Peptidomics of Prolyl Endopeptidase in the Central Nervous System

Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Biochemistry (Impact Factor: 3.38). 11/2009; 48(50):11971-81. DOI: 10.1021/bi901637c
Source: PubMed

ABSTRACT Prolyl endopeptidase (Prep) is a member of the prolyl peptidase family and is of interest because of 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-beta4, 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.

1 Bookmark
  • [Show abstract] [Hide abstract]
    ABSTRACT: Genome sequencing efforts have revealed a strikingly large number of uncharacterized genes, including poorly or uncharacterized metabolic enzymes, metabolites, and metabolic networks that operate in normal physiology, and those enzymes and pathways that may be rewired under pathological conditions. Although deciphering the functions of the uncharacterized metabolic genome is a challenging prospect, it also presents an opportunity for identifying novel metabolic nodes that may be important in disease therapy. In this review, we will discuss the chemoproteomic and metabolomic platforms used in identifying, characterizing, and targeting nodal metabolic pathways important in physiology and disease, describing an integrated workflow for functional mapping of metabolic enzymes.
    Chemistry & Biology 09/2014; 21(9):1171-1184. DOI:10.1016/j.chembiol.2014.07.007 · 6.59 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Several investigative prolyl oligopeptidase (PREP) inhibitors have been shown to improve learning and memory in various preclinical trials but the mechanism of action behind these effects remains unclear. Since hippocampal and cortical acetylcholine (ACh) is known to play an important role in cognitive processes, the effects of two potent PREP inhibitors, JTP-4819 and KYP-2047, on extracellular ACh levels in hippocampus and medial prefrontal cortex were assessed using in vivo microdialysis. Conscious rats were treated with a single dose (15 or 50μmol/kg i.p.) of JTP-4819, KYP-2047 or vehicle, and extracellular ACh levels were monitored for 5hours after treatment. In hippocampus, KYP-2047 had no significant effect on the ACh levels, although a trend towards decreased levels was observed at the higher dose. JTP-4819 had no significant effect on the hippocampal ACh levels at the lower dose (15μmol/kg), but the higher dose (50μmol/kg) significantly decreased ACh levels in hippocampus by about 25%. In cortex, the smaller dose (15μmol/kg) of KYP-2047 decreased ACh levels maximally by 25%, and a similar (ns) effect was also observed after the higher dose. JTP-4819 had no effect at the lower dose, but the higher dose decreased ACh levels maximally by about 30%. In conclusion, the present results suggest that the cognition-enhancing effects of investigative PREP inhibitors are not due to enhanced cholinergic transmission in hippocampus or cortex.
    Neuroscience Letters 07/2014; DOI:10.1016/j.neulet.2014.07.026 · 2.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.
    PLoS ONE 07/2014; 9(7):e103604. DOI:10.1371/journal.pone.0103604 · 3.53 Impact Factor


1 Download
Available from

Whitney Nolte