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PubMed2002

Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase.

Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria. The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes. Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP). Some precursors undergo additional processing steps carried out by specialized processing peptidases. For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form. We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription-translation. We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.

Animals↗
PubMed2005

(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine: a potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

A novel series of beta-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC(50) = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.

Administration, Oral↗
PubMed2006

Discovery, structure-activity relationship, and pharmacological evaluation of (5-substituted-pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidines as potent dipeptidyl peptidase IV inhibitors.

A series of (5-substituted pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidine (C5-Pro-Pro) analogues was discovered as dipeptidyl peptidase IV (DPPIV) inhibitors as a potential treatment of diabetes and obesity. X-ray crystallography data show that these inhibitors bind to the catalytic site of DPPIV with the cyano group forming a covalent bond with the serine residue of DPPIV. The C5-substituents make various interactions with the enzyme and affect potency, chemical stability, selectivity, and PK properties of the inhibitors. Optimized analogues are extremely potent with subnanomolar K(i)'s, are chemically stable, show very little potency decrease in the presence of plasma, and exhibit more than 1,000-fold selectivity against related peptidases. The best compounds also possess good PK and are efficacious in lowering blood glucose in an oral glucose tolerance test in ZDF rats.

Animals↗
PubMed2006

cis-2,5-dicyanopyrrolidine inhibitors of dipeptidyl peptidase IV: synthesis and in vitro, in vivo, and X-ray crystallographic characterization.

Inhibitors of the glucagon-like peptide-1 (GLP-1) degrading enzyme dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes in animal models and in human subjects. A novel series of cis-2,5-dicyanopyrrolidine alpha-amino amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. 1-({[1-(Hydroxymethyl)cyclopentyl]amino}acetyl)pyrrolidine-2,5-cis-dicarbonitrile (1c) is an achiral, slow-binding (time-dependent) inhibitor of DPP-IV that is selective for DPP-IV over other DPP isozymes and proline specific serine proteases, and which has oral bioavailability in preclinical species and in vivo efficacy in animal models. The mode of binding of the cis-2,5-dicyanopyrrolidine moiety was determined by X-ray crystallography. The hydrochloride salt of 1c was further profiled for development as a potential new treatment for type 2 diabetes.

Adenosine Deaminase↗
PubMed1998

Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26.

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.

Animals↗
PubMed2003

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog.

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.

Binding Sites↗
PubMed2002

Membrane-associated dipeptidyl peptidase IV is involved in encystation-specific gene expression during Giardia differentiation.

Giardia is a flagellated protozoan that resides in the upper small intestine of its vertebrate host and is the most common cause of defined waterborne diarrhoea worldwide. Giardia trophozoites undergo significant biological changes to survive outside the host by differentiating into infective cysts. Encystation is thus essential for transmission of the parasite among susceptible hosts. In the present study, we report that bestatin, a competitive inhibitor of aminopeptidases, blocks cyst formation in vitro by abolishing the expression of encystation-specific genes, such as those coding for cyst wall proteins. Bestatin does not affect proliferating trophozoites, indicating that its effect is encystation-specific. Using biochemical and molecular biological approaches, we identified the enzyme inhibited by bestatin and cloned its corresponding gene. Sequence similarity indicated that this enzyme belongs to a family of dipeptidyl peptidases. Our results suggest that a specific proteolytic event caused by a constitutively expressed membrane-associated dipeptidyl peptidase IV is necessary for encystation of Giardia.

Amino Acid Sequence↗
PubMed2004

Lower activities of serum peptidases predict higher depressive and anxiety levels following interferon-alpha-based immunotherapy in patients with hepatitis C.

OBJECTIVE: There is evidence that in patients with chronic hepatitis C, immunotherapy with interferon-alpha (IFN alpha) may induce depression. A lowered activity of peptidases, such as prolylendopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV), occurs in depression. This study examines whether lowered serum PEP or DPP IV activity before starting IFN alpha-based immunotherapy predicts the increase in depressive symptoms during immunotherapy. METHOD: Serum PEP and DPP IV activities are measured in patients with hepatitis C before and 2, 4 and 16 weeks after starting IFN alpha-based immunotherapy. The Montgomery Asberg Depression Rating Scale (MADRS) and the Hamilton Anxiety Rating Scale (HAM-A) are completed. RESULTS: Patients with lower baseline PEP or DPP IV had significantly higher MADRS and HAM-A scores both at baseline and during immunotherapy. Patients with lower baseline DPP IV had significantly higher increases in the MADRS following IFN alpha treatment. CONCLUSION: Lower baseline PEP and DPP IV predict higher depressive and anxiety ratings during IFN alpha-based immunotherapy.

Adult↗
PubMed1999

Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted beta propeller domain.

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.

Adenosine Deaminase↗
PubMed2000

Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity.

The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure-function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half-life of these mutants is reduced to < 10% compared with wild-type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell-surface expression, dimerization and the existence of a 150-kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.

Amino Acid Substitution↗
PubMed2003

Mice lacking dipeptidyl peptidase IV are protected against obesity and insulin resistance.

Dipeptidyl peptidase IV (DP-IV), a member of the prolyl oligopeptidase family of peptidases, is involved in the metabolic inactivation of a glucose-dependent insulinotropic hormone, glucagon-like peptide 1 (GLP-1), and other incretin hormones. Here, we investigated the impact of DP-IV deficiency on body weight control and insulin sensitivity in mice. Whereas WT mice displayed accelerated weight gain and hyperinsulinemia when fed a high-fat diet (HFD), mice lacking the gene encoding DP-IV (DP-IV-/-) are refractory to the development of obesity and hyperinsulinemia. Pair-feeding and indirect calorimetry studies indicate that reduced food intake and increased energy expenditure accounted for the resistance to HFD-induced obesity in the DP-IV-/- mice. Ablation of DP-IV also is associated with elevated GLP-1 levels and improved metabolic control in these animals, resulting in improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin-induced loss of beta cell mass and hyperglycemia. Together, these observations suggest that chronic deletion of DP-IV gene has significant impact on body weight control and energy homeostasis, providing validation of DP-IV inhibition as a viable therapeutic option for the treatment of metabolic disorders related to diabetes and obesity.

Animals↗
PubMed1995

Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats.

To analyze mechanisms of liver repopulation, we transplanted normal hepatocytes into syngeneic rats deficient in dipeptidyl peptidase IV activity. When isolated hepatocytes were injected into splenic pulp, cells promptly migrated into hepatic sinusoids. To examine whether transplanted hepatocytes entered liver plates and integrated with host hepatocytes, we analyzed sharing of hepatocyte-specific gap junctions and bile canaliculi. Colocalization studies showed gap junctions uniting adjacent transplanted and host hepatocytes in liver plates. Visualization of bile canalicular domains in transplanted and host hepatocytes with dipeptidyl peptidase IV and ATPase activities, respectively, demonstrated hybrid bile canaliculi, which excreted a fluorescent conjugated bile acid analogue. These results indicate that transplanted hepatocytes swiftly overcome mechanical barriers in hepatic sinusoids to enter liver plates and join host cells. Integration into liver parenchyma should physiologically regulate the function or disposition of transplanted hepatocytes and benefit applications such as gene therapy.

Adenosine Triphosphatases↗
PubMed1998

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics.

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-psi[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.

Crystallography, X-Ray↗
PubMed1998

Activation of a matrix processing peptidase from the crystalline cytochrome bc1 complex of bovine heart mitochondria.

No mitochondrial processing peptidase (MPP) activity is detected in crystalline bovine heart mitochondrial cytochrome bc1 complex, which possesses full electron transfer activity. However, when the complex is treated with increasing concentrations of Triton X-100 at 37 degreesC, the electron transfer activity decreases, whereas peptidase activity increases. Maximum MPP activity is obtained when the electron transfer activity in the complex is completely inactivated with 1.5 mM of Triton X-100. This result supports our suggestion that the lack of MPP activity in the mammalian cytochrome bc1 complex is because of binding of an inhibitor polypeptide to the active site of MPP located at the interface of core subunits I and II. This suggestion is based on the three-dimensional structural information for the bc1 complex and the sequence homology between subunits of MPP and the core subunits of the beef complex. Triton X-100, at concentrations that disrupt the structural integrity of the bc1 complex as indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide to the active site of MPP in core subunits, thus activating MPP. The Triton X-100-activated MPP is pH-, buffer system-, ionic strength-, and temperature-dependent. Maximum activity is observed with an assay mixture containing 15 mM Tris-HCl buffer at neutral pH (6.5-8.5) and at 37 degreesC. Activated MPP is completely inhibited by metal ion chelators such as EDTA and o-phenanthroline and partially inhibited by myxothiazol (58%), ferricyanide (28%), and dithiothreitol (81%). The metal ion chelator-inhibited activity can be partially restored by the addition of divalent cations such as Zn2+ (68%), Mg2+ (44%), Mn2+ (54%), Co2+ (62%), and Fe2+ (92%), indicating that metal ion is required for MPP activity. The cleavage site specificity of activated MPP depends more on the length of amino acid sequence from the mature protein portion and less on the presequence portion, when a synthetic peptide composed of NH2-terminal residues of a mature protein and the COOH-terminal residues of its presequence is used as a substrate.

Amino Acid Sequence↗
PubMed1998

Role of alpha-subunit of mitochondrial processing peptidase in substrate recognition.

Mitochondrial processing peptidase is a heterodimer consisting of alpha-mitochondrial processing peptidase (alpha-MPP) and beta-MPP. We investigated the role of alpha-MPP in substrate recognition using a recombinant yeast MPP. Disruption of amino acid residues between 10 and 129 of the alpha-MPP did not essentially impair binding activity with beta-MPP and processing activity, whereas truncation of the C-terminal 41 amino acids led to a significant loss of binding and processing activity. Several acidic amino acids in the region conserved among the enzymes from various species were mutated to asparagine or glutamine, and effects on processing of the precursors were analyzed. Glu353 is required for processing of malate dehydrogenase, aspartate aminotransferase, and adrenodoxin precursors. Glu377 and Asp378 are needed only for the processing of aspartate aminotransferase and adrenodoxin precursors, both of which have a longer extension peptide than the others studied. However, processing of the yeast alpha-MPP precursor, which has a short extension peptide of nine amino acids, was not affected by these mutations. Thus, effects of substitution of acidic amino acids on the processing differed with the precursor protein and depended on length of the extension peptides. alpha-MPP may function as a substrate-recognizing subunit by interacting mainly with basic amino acids at a region distal to the cleavage site in precursors with a longer extension peptide.

Amino Acid Sequence↗
PubMed1999

Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase.

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.

Amino Acid Sequence↗
PubMed2001

The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis.

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O.H-N:Inh and Gln71:N-H.OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k(cat) value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k(cat)/K(m)) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.

Amino Acid Sequence↗
PubMed2001

Association of Na(+)-H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule.

In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.

Amino Acid Sequence↗