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PubMed2005

Identification and characterization of the promoter for the gene encoding human tripeptidyl-peptidase II.

Tripeptidyl-peptidase II (TPP II) is a ubiquitously expressed exopeptidase. The expression of this enzyme is increased, e.g. in some tumor cells, but the regulation of the expression of the gene has not been investigated previously. The gene encoding human TPP II (TPP2) is 82 kb and consists of 30 exons. An 8 kb NcoI fragment covering the 5'-flanking region of the TPP2 gene, including the initiation codon, was cloned into a luciferase-containing reporter vector. Human embryonic kidney cells (HEK-293 cells) and murine fibroblasts (NIH3T3 cells) were transiently transfected with the construct. Through sequential deletions and analysis of short PCR-fragments, the promoter could be localized to a 215 bp fragment upstream of the initiation codon. This region is GC-rich, lacks a TATA-box and contains two inverted CCAAT-boxes and a GC-box. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoter fragment. The 85 bp 5'-end of the promoter fragment is essential for transcriptional activation. Out of this a 44 bp fragment suffices to compete with binding of nuclear proteins to the 215 bp fragment. Supershift assays demonstrated that the CCAAT-binding factor (CBF; NF-Y) is involved in the formation of a complex with the 215 bp fragment. Although Sp1 binds to the promoter fragment in vitro, it was found to bind to the 3'-end of the 215 bp fragment which is not essential for transcription. The potential role of Sp1 in transcription of TPP2 therefore remains to be established.

Aminopeptidases↗
PubMed2005

Inhibitor-based validation of a homology model of the active-site of tripeptidyl peptidase II.

A homology model of the active site region of tripeptidyl peptidase II (TPP II) was constructed based on the crystal structures of four subtilisin-like templates. The resulting model was subsequently validated by judging expectations of the model versus observed activities for a broad set of prepared TPP II inhibitors. The structure-activity relationships observed for the prepared TPP II inhibitors correlated nicely with the structural details of the TPP II active site model, supporting the validity of this model and its usefulness for structure-based drug design and pharmacophore searching experiments.

Amino Acid Sequence↗
PubMed1997

Structure-function studies of recombinant murine tripeptidyl-peptidase II: the extra domain which is subject to alternative splicing is involved in complex formation.

Tripeptidyl-peptidase II (TPP II) is an exopeptidase with a remarkably high native Mr (> 10(6)). Recently, an alternatively spliced, murine cDNA variant was identified which contains an additional 39 bp, encoding 13 amino acids in the C-terminal end of the protein. The two enzyme variants were expressed in human kidney 293 cells. Both types of subunit were found to form the active oligomers. In addition, subunits containing the extra 13 amino acids formed an even larger complex eluting in the void volume of a Sepharose CL-4B column. Thus, it appears that this sequence is important for aggregation of subunits.

Alternative Splicing↗
PubMed1991

Histochemical and biochemical studies of dipeptidyl peptidase I (DPP I) in laboratory rodents.

Different from other proteases the halide- and thiol-dependent lysosomal dipeptidyl peptidase I (DPP I, cathepsin C, EC 3.4.14.1.) was not thoroughly analysed up to now. Therefore, using complementary methods of catalytic histochemistry and biochemistry, DPP I was studied in many rat and mouse organs and compared with DPP II, another lysosomal protease, whose biochemical and histochemical behaviour is known. Continuous (kinetic) fluorometric measurements revealed Gly-Arg naphthylamine (NA) as the substrate with the highest rate of hydrolysis in which, however, also microsomal alanyl aminopeptidase may participate. A more specific substrate appeared to be Pro-Arg-NA; Gly-Phe-NA, and Ser-Tyr-NA were inferior substrates. The Km values were 0.1 mmol/l and 0.2 mmol/l for Gly-Arg-NA and Pro-Arg-NA. The optimal substrate concentration was between 1 and 2.5 mmol/l and the optimal pH value between 5 less than or equal to pH less than or equal to 5.5. The highest reaction velocity was measured with cacodylate or phosphate buffer. Chloride ions and thiol reagents increased the rate of hydrolysis, but an absolute chloride or thiol dependence was not found. Formaldehyde or glutaraldehyde inhibited DPP I depending on the type and concentration of aldehyde. The DPP I activities in rat and mouse organs varied species-dependently and were highest in the extraorbital gland, liver, jejunum, and kidney. In many of the organs, DPP I activity differed considerably from that of DPP II. For catalytic DPP I histochemistry and localization of the enzyme in lysosomes and secretion granules freeze-dried celloidin-mounted cryosections incubated with Gly-Arg-4-methoxy-2-naphthylamine in the presence of Fast Garnet GBC or Fast Blue B were the method of choice in comparison with other tissue pretreatments, Pro-Arg-MNA as substrate and other coupling agents. Use of thiol reagents and chloride ions did not improve the histochemical results. Using this method DPP I was visualized in many cells of rat and mouse organs where its existence was not yet known before. At many sites, DPP I and DPP II distribution patterns differed considerably. In conclusion, a histochemical chromogenic method is now available, which allows the reliable detection of DPP I as was already possible for DPP II using an MNA substrate.

Animals↗
PubMed2003

Tumor cytosol dipeptidyl peptidase III activity is increased with histological aggressiveness of ovarian primary carcinomas.

OBJECTIVE: Proteolytic enzymes have been implicated in the progression of various human malignancies, including ovarian cancer. The enhanced expression of dipeptidyl peptidase III (DPP III) was found in endometrial carcinomas of various histological types and grade. The aim of this study was to assess activity of DPP III in ovarian tissue specimens and to correlate it with clinico-pathological data. METHODS: DPP III hydrolytic activity toward Arg-Arg-2-naphthylamide was determined in 108 ovarian tissue cytosol specimens of 79 patients. The data obtained for 41 ovarian primary carcinoma specimens were stratified according to clinical stage, histological grade and type, and age of the patients. RESULTS: Median DPP III activity expressed as milliunits per milligram protein was 6 in normal ovarian tissues (n = 29), 6.5 in benign ovarian tumors (n = 19), 19.5 in primary ovarian carcinomas (n = 41), 12.5 in nonepithelial primary ovarian tumors (n = 7), and 22.1 in metastatic ovarian malignancies (n = 12). A significant rise in median DPP III specific activity was observed in malignant ovarian tumors (of epithelial, nonepithelial, and metastatic origin), but not in benign ovarian tumors, compared to the activity in normal tissue. A significant difference of DPP III expression was found between the group of normal tissues and tumors of clinical stage I and II, of grade 2 and 3, of serous and mucinous histologic type. CONCLUSIONS: DPP III activity of benign ovarian tumors equaled that in normal ovarian tissue. In malignant neoplasms of the ovary it increased with growing histologic grade.

Adult↗
PubMed2006

The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes.

Glucagon-like peptide 1 (GLP-1) is a gut-derived incretin hormone that stimulates insulin and suppresses glucagon secretion, inhibits gastric emptying, and reduces appetite and food intake. Therapeutic approaches for enhancing incretin action include degradation-resistant GLP-1 receptor agonists (incretin mimetics), and inhibitors of dipeptidyl peptidase-4 (DPP-4) activity (incretin enhancers). Clinical trials with the incretin mimetic exenatide (two injections per day or long-acting release form once weekly) and liraglutide (one injection per day) show reductions in fasting and postprandial glucose concentrations, and haemoglobin A1c (HbA1c) (1-2%), associated with weight loss (2-5 kg). The most common adverse event associated with GLP-1 receptor agonists is mild nausea, which lessens over time. Orally administered DPP-4 inhibitors, such as sitagliptin and vildagliptin, reduce HbA1c by 0.5-1.0%, with few adverse events and no weight gain. These new classes of antidiabetic agents, and incretin mimetics and enhancers, also expand beta-cell mass in preclinical studies. However, long-term clinical studies are needed to determine the benefits of targeting the incretin axis for the treatment of type 2 diabetes.

Adamantane↗
PubMed2003

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides.

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.

Animals↗
PubMed1982

Serum dipeptidyl peptidase (DPP) IV activities in oral cancer patients.

Dipeptidyl peptidase (DPP) IV activities in normal human sera and in the sera of patients with malignant tumors of the oral region were assayed. Patients with oral squamous cell carcinoma had significantly lower enzyme activity than that of normal controls (p less than 0.001). Patients with advanced carcinoma showed a more significant reduction of serum enzyme activity. On the other hand, in oral squamous cell carcinoma patients with hepatic dysfunction, the serum enzyme activity was significantly increased (p less than 0.02).

Adult↗
PubMed2000

Molecular cloning and immunohistochemical localization of rat dipeptidyl peptidase III.

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS, and 82000 on non-denatured PAGE and 82000 on SDS-PAGE in the absence or presence of beta-ME. These findings suggest that the enzyme assumes a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Ala-Arg-MCA in a pH range of 7. 5 to 9.5. The K(in), K(cat) and K(cat)/K(m) values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 6.21x10(4) s(-1)M(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 3.62x10(4) s(-1)M(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS, NEM, beta-ME and iodoacetamide. Furthermore, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG, and we determined the cDNA structure and deduced the amino acid sequence. The cDNA designated as lambdaRDIII-11 is composed of 2640 bp of nucleotides in length and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH in structure, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. These findings suggest that DPP III is a metalloprotease that is probably regulated by SH modification. The DPP III antigen was extensively detected in the cytosol of various rat tissues by the immunohistochemical examination of the protein.

Amino Acid Sequence↗
PubMed1998

Dipeptidyl peptidase III in malignant and non-malignant gynaecological tissue.

Exopeptidases, in contrast to endopeptidases (proteinases) have been much less studied in relation to cancer. The aim of this study was to investigate one such enzyme, dipeptidyl peptidase III (DPP III), in gynaecological tissues, by measuring both the enzyme activity and enzyme content. DPP III activity was assessed in normal (n = 65), benign (n = 9) and malignant (n = 51) gynaecological tissues. A statistically significant higher DPP III activity was observed in endometrial (n = 40, P = 4.6 x 10(-7)) and ovarian (n = 11, P = 8.1 x 10(-4)) malignant tumours, whereas no significant difference was detected for leiomyomas (n = 8), if compared to the activity in normal tissue. A matched pair analysis of normal and cancerous endometrial tissue confirmed the significance of the DPP III activity increase in the transformed tissue (n = 7, P = 0.022). Western blot analysis revealed a significantly (P = 0.014) increased level of DPP III in endometrial cancer. Further, regression analysis showed a positive correlation between the activity and the content of DPP III in normal tissue (r = 0.637, P = 0.047) and in endometrial cancer (r = 0.574, P < 0.007). The increase of the DPP III activity was observed in the endometrial carcinomas of various histological types, grade or the depth of myometrial invasion. The easy-to-perform determination of this exopeptidase activity may serve as a potential indicator of endometrial and ovarian malignancies.

Adolescent↗
PubMed2004

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III.

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance. The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme's residual activity with fluorimetric detection. Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523+/-567M(-1)min(-1). Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540M(-1)s(-1) for slower reacting sulfhydryl, and 21,855M(-1)s(-1) for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H(2)O(2) inactivated the rat DPP III. Human enzyme was resistant to H(2)O(2) attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues. These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.

Amino Acid Sequence↗
PubMed1999

Dipeptidyl peptidase I and III activities of adult schistosomes.

Soluble extracts of adult Schistosoma japonicum and S. mansoni were examined for the presence of proteolytic activities ascribable to dipeptidyl peptidases (DPPs) at a range of pH from 4 to 11 using synthetic peptidyl substrates diagnostic of DPPs I, II, III and IV. Activity capable of cleaving the DPP I-specific substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec which exhibited a pH optimum of 5.5 was observed in extracts of schistosomes. Female schistosomes exhibited greater DPP I activity than male schistosomes, while female S. japonicum showed substantially more activity than female S. mansoni. The specific activities against H-Gly-Arg-NHMec were 21.5 and 1.9 nmoles NHMec/min/mg protein for female and male S. japonicum and 8.5 and 1.9 nmoles NHMec/min/mg for female and male S. mansoni. The biochemical properties of schistosome DPP I were similar to mammalian DPP I (= cathepsin C) in that schistosome DPP I was only slowly inhibited by the cysteine protease inhibitor trans-epoxysuccinyl-1-leucylamido (4-guanidino)-butane, partly inhibited by the blocked diazomethyl ketones Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2, but enhanced by halide ions. At pH 8.5, activity against the DPP III-specific substrate H-Arg-Arg-NHMec was evident in schistosome extracts, and this activity appeared to be due to a zinc metallo-exopeptidase because it was inhibited by 1,10-phenathroline and by EDTA. DPP II or DPP IV activity was not detected in the schistosome extracts.

Animals↗
PubMed2001

Characterization of the metal-substituted dipeptidyl peptidase III (rat liver).

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.

Animals↗
PubMed1999

The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme.

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.

Amino Acid Sequence↗
PubMed2002

Design, synthesis, and tripeptidyl peptidase II inhibitory activity of a novel series of (S)-2,3-dihydro-2-(4-alkyl-1H-imidazol-2-yl)-1H-indoles.

Butabindide, 1, was previously reported as a potent inhibitor (IC50 = 7 nM) of the serine protease enzyme tripeptidyl peptidase II (TPPII), an endogenous protease that degrades cholecystokinin-8 (CCK-8). We found that 1 has some inherent chemical instability, yielding diketopiperazine 2 fairly readily under mimicked physiological conditions. We therefore prepared imidazoles 3, which are void of 1's inherent instability, and have found that our novel analogues maintained comparable TPPII inhibitory activity (e.g.,for 3c, IC50 = 4 nM) as 1.

Aminopeptidases↗
PubMed2006

Discovery of 2-[4-{{2-(2S,5R)-2-cyano-5-ethynyl-1-pyrrolidinyl]-2-oxoethyl]amino]- 4-methyl-1-piperidinyl]-4-pyridinecarboxylic acid (ABT-279): a very potent, selective, effective, and well-tolerated inhibitor of dipeptidyl peptidase-IV, useful for the treatment of diabetes.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are poised to be the next major drug class for the treatment of type 2 diabetes. Structure-activity studies of substitutions at the C5 position of the 2-cyanopyrrolidide warhead led to the discovery of potent inhibitors of DPP-IV that lack activity against DPP8 and DPP9. Further modification led to an extremely potent (Ki(DPP)(-)(IV) = 1.0 nM) and selective (Ki(DPP8) > 30 microM; Ki(DPP9) > 30 microM) clinical candidate, ABT-279, that is orally available, efficacious, and remarkably safe in preclinical safety studies.

Adenosine Deaminase↗
PubMed1996

Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms.

Boronic Acids↗