyme activity, the bioactive peptide will be degraded less and so be present at higher levels. In the case of CGRP, where IDE had been identified as the candidate enzyme capable of performing both of the two biologically relevant cleavages, it was in fact observed that CGRP levels were significantly higher in the IDE knockout mouse. Additionally, capsaicin, which leads to CGRP release into the bloodstream, was administered to the mice to test IDEs ability to regulate CGRP under different physiological conditions and it was found that IDE still regulated CGRP levels under these conditions, with significantly higher levels of CGRP observed in the IDE knockout mice than in their wildtype littermates. Using this novel approach, an entirely new model for CGRP regulation has been established, with IDE as a key regulator of this peptide’s levels in vivo(D).
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