Peptide metabolism is a complex process involving many proteins working in concert. Mass spectrometry (MS)-based global peptide profiling of mice lacking dipeptidyl peptidase 4 (DPP4) identified endogenous DPP4 substrates and revealed an unrecognized pathway during proline peptide catabolism that interlinks aminopeptidase and DPP4 activities. Together, these studies elucidate specific aspects of DPP4-regulated metabolism and, more generally, highlight the utility of global peptide profiling for studying peptide metabolism in vivo.

In recent years, the biological sciences have seen a surge in the development of methods, including high-throughput global methods, for the quantitative measurement of biomolecule levels (i.e., RNA, proteins, metabolites) from cells and tissues. Just as important as quantitation of biomolecules has been the creation of approaches that uncover the regulatory and signaling connections between biomolecules. Our specific interest is in understanding peptide metabolism in a physiological setting, and this has led us to develop a multidisciplinary approach that integrates genetics, analytical chemistry, synthetic chemistry, biochemistry, and chemical biology to identify the substrates of peptidases in vivo. To accomplish this we utilize a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics platform to measure changes in the peptidome-all peptides in a cell, tissue, or organism-as a function of peptidase activity. Previous analysis of mice lacking the enzyme dipeptidyl peptidase 4 (DPP4 −/− mice), a biomedically relevant peptidase, using this approach identified a handful of novel endogenous DPP4 substrates. Here, we utilize these substrates and tissues from DPP4 −/− mice to improve the coverage of the peptidomics platform by optimizing the key steps in the workflow, and in doing so, discover a total of 70 renal DPP4 substrates (up from 7 at the beginning of our optimization), a 10-fold improvement in our coverage. The sequences of these DPP4 peptide substrates support a broad role for DPP4 in proline-containing peptide catabolism and strengthen a biochemical model that interlinks aminopeptidase and DPP4 activities. Moreover, the improved peptidome coverage also led to the detection of greater numbers of known bioactive peptides (e.g., peptide hormones) during the analysis of gut samples suggesting additional uses for this optimized workflow. Together these results strengthen our ability to identify endogenous peptide substrates through improved peptidome coverage and demonstrate a broader potential of this peptidomics platform.

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...

A supramolecular assembly containing an isoguanosine pentaplex with both a "protein-binding" face and a "reporter" face has been generated. When phosphocholine is appended to the protein-binding face this supramolecular assembly binds multivalently to the pentameric human C-reactive protein, a biomolecule implicated in inflammation and heart disease.

The toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system, responsible for the early detection of foreign or endogenous threat signals. In the context of autoimmunity, the unintended recognition of self-motifs as foreign promotes initiation or propagation of disease. Overactivation of TLR7 and TLR9 have been implicated as factors contributing to autoimmune disorders such as psoriasis, arthritis, and lupus. In our search for small molecule antagonists of TLR7/9, 7f was identified as possessing excellent on-target potency for human TLR7/9 as well as for TLR8, with selectivity against other representative TLR family members. Good pharmacokinetic properties and a relatively balanced potency against TLR7 and TLR9 in mouse systems (systems which lack functional TLR8) made this an excellent in vivo tool compound, and efficacy from oral dosing in preclinical models of autoimmune disease was demonstrated.