Peptidomics of Prolyl Endopeptidase in the Central Nervous System

Abstract

Prolyl endopeptidase (Prep) is a member of the prolyl peptidase family and is of interest due to its unique biochemistry and connections to cognitive function. Using an unbiased mass spectrometry (MS)-based peptidomics platform, we identified Prep regulated peptides in the central nervous system (CNS) of mice by measuring changes in the peptidome as a function of Prep activity. This approach was validated by the identification of known Prep substrates, such as the neuropeptide substance P and thymosin-β4, the precursor to the bioactive peptide Ac-SDKP. In addition to these known substrates, we also discovered that Prep regulates many additional peptides, including additional bioactive peptides and proline rich peptides (PRPs). Biochemical experiments confirmed that some of these Prep regulated peptides are indeed substrates of the enzyme. Moreover, these experiments also supported the known preference of Prep for shorter peptides, while revealing a previously unknown cleavage site specificity of Prep when processing certain multi-proline containing peptides, including PRPs. The discovery of Prep regulated peptides implicates Prep in new biological pathways and provides insights into the biochemistry of this enzyme.Prolyl endopeptidase (Prep), also commonly referred to as prolyl oligopeptidase (POP) (EC 3.4.21.26), was first identified as an oxytocin cleaving activity in the human uterus (1) and is part of the prolyl peptidase family of enzymes (2,3). Other mammalian members of the prolyl peptidase family include the dipeptidyl peptidases, such as the anti-diabetic target dipeptidyl peptidase 4 (DPP4) (4), and the recently characterized prolyl endopeptidase-like (PrepL) (5), which has been genetically linked to hypotoniacystinuria syndrome (HCS) (6)(7)(8). Prep has been of general interest because of its unique biochemical activity as a proline endopeptidase. Unlike the dipeptidyl peptidases, which are restricted to N-terminal dipeptide cleavage (3,9), Prep proteolysis occurs at internal prolines in a peptide (10)(11)(12). On the basis of the known preference of Prep for cleavage at a proline, many proline-containing bioactive peptides have been tested, and identified, as Prep substrates in vitro (12). These substrates range from the tripeptide, thyrotropin-releasing hormone, to a 31 amino acid peptide, beta endorphin (2,13).Correspondence to: Alan Saghatelian, saghatelian@chemistry.harvard.edu. *To whom correspondence should be addressed. Phone: (617) . saghatelian@chemistry.harvard.edu. Supporting Information. ABPP gels with prolyl peptidases and S17092, Prep activity assay showing heat inactivation, graph with fold changes of peptides at one hour and 4 hours of inhibition, graph of CGRP(1-37) levels after one hour of inhibition, kinetic plots of CGRP analogs with wild type and mutant Prep, table with kinetic parameters of CGRP analogs with wild type and mutant Prep with kcat/Km calculated as second order rate constant, MALDI-MS spectrum of the reaction of unacetylated Hsp12a(2-23) with Prep, circula...

Peptidomics of the Prolyl Peptidases

“…For example, in vitro assays with recombinant enzyme cannot account for protein-protein interactions or spatial localization that can promote or prevent interactions between peptidases and candidate substrates (22). To overcome these challenges, recent studies have begun to look at the direct impact of peptidase activity on the peptides in cells and tissues through the application of mass spectrometry (MS)-based peptidomics (23)(24)(25)(26)(27)(28). In these experiments, changes in the peptide profile as a function of peptidase activity can reveal substrates, products, and pathways regulated by a peptidase in vivo.…”

“…Through liquid chromatography (LC)-MS-based peptidomics experiments, the necessary protocols for the isolation and detection of peptides from tissues have been developed (23,24,26,(29)(30)(31)(32). In such experiments, known endogenous peptides and, importantly, previously unidentified peptides, including posttranslationally modified versions of known peptides, can be measured (29).…”

“…In such experiments, known endogenous peptides and, importantly, previously unidentified peptides, including posttranslationally modified versions of known peptides, can be measured (29). Recently, peptidomics has been applied to identify peptidase-regulated peptides, including substrates, by quantifying changes in the tissue peptidome as a function of peptidase activity (23)(24)(25)27,28,31,32) (Fig. 2).…”

“…2). Since these initial efforts, similar approaches have been used to study other peptidases as well (23)(24)(25)(26)(27)(28)31,32). Fig.…”

“…In the analysis of prolyl peptidases, pharmacology (23,28) and genetics (23,24) have both been employed to inactivate the enzymes. The benefit of genetic knockouts is that they are highly specific, effectively remove all enzyme activity, and can be used to target any peptidase without needing any specific reagents.…”