Cathepsins L and S are not required for activation of dipeptidyl peptidase I (cathepsin C) in mice

The cysteine protease dipeptidyl peptidase I (DPPI) activates granule-associated immune-cell serine proteases. The in vivo activator of DPPI itself is unknown; however, cathepsins L and S are candidates because they activate pro-DPPI in vitro. In this study, we tested whether cathepsins L and S activate pro-DPPI in vivo by characterizing DPPI activity and processing in cells lacking cathepsins L and S. DPPI activity, and the relative size and amounts of DPPI heavy and light chains, were identical in mast cells from wild-type and cathepsin L/S double-null mice. Furthermore, the activity of DPPI-dependent chymase was preserved in tissues of cathepsin L/S double-null mice. These results show that neither cathepsin L nor S is required for activation of DPPI and suggest that one or more additional proteases is responsible.     

The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI-inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 A (1 A=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 A resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme-substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.     

Design and evaluation of inhibitors for dipeptidyl peptidase I (Cathepsin C)

Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.     

Novel semicarbazide-derived inhibitors of human dipeptidyl peptidase I (hDPPI)

Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC 3.4.14.1) is a novel putative drug target for the treatment of inflammatory diseases. Using 1 as a starting point (IC50>10 microM), we have improved potency by more than 500-fold and successfully identified novel inhibitors of DPPI via screening of a one-bead-two-compounds library of semicarbazide derivatives. Selected compounds were shown to inhibit intracellular DPPI in RBL-2H3 cells. These compounds were further characterized for adverse effects on HepG2 cells (cytotoxicity and viability) and their metabolic stability in rat liver microsomes was estimated. One of the most potent inhibitors, 8 (IC50=31+/-3 nM; Ki=45+/-2 nM, competitive inhibition), is selective for DPPI over other cysteine and serine proteases, has a half-life of 24 min in rat liver microsomes, shows approximately 50% inhibition of intracellular DPPI at 20 microM and is noncytotoxic.     

Inhibition of the activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing

Cathepsin C is a cysteine protease required for the activation of several pro-inflammatory serine proteases and, as such, is of interest as a therapeutic target. In cathepsin C-deficient mice and humans, the N-terminal processing and activation of neutrophil elastase, cathepsin G, and proteinase-3 is abolished and is accompanied by a reduction of protein levels. Pharmacologically, the consequence of cathepsin C inhibition on the activation of these serine proteases has not been described, due to the lack of stable and non-toxic inhibitors and the absence of appropriate experimental cell systems. Using novel reversible peptide nitrile inhibitors of cathepsin C, and cell-based assays with U937 and EcoM-G cells, we determined the effects of pharmacological inhibition of cathepsin C on serine protease activity. We show that indirect and complete inhibition of neutrophil elastase, cathepsin G, and proteinase-3 is achievable in intact cells with selective and non-cytotoxic cathepsin C inhibitors, at concentrations approximately 10-fold higher than those required to inhibit purified cathepsin C. The concentration of inhibitor needed to block processing of these three serine proteases was similar, regardless of the cell system used. Importantly, cathepsin C inhibition must be sustained to maintain serine protease inhibition, because removal of the reversible inhibitors resulted in the activation of pro-enzymes in intact cells. These findings demonstrate that near complete inhibition of multiple serine proteases can be achieved with cathepsin C inhibitors and that cathepsin C inhibition represents a viable but challenging approach for the treatment of neutrophil-based inflammatory diseases.     

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.     

Potent peptide agonists for human melanocortin 3 and 4 receptors derived from enzymatic cleavages of human beta-MSH(5-22) by dipeptidyl peptidase I and dipeptidyl peptidase IV

Human beta-MSH(1-22) was first isolated from human pituitary as a 22-amino acid (aa) peptide derived from a precursor protein, pro-opiomelanocortin (POMC). However, Bertagna et al. demonstrated that a shorter human beta-MSH(5-22), (DEGPYRMEHFRWGSPPKD), is a true endogenous peptide produced in human hypothalamus. In this report, we demonstrated that in vitro enzymatic cleavage of native human beta-MSH(5-22) with two ubiquitous dipeptidyl peptidases (DPP), DPP-I and DPP-IV, generated two potent MC3/4R peptide analogues, beta-MSH(7-22) (GPYRMEHFRWGSPPKD) and beta-MSH(9-22) (YRMEHFRWGSPPKD). In fact, the MC4R binding affinity and functional potency of beta-MSH(7-22) (Ki=4.6 nM, EC50=0.6 nM) and beta-MSH(9-22) (Ki=5.7 nM, EC50=0.6 nM) are almost an order of magnitude greater than those of their parent peptide, beta-MSH(5-22) (MC4R, Ki=23 nM, EC50= 3nM). Furthermore, the DPP-I/DPP-IV cleaved peptide, beta-MSH(9-22), when administered intracerebroventricularly (ICV) at a dose of 3 nmol/rat, potently induced an acute negative energy balance in a diet-induced obese rat model, while its parent molecule, beta-MSH(5-22), administered at the same dose did not have any effect. These data suggest that DPP-I and DPP-IV may play a role in converting the endogenous beta-MSH(5-22) to more potent peptides that regulate energy homeostasis in the hypothalamus.     

Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii

The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.     

The specificity of dipeptidyl aminopeptidase I (cathepsin C) and its use in peptide sequence studies

1. The characterization of cathepsin C as an aminodipeptidase has been confirmed. 2. An extra limitation on its specificity, namely that peptides involving proline as the third residue are immune to the enzyme, has been found. 3. A novel technique of using the enzyme for amino acid sequence determination has been shown to be feasible.     

Functional expression of dipeptidyl peptidase I (Cathepsin C) of the oriental blood fluke Schistosoma japonicum in Trichoplusia ni insect cells

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel-nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS-PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH(2)-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues -180 to -175, NH(2)-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH(2)-terminal sequence, LDXNQLY, corresponding to residues -73 to -67 of the proregion peptide and thus were generated by removal of 126 residues from the NH(2)-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37 degrees C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k(cat)/K(m)) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM(-)1 s(-1), respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) High Five cells.     

Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes.

In the present report, we demonstrated that modulation of CD26 from T cell surface induced by antiCD26 (1F7) led to enhanced phosphorylation of CD3 zeta tyrosine residues and increased CD4 associated p56lck tyrosine kinase activity. We further showed that CD26 was comodulated on the T cell surface with CD45, a known membrane-linked protein tyrosine phosphatase and that anti-CD26 was capable of precipitating CD45 from T cell lysates. These findings strongly suggest that CD26 may be closely associated with the CD45 protein tyrosine phosphatase on T cell surface and further support the notion that the interaction of CD26 with CD45 results in enhanced tyrosine kinase activity, zeta chain phosphorylation, and T cell activation.     

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.     

Structure of human protein kinase C eta (PKCeta) C2 domain and identification of phosphorylation sites

Protein kinase C eta (PKCeta) is one of several PKC isoforms found in humans. It is a novel PKC isoform in that it is activated by diacylglycerol and anionic phospholipids but not calcium. The crystal structure of the PKCeta-C2 domain, which is thought to mediate anionic phospholipid sensing in the protein, was determined at 1.75 A resolution. The structure is similar to that of the PKC epsilon C2 domain but with significant variations at the putative lipid-binding site. Two serine residues within PKC eta were identified in vitro as potential autophosphorylation sites. In the unphosphorylated structure both serines line the putative lipid-binding site and may therefore play a role in the lipid-regulation of the kinase.     

Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I (cathepsin C) and the elimination of their activities from preparations used to sequence peptides

The best preparations of dipeptidyl aminopeptidase I (DAP I) from beef spleen and rat liver were found to contain a carboxypeptidase (“catheptic carboxypeptidase C”) and a dipeptidase (“Ser-Met dipeptidase”). Each had a pH optimum near 5.5, a resistance to sulfhydryl inhibitors, and a lysosomal origin. The carboxypeptidase, which was inhibited by diisopropylphosphorofluoridate (DFP), preferentially cleaved COOH-terminal residues adjacent to proline, as in angiotensin II and Z-Pro-Phe. No action was detected on Z-Pro-Phe-NH₂. The dipeptidase, which was separable by electrofocusing, was most active on Ser-Met, and showed no action on Z-Ser-Met, Ser-Met-NH₂, Ser-Met-Glu, Gly-Gly or Gly-Leu. Ser-Met dipeptidase was unaffected by DFP, but was strongly inhibited by EDTA. A metal requirement was not apparent, however. A simplified method is described for preparing DAP I as a sequencing reagent free of these contaminating activities.     

Development of a robust QSAR model to predict the affinity of pyrrolidine analogs for dipeptidyl peptidase IV (DPP- IV)

QSAR analysis using multiple linear regression and partial least squares methods were conducted on a data set of 47 pyrrolidine analogs acting as DPP IV inhibitors. The QSAR models generated (both MLR and PLS) were robust with statistically significant s, F, r, r(2) and r(2) (CV) values. The analysis helped to ascertain the role of shape flexibility index, Ipso atom E-state index and electrostatic parameters like dipole moment, in determining the activity of DPP IV inhibitors. In addition to QSAR modeling, Lipinski's rule of five was also employed to check the pharmacokinetic profile of DPP IV inhibitors. Since none of the compounds violated the Lipinski's rule of five indicating that the DPP IV inhibitors reported herein have sound pharmacokinetic profile and can be considered as potential drug candidates for diabetes mellitus Type II.     

Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption.

We screened a human osteoclast (OCL) cDNA expression library for OCL inhibitory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and parathyroid hormone-related protein (PTHrP) in mouse and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly isolated marrow plasma from normal donors and conditioned media from human marrow cultures. Furthermore, treatment of human marrow cultures with an antibody to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH)(2)D(3). Implantation in nude mice of 293 cells transfected with the legumain cDNA and constitutively expressing high levels of the protein significantly reduced hypercalcemia induced by PTHrP by about 50%, and significantly inhibited the increase in OCL surface and in OCL number expressed per mm(2) bone area and per mm bone surface induced by PTHrP. These results suggest that legumain may be a physiologic local regulator of OCL activity that can negatively modulate OCL formation and activity.