The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 Å crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated -propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed -propeller with open Velcro topology and the C-terminal ␣͞-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the -propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P 2-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.serine protease ͉ oxyanion hole ͉ substrate channeling ͉ drug design ͉ diabetes mellitus
Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE1-CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.
The incretins glucose-dependent insulinotropic polypeptide (GIP 1-42 ) and glucagon-like peptide-1-(7-36)-amide (GLP-1 7-36 ), hormones that potentiate glucose-induced insulin secretion from the endocrine pancreas, are substrates of the circulating exopeptidase dipeptidyl peptidase IV and are rendered biologically inactive upon cleavage of their N-terminal dipeptides. This study was designed to determine if matrix-assisted laser desorption/ionization-time of flight mass spectrometry is a useful analytical tool to study the hydrolysis of these hormones by dipeptidyl peptidase IV, including kinetic analysis. Spectra indicated that serumincubated peptides were cleaved by this enzyme with only minor secondary degradation due to other serum protease activity. Quantification of the mass spectrometric signals allowed kinetic constants for both porcine kidney-and human serum dipeptidyl peptidase IVcatalyzed incretin hydrolysis to be calculated. The binding constants (K m ) of these incretins to purified porcine kidney-derived enzyme were 1.8 ؎ 0.3 and 3.8 ؎ 0.3 M, whereas the binding constants observed in human serum were 39 ؎ 29 and 13 ؎ 9 M for glucose-dependent-insulinotropic polypeptide and glucagon-like peptide-1-(7-36)-amide respectively. The large range of K m values found in human serum suggests a heterogeneous pool of enzyme. The close correlation between the reported kinetic constants and those previously described validates this novel approach to kinetic analysis.Incretins are hormones of the enteroinsular axis, which potentiate the actions of glucose on the endocrine pancreas (1). The most potent known incretins are glucose-dependent insulinotropic polypeptide (GIP 1 and truncated forms of glucagon-like peptide-1 (GLP-1 7-36 -amide and GLP-1 7-37 ); both are members of the glucagon family of hormones sharing considerable N-terminal sequence homology (2, 3). Both hormones are released from the gut in response to ingested nutrients and were recently shown to be substrates of the circulating exopeptidase dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) (4, 5). This enzyme is a highly specific protease, preferentially hydrolyzing peptides with N-terminal Xaa-Pro and Xaa-Ala motifs (6). Hydrolysis of GIP 1-42 and GLP-1 7-36 by DP IV yields GIP 3-42 and GLP-1 9 -36 and the dipeptides Tyr-Ala and His-Ala, respectively. Activation or inactivation of biologically active peptides is frequently associated with DP IV catalysis. Work by ourselves and others (7,8) has demonstrated that GIP 3-42 and GLP-1 9 -36 are biologically inactive, and it has been hypothesized that serum degradation of GIP 1-42 and GLP-1 7-36 by DP IV is the primary step in the metabolism of these hormones in the circulation (4, 5, 9). In 1993 Mentlein and co-workers (4) reported on the kinetics of enzymatic degradation of GIP 1-42 and GLP-1 7-36 by purified human placental DP IV, as determined by high performance liquid chromatography (HPLC), and suggested that this may be a physiologically important pathway for the degradation of these hormones. This ...
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