Evolutionary Analysis of Dipeptidyl Peptidase I.

Human dipeptidyl peptidase I (DPPI) belongs to the family of papain-like cysteine peptidases. Its distinctive features are the unique exclusion domain which enables the eponymous activity and homotetramerization of DPPI, and its dependence on chloride ions for enzymatic activity. The oligomeric state of DPPI is unique in this family of predominantly monomeric peptidases. However, a distant DPPI ortholog from Plasmodium falciparum has been shown to be monomeric, indicating that the oligomeric state of DPPI varies between lineages. The aim of this work was to study the evolution of DPPI, with particular attention to the structural features that determine its characteristic enzymatic activity and preferences, and to reconstruct the evolution of its oligomerization. We analyzed fifty-seven selected sequences of DPPI and confirmed its presence in three lineages, namely, Amorphea (including animals and Amoebozoa), Alveolates and the metamonad Giardia. The amino acid residues that bind the chloride ion are highly conserved in all species, indicating that the dependence on chloride ions for activity is an evolutionarily conserved feature of DPPI. The number of N-glycosylation sites is significantly increased in animals, particularly vertebrates. Analysis of homology models and subunit contacts suggests that oligomerization is likely restricted to DPPIs in the Amorphea group.

Exome sequencing identifies a novel missense variant in CTSC causing nonsyndromic aggressive periodontitis.

Cathepsin C (CatC) is a cysteine protease involved in a variety of immune and inflammatory pathways such as activation of cytotoxicity of various immune cells. Homozygous or compound heterozygous variants in the CatC coding gene CTSC cause different conditions that have in common severe periodontitis. Periodontitis may occur as part of Papillon-Lefèvre syndrome (PLS; OMIM#245000) or Haim-Munk syndrome (HMS; OMIM#245010), or may present as an isolated finding named aggressive periodontitis (AP1; OMIM#170650). AP1 generally affects young children and results in destruction of the periodontal support of the primary dentition. In the present study we report exome sequencing of a three generation consanguineous Turkish family with a recessive form of early-onset AP1. We identified a novel homozygous missense variant in exon 2 of CTSC (NM_148170, c.G302C, p.Trp101Ser) predicted to disrupt protein structure and to be disease causing. This is the first described CTSC variant specific to the nonsyndromic AP1 form. Given the broad phenotypic spectrum associated with CTSC variants, reporting this novel variant gives new insights on genotype/phenotype correlations and might improve diagnosis of patients with early-onset AP1.

Interplay between tetrameric structure, enzymatic activity and allosteric regulation of human dipeptidyl-peptidase I.

Human dipeptidyl-peptidase I (DPPI) is a tetrameric enzyme from the family of papain-like cysteine peptidases. It is ubiquitously expressed and plays important roles in general protein turnover, skin homeostasis and proteolytic processing of effector peptidases in immune cells. In this work we investigate allosteric regulation of DPPI and its relation to the oligomeric structure. First, we investigate the functional significance of the tetrameric state by comparing the kinetic properties of the tetrameric form (DPPItet) with a recombinant monomeric form (DPPImono). We find that both forms have very similar kinetic properties for the hydrolysis of a commonly used synthetic substrate. In agreement with previous studies, no cooperativity is observed in the tetramer. The only significant difference between both forms is a higher catalytic rate of DPPImono. We then characterize three compounds, 3'-nitrophthalanilic acid, chlorogenic acid and caffeic acid that affect DPPI activity via kinetic mechanisms consistent with binding outside of the active site. These compounds are the first known modifiers of DPPI that do not act as specific inhibitors. Chlorogenic acid and caffeic acid act as linear mixed and linear catalytic inhibitors, respectively, and do not discriminate between both forms. In contrast, 3'-nitrophthalanilic acid is a hyperbolic inhibitor that binds DPPItet and DPPImono with different affinities and inhibits their activities via different kinetic mechanisms. Altogether, these results show that the tetrameric structure of DPPI is not necessary for enzymatic activity, however, oligomerization-related structural features can play a role in its regulation.

Clinical and molecular analysis in Papillon-Lefèvre syndrome.

Papillon-Lefèvre syndrome (PLS; MIM#245000) is a rare recessive autosomal disorder characterized by palmar and plantar hyperkeratosis, and aggressively progressing periodontitis leading to premature loss of deciduous and permanent teeth. PLS is caused by loss-of-function mutations in the CTSC gene, which encodes cathepsin C. PLS clinical expressivity is highly variable and no consistent genotype-phenotype correlation has been demonstrated yet. Here we report the clinical and genetic features of five PLS patients presenting a severe periodontal breakdown in primary and permanent dentition, hyperkeratosis over palms and soles, and recurrent sinusitis and/or tonsillitis. Mutation analysis revealed two novel homozygous recessive mutations (c.947T>C and c.1010G>C) and one previous described homozygous recessive mutation (c.901G>A), with parents carrying them in heterozygous, in three families (four patients). The fourth family presented with the CTSC c.628C>T mutation in heterozygous, which was inherited maternally. Patient carrying the CTSC c.628C>T mutation featured classical PLS phenotype, but no PLS clinical characteristics were found in his carrier mother. All mutations were found to affect directly (c.901G>A, c.947T>C, and c.1010G>C) or indirectly (c.628C>T, which induces a premature termination) the heavy chain of the cathepsin C, the region responsible for activation of the lysosomal protease. Together, these findings indicate that both homozygous and heterozygous mutations in the cathepsin C heavy chain domain may lead to classical PLS phenotype, suggesting roles for epistasis or gene-environment interactions on determination of PLS phenotypes.

The catalytic domain of cathepsin C (dipeptidyl-peptidase I) alone is a fully functional endoprotease.

Cathepsin C is a tetrameric lysosomal protease that acts as a dipeptidyl-peptidase due to the presence of the exclusion domain that is unique among papain-like cysteine proteases. Here we describe a recombinant form of cathepsin C lacking its exclusion domain (CatCΔEx) produced in a bacterial expression system (E. coli). CatCΔEx is a monomer with endoprotease activity and affinity for hydrophobic residues such as Phe, Leu or Pro, but not Val, in the P2 position. As opposed to cathepsin C, it does not require chloride ions for its activity. Despite lower turnover rates of hydrolysis of synthetic substrates, CatCΔEx has elastolytic and gelatinolytic activity comparable to other cysteine cathepsins.

Characterization of cathepsin C from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

The lysosomal cysteine protease cathepsin C plays a pivotal role in regulation of inflammatory and immune responses. However, the function of fish cathepsin C in virus replication remains largely unknown. In this study, cathepsin C gene (Ec-CC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CC cDNA was composed of 2077 bp. It contained an open reading frame (ORF) of 1374 bp and encoded a 458-amino acid protein which shared 89% identity to cathepsin C from bicolor damselfish (Stegastes partitus). Amino acid alignment analysis showed that Ec-CC contained an N-terminal signal peptide, the propeptide region and the mature peptide. RT-PCR analysis showed that Ec-CC transcript was expressed in all the examined tissues which abundant in spleen and head kidney. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the relative expression of EC-CC was significantly increased at 24 h post-infection. Subcellular localization analysis revealed that Ec-CC was distributed mainly in the cytoplasm. Further studies showed that overexpression of Ec-CC in vitro significantly delayed the cytopathic effect (CPE) progression evoked by SGIV and inhibited the viral genes transcription. Moreover, overexpression of Ec-CC significantly increased the expression of proinflammatory cytokines during SGIV infection. Taken together, our results demonstrated that Ec-CC might play a functional role in SGIV infection by regulating the inflammation response.

CTSC compound heterozygous mutations in two Chinese patients with Papillon-Lefèvre syndrome.

OBJECTIVES: To identify the molecular basis of Papillon-Lefèvre syndrome in two Chinese families. METHODS: Peripheral blood and mouth swab samples were obtained, from which genomic DNA and RNA were isolated. Sanger sequencing was employed to identify the mutations. mRNA expression was tested by real-time quantitative PCR. Evolutionary conservation, pathogenicity prediction and impact of protein structures of the mutations were conducted with bioinformatics tools and homology modelling. HEK293 cells were transfected with plasmids expressing wild-type or mutated CTSC. CTSC protein expression level and enzyme activity were explored. RESULTS: Mutation analysis revealed two novel compound heterozygous mutations, the c.190-191insA and c.1211-1212delA in patient 1 and the c.716A>G and c.757+1G>A in patient 2. In both patients, the levels of CTSC mRNA were significantly lower than in their relatives. Homology modelling analysis predicted that the mutations affect the structure and stability of the protein, and in vitro study showed that the CTSC proteins containing the mutations c.190-191insA and c.1211-1212delA, which result in truncated versions of protein, display impaired enzyme activity. The protein containing c.716A>G mutation showed quite similar enzyme activity compared to wild-type CTSC. CONCLUSION: Our data support the molecular mechanism of PLS and enlarge the scope of CTSC gene mutations related to PLS.

Processing and Maturation of Cathepsin C Zymogen: A Biochemical and Molecular Modeling Analysis.

Cysteine cathepsin C (CatC) is a ubiquitously expressed, lysosomal aminopeptidase involved in the activation of zymogens of immune-cell-associated serine proteinases (elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, lymphocyte granzymes, and mast cell chymases). CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages. A molecular modeling analysis of proCatC indicated that its propeptide displayed a similar fold to those of other lysosomal cysteine cathepsins, and could be involved in dimer formation. Our in vitro experiments revealed that human proCatC was processed and activated by CatF, CatK, and CatV in two consecutive steps of maturation, as reported for CatL and CatS previously. The unique positioning of the propeptide domains in the proCatC dimer complex allows this order of cleavages to be understood. The missense mutation Leu172Pro within the propeptide region associated with the Papillon-Lefèvre and Haim-Munk syndrome altered the proform stability as well as the maturation of the recombinant Leu172Pro proform.

Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples.

Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S' specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO2)-NH2, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

Papillon-Lefevre syndrome in twelve Egyptian patients: Five novel CTSC variants and functional characterization of a missense variant and its effect on splicing.

OBJECTIVES: describing the clinical features of twelve Egyptian patients with Papillon-Lefever syndrome (PLS). Five novel mutations in the cathepsin C (CTSC) gene are introduced and the phenotype of the syndrome is expanded by the identification of new clinical features. DESIGN: the clinical, oro-dental data of twelve Egyptian patients from seven unrelated families are described. Sequence analysis of the CTSC gene was performed to identify the causative mutaions. RESULTS: Typical PLS features were presented in all patints but with variable severity. One patient showed atypical dental features including dental structural defect, minimal periodontitis, severe gingivitis, and delayed closure of root apices. Another patient presented with arachnodactyly, dystrophic nails, and buphthalmos in the right eye secondary to uncontrolled congenital glaucoma. Mutational analysis of CTSC gene revealed seven distinct homozygous variants including five novel ones: c.285_286delGT (p.Leu96GlufsTer2), c .302 G>C (p.Trp101Ser), c.622_628delCACAGTC (p.H208Efs*11), c.1331delinsAAAAA (p.G444Efs*4) and c .1343 G>A (p.Cys448Tyr). The previously reported missense variant c .757 G>A (p.Ala253Thr) was found in one patient. This variant is very close to the splice region and by functional studies, we proved that it results in exon skipping and early protein truncation (p.R214Sfs*46). CONCLUSION: We report five novel CTSC variants and describe rare and unusual associated clinical and dental findings such as dental structural defects, delayed closure of root apices, and congenital glaucoma. Therefore, our results expand both the phenotypic and mutational spectrum of PLS.

The amino-acid substituents of dipeptide substrates of cathepsin C can determine the rate-limiting steps of catalysis.

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k(cat)/K(a) and k(cat) values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k(cat)/K(a) (1.6 ± 0.09 µM(-1) s(-1)) and k(cat) (255 ± 6 s(-1)), an inverse sKIE on k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.6 ± 0.15), a modest, normal sKIE on k(cat) ((D)k(cat) = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s(-1)). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k(ac)) and deacylation (k(dac)) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697-8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k(cat)/K(a) values of 0.49 ± 0.07 µM(-1 )s(-1) and 5.3 ± 0.5 µM(-1 )s(-1), and k(cat) values of 28 ± 1 s(-1) and 25 ± 0.5 s(-1). Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.74 ± 0.05) and normal for k(cat) ((D)k(cat) = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s(-1) and 390 s(-1), respectively), from which it was determined that k(ac) ≫ k(dac) ∼ k(cat). The third substrate type, Gly-Ile-AMC, gave very low values of k(cat)/K(a) (0.0015 ± 0.0001 µM(-1) s(-1)) and k(cat) (0.33 ± 0.02 s(-1)), no sKIEs, ((D)(k(cat)/K(a)) = 1.05 ± 0.5 and (D)k(cat) = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which k(dac) > k(ac) ∼ k(cat), and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P(1) position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases.

Involvement of Fenneropenaeus chinensis Cathepsin C in antiviral immunity.

Cathepsin C (Cath C) is a lysosomal cysteine protease that belongs to the papain superfamily. Cath C is capable of activating many chymotrypsin-like serine proteases and is reported to be a central coordinator for the activation of many serine proteinases in immune and inflammatory cells. In this study, Cath C cDNA was cloned from Fenneropenaeus chinensis (Fc). The complete cDNA of Fc-Cath C in Chinese white shrimp was found to be 1445-base pairs (bp) long. It contained an open reading frame (ORF) 1356-bp long and encoded a 451-amino acid residue protein, including a 17-amino acid residue signal peptide. Real-time PCR analysis results indicated that Fc-Cath C was present in all the tissues detected and exhibited high level of transcription in the hepatopancreas. In hemocytes, hepatopancreas, gills and intestine, Fc-Cath C was upregulated after stimulation by the Vibrio anguillarum and the white spot syndrome viruses (WSSVs). Replication of the WSSV increased after the injection of Fc-Cath C antiserum or knockdown Cath C by RNA interference. These results implied that Cath C might play a crucial role in the antiviral immune response of shrimp.

High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells.

Dipeptidyl peptidase I (DPPI) plays a crucial role in maturation of many regulatory peptides and has been suggested as a pharmaceutical target in several inflammatory diseases. It is also a useful processing enzyme for the generation of authentic protein products by catalyzing the removal of N-terminal fusion peptides. We used a robust transient transfection system in human embryonic kidney 293 cells to exploit expression and activation of DPPI from chicken, rat and man for the development of an industrial production process. The expression of human and rat DPPI was significantly higher in the human HEK293 cell line than that obtained with avian DPPI. A CHO K1SV stable cell line was selected as the optimal stable host system for production of human DPPI yielding expression levels higher than 1.5 g/L. The secreted pro-DPPI underwent auto-maturation during defined buffer conditions during the purification steps. Active human DPPI was purified with a three-step purification strategy employing: Butyl Sepharose 4 Fast Flow, Sephadex G-25 Medium and Q Sepharose Fast Flow chromatography. The final yield of active enzyme was approximately 1 g/L cell culture. The enzyme exhibited exopeptidase activity against both a dipeptide-p-nitroanilide substrate and N-terminally extended MEAE-hGH (Met-Glu-Ala-Glu-human growth hormone). In conclusion, an efficient production process for recombinant human DPPI has been developed including a highly efficient and stable CHO cell system and an efficient purification procedure, which is simple and easy to scale for industrial purposes. The present data facilitates not only industrial applications of DPPI as a processing enzyme, but also provides active enzyme useful in the identification of small molecule inhibitors.

Cathepsin C inhibition as a potential treatment strategy in cancer.

Epidemiological studies established an association between chronic inflammation and higher risk of cancer. Inhibition of proteolytic enzymes represents a potential treatment strategy for cancer and prevention of cancer metastasis. Cathepsin C (CatC) is a highly conserved lysosomal cysteine dipeptidyl aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases (NSPs, elastase, proteinase 3, cathepsin G and NSP-4). NSPs are locally released by activated neutrophils in response to pathogens and non-infectious danger signals. Activated neutrophils also release neutrophil extracellular traps (NETs) that are decorated with several neutrophil proteins, including NSPs. NSPs are not only NETs constituents but also play a role in NET formation and release. Although immune cells harbor large amounts of CatC, additional cell sources for this protease exists. Upregulation of CatC expression was observed in different tissues during carcinogenesis and correlated with metastasis and poor patient survival. Recent mechanistic studies indicated an important interaction of tumor-associated CatC, NSPs, and NETs in cancer development and metastasis and suggested CatC as a therapeutic target in a several cancer types. Cancer cell-derived CatC promotes neutrophil recruitment in the inflammatory tumor microenvironment. Because the clinical consequences of genetic CatC deficiency in humans resulting in the elimination of NSPs are mild, small molecule inhibitors of CatC are assumed as safe drugs to reduce the NSP burden. Brensocatib, a nitrile CatC inhibitor is currently tested in a phase 3 clinical trial as a novel anti-inflammatory therapy for patients with bronchiectasis. However, recently developed CatC inhibitors possibly have protective effects beyond inflammation. In this review, we describe the pathophysiological function of CatC and discuss molecular mechanisms substantiating pharmacological CatC inhibition as a potential strategy for cancer treatment.

Identification and Optimization of Novel Cathepsin C Inhibitors Derived from EGFR Inhibitors.

In the course of developing the biochemistry to chemistry activity-based protein profiling (BTC-ABPP) method, we herein unexpectedly discovered that the epidermal growth factor receptor irreversible inhibitor WZ4002 also functioned as a low micromolar inhibitor of cathepsin C (CatC), a promising target for the treatment of numerous inflammatory and autoimmune diseases. Building on from this discovery, and following structure-activity relationship investigations guided by computational modeling, a novel series of pyridine scaffold compounds were developed as irreversible CatC inhibitors, further culminated in identifying a highly potent and selective inhibitor 22, which displays good metabolic stability and oral bioavailability. In vivo studies revealed that compound 22 clearly displays the ability to inhibit CatC, consequently leading to efficient inhibition of downstream neutrophil serine proteases in both bone marrow and blood. The overall excellent profile of compound 22 made it an interesting candidate for further preclinical investigation.

Chemical mechanism of a cysteine protease, cathepsin C, as revealed by integration of both steady-state and pre-steady-state solvent kinetic isotope effects.

Cathepsin C, or dipeptidyl peptidase I, is a lysosomal cysteine protease of the papain family that catalyzes the sequential removal of dipeptides from the free N-termini of proteins and peptides. Using the dipeptide substrate Ser-Tyr-AMC, cathepsin C was characterized in both steady-state and pre-steady-state kinetic modes. The pH(D) rate profiles for both log k cat/ K m and log k cat conformed to bell-shaped curves for which an inverse solvent kinetic isotope effect (sKIE) of 0.71 +/- 0.14 for (D)( k cat/ K a) and a normal sKIE of 2.76 +/- 0.03 for (D) k cat were obtained. Pre-steady-state kinetics exhibited a single-exponential burst of AMC formation in which the maximal acylation rate ( k ac = 397 +/- 5 s (-1)) was found to be nearly 30-fold greater than the rate-limiting deacylation rate ( k dac = 13.95 +/- 0.013 s (-1)) and turnover number ( k cat = 13.92 +/- 0.001 s (-1)). Analysis of pre-steady-state burst kinetics in D 2O allowed abstraction of a normal sKIE for the acylation half-reaction that was not observed in steady-state kinetics. Since normal sKIEs were obtained for all measurable acylation steps in the presteady state [ (D) k ac = 1.31 +/- 0.04, and the transient kinetic isotope effect at time zero (tKIE (0)) = 2.3 +/- 0.2], the kinetic step(s) contributing to the inverse sKIE of (D)( k cat/ K a) must occur more rapidly than the experimental time frame of the transient kinetics. Results are consistent with a chemical mechanism in which acylation occurs via a two-step process: the thiolate form of Cys-234, which is enriched in D 2O and gives rise to the inverse value of (D)( k cat/ K a), attacks the substrate to form a tetrahedral intermediate that proceeds to form an acyl-enzyme intermediate during a proton transfer step expressing a normal sKIE. The subsequent deacylation half-reaction is rate-limiting, with proton transfers exhibiting normal sKIEs. Through derivation of 12 equations describing all kinetic parameters and sKIEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKIEs allowed the provision of at least one self-consistent set of values for all 13 rate constants in this cysteine protease's chemical mechanism. Simulation of the resulting kinetic profile showed that at steady state approximately 80% of the enzyme exists in an active-site cysteine-acylated form in the mechanistic pathway. The chemical and kinetic details deduced from this work provide a potential roadmap to help steer drug discovery efforts for this and other disease-relevant cysteine proteases.

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.

Molecular cloning and mRNA expression of cathepsin C gene in black tiger shrimp (Penaeus monodon).

Cathepsin C (dipeptidyl-peptidase I, DPPI) is a lysosomal cysteine proteinase belonging to the papain superfamily, which is capable of removing dipeptides sequentially from the amino terminus of peptide and protein substrates. In the present study, the cDNA of a cathepsin C was cloned from black tiger shrimp Penaeus monodon (designated PmcathepsinC) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of PmcathepsinC consisted of 2051 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 1350 bp encoding a polypeptide of 449 amino acid residues with a predicted molecular weight of 50.0 kDa and theoretical isoelectric point of 5.65. The high identity of PmcathepsinC with Cathepsin C in other organisms indicated that PmcathepsinC should be a new member of the Cathepsin C family. By fluorescent quantitative real-time PCR, mRNA transcript of PmcathepsinC was detectable in all the examined tissues with higher level in ovary and heart. The temporal expression of PmcathepsinC mRNA in the hepatopancreas was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at 4 h post-stimulation, and then dropped back to the original level gradually. These results indicated that PmcathepsinC was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of P. monodon.