Potato Carboxypeptidase Inhibitor Research Articles

A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders.

Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely Momordica charantia 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of de novo binders to drug targets of therapeutic relevance.

Structure of Aedes aegypti carboxypeptidase B1-inhibitor complex uncover the disparity between mosquito and non-mosquito insect carboxypeptidase inhibition mechanism.

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and β8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki =14.7nM) is marginally less than that of bovine CPA1 (Ki =5nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.

Abstract 3240: Designer peptides targeting epidermal growth factor as novel anti-breast cancer agents

Abstract Triple negative breast cancer (TBNC) is the only major type of breast cancer for which no specific FDA approved target therapy is available to improve patient outcomes. TNBC lacks the usual breast cancer targets estrogen receptor (ER), progesterone receptor (PR) and HER2/neu receptor. Studies have shown that TBNC does express the epidermal growth factor receptor and has recently emerged as one of the potential targets in TBNC. Epidermal growth factor (EGF) mediates several cellular functions and processes such as survival, motility, differentiation, proliferation and death. When it is released by epidermal tissues, EGF binds to cell surface receptors such as the Epidermal growth factor receptor (EGFR) causing a conformational change which facilitates its dimerization. This receptor dimerization triggers its tyrosine kinase-mediated signal transduction activity. Apart from EGF, EGFR is reported to bind to Transforming growth factor alpha (TGF-α), Argos and Potato Carboxypeptidase inhibitor (PCI). All of these proteins have unique structural motifs which targets EGFR. PCI’s structure consisting of three disulfide bridges presents a unique knot like binding motif resembling EGF and is able to compete with EGF for its binding site on EGFR preventing receptor dimerization thereby antagonizing its action. We have used an integrated computational drug discovery approach to generate several PCI derived peptidic ligands targeting EGFR protein-protein interactions. PCI- peptides bind to EGFR in silico and block EGFR-mediated cell growth, proliferation and migration in vitro. We believe these designer peptidic inhibitors will provide structural and mechanistic clues towards novel therapeutics to combat TNBC. Citation Format: Emmanuel Yawson, Angel Garcia, Husamuldeen Dhari, Rajendram V. Rajnarayanan. Designer peptides targeting epidermal growth factor as novel anti-breast cancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2017-3240

Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes

Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2–4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.

Ion mobility and Top-down MS complementary approaches for the structural analysis of protein models bound to anticancer metallodrugs

Interactions between proteins and platinum (Pt) compounds lie at the heart of both the effectiveness of Pt-based drugs and their side effects. A greater understanding of such interactions at the molecular level would contribute to the design of Pt compounds for future clinical use. In this regard, here we used ion mobility mass spectrometry (IM-MS) to study the structural behavior of three dicarboxylate platinum compounds, namely [Ptcbdca(dach)] (trans-1,2-cyclohexanediamine)(1,1-cyclobutanedicarbo-xylato)platinum(II), [Pt(4-hmpy)2mdbs]bis[(4-pyridinemethanol)(meso-2,3-dibromosuccinato)]platinum(II) and [Ptdcch(4-hmpy)2]bis-[(4-pyridinemethanol)(trans-1,2-cyclo-hexanedicarboxylato)]platinum(II), upon binding to model proteins involved in carcinogenic or anti-carcinogenic processes (Ubiquitin, Cytochrome C and potato carboxypeptidase inhibitor (PCI)). We found that the three Pt compounds bound Ubiquitin, PCI and Cytochrome C, highlighting the formation of biadducts and triadducts with the latter. Using a competitive assay with glutathione, we also explored the potential transfer of Pt to biological nucleophiles and particularly to DNA in protein-containing methionine-Pt adducts. HPLC-MS [ESI(+)] studies demonstrated the capacity of glutathione to scavenge the Pt attached to the protein moiety. Furthermore, using targeted Top-down mass spectrometry, we identified N-terminal Met1 and C-terminal His68 residues as the preferential binding of the dicarboxylate platinum compounds sites on Ubiquitin.

Pivotal role of mast cell carboxypeptidase A in mediating protection against small intestinal ischemia–reperfusion injury in rats after ischemic preconditioning

Aim of the studyMast cell (MC) degranulation contributes to the protection mediated by ischemic preconditioning (IPC); however, the precise mechanisms underlying this protection remain largely unknown. Mast cell carboxypeptidase A (MC-CPA) is released solely from MCs and plays a critical role in degrading toxins and endothelin 1 (ET-1). The present study sought to explore whether MC-CPA is involved in the process of IPC in a rodent model of small intestinal ischemia reperfusion (IIR) injury. Materials and methodsIIR injuries were induced in Sprague–Dawley rats by clamping the superior mesenteric artery for 60 min followed by reperfusion for 2 h. One cycle of 10 min intestinal ischemia and 10 min of reperfusion was used in the IPC group, and the MC stabilizer cromolyn sodium and MC potato carboxypeptidase inhibitor were administered before the start of IPC. At the end of experiment, intestine tissue was obtained for assays of the MC-CPA3, tumor necrosis factor-α, interleukin-6, and ET-1 contents and myeloperoxidase activities. Intestinal histologic injury scores and MC degranulation were assessed. Apoptosis indices and cleaved caspase- 3 protein expressions were quantified. ResultsIIR resulted in severe injury, as evidenced by significant increases in injury scores and MC-CPA3, tumor necrosis factor-α, interleukin-6, and ET-1 contents that were accompanied with concomitant elevations in cleaved caspase 3 expression, apoptosis indices, and myeloperoxidase activities. IPC induced a significant increase in MC-CPA3, induced MC degranulation, and attenuated IIR injury by downregulating IIR-induced biochemical changes, whereas cromolyn sodium and potato carboxypeptidase inhibitor abolished the IPC-mediated changes. ConclusionsThese data suggest that IPC protected against IIR injury via the MC degranulation-mediated release of MC-CPA.

Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

Influence of PPh3 moiety in the anticancer activity of new organometallic ruthenium complexes

The effect of the PPh3 group in the antitumor activity of some new organometallic ruthenium(II) complexes has been investigated. Several complexes of the type [Ru(II)(Cl)(PPh3)(Lig-N)], [Ru(II)(Cl)2(Lig-N)] (where Lig-N=pyridine derivate) and [Ru(II)(Cl)(PPh3)2], have been synthesized and characterized. A noticeable increment of the antitumor activity and cytotoxicity of the complexes due to the presence of PPh3 moiety has also been demonstrated, affording IC50 values of 5.2μM in HL-60 tumor cell lines. Atomic force microscopy, circular dichroism and electrophoresis experiments have proved that these complexes can bind DNA resulting in a distortion of both secondary and tertiary structures. Ethidium bromide displacement fluorescence spectroscopy studies and viscosity measurements support that the presence of PPh3 group induces intercalation interactions with DNA. Indeed, crystallographic analysis, suggest that intra-molecular π–π interactions could be involved in the intercalation within DNA base pairs. Furthermore, high performance liquid chromatography mass spectrometry (HPLC–MS) studies have confirmed a strong interaction between ruthenium complexes and proteins (ubiquitin and potato carboxypeptidase inhibitor — PCI) including slower kinetics due to the presence of PPh3 moiety, which could have an important role in detoxification mechanism and others. Finally, ion mobility mass spectrometry (IMMS) experiments have proved that there is no significant change in the gas phase structural conformation of the proteins owing to their bonding to ruthenium complexes.

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C.suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Detection and characterisation of a new metallocarboxypeptidase inhibitor from Solanum tuberosum cv. Desirèe using proteomic techniques

Abstract Protease inhibitors have been considered only as long as anti-nutritional factors, but have regained much interest in recent years because of their potential anti-cancer action and also because of its positive dietary effects. A new potato carboxypeptidase inhibitor (PCI) was isolated using proteomics techniques; the inhibitor was detected in an extract of Solanum tuberosum cv. Desiree by intensity fading MALDI-TOF mass spectrometry, and then was purified and characterised. The mass spectral from tryptic digestion (peptide mass fingerprint) was analysed with the “MASCOT search tool” and did not match any of the inhibitors of other plants. Protein identification and differentiation by peptide mass fingerprinting (PMF) has been adopted in our group as an excellent tool to differentiate, in fast an unequivocal way, proteins with very similar physicochemical and functional properties. The new PCI exhibited a molecular mass of 4218 Da and consists of a single polypeptide chain with an isoelectric point of 6.5. MALDI-TOF/TOF sequence analysis allowed determines a sequence belonging to the inhibitor.

Thrombomodulin-dependent effect of factor V Leiden mutation on the cross-linking of α2-plasmin inhibitor to fibrin and its consequences on fibrinolysis

IntroductionIt has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FVLeiden) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FVLeiden carriers. MethodsIn the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FVLeiden coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α2-PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method. ResultsrhTM significantly delayed the formation of α2-PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FVLeiden. rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FVLeiden. When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α2-PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FVLeiden. ConclusionFVLeiden abrogates the delaying effect of rhTM on α2-PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FVLeiden carriers.

Molecular dynamics and free energy studies on the carboxypeptidases complexed with peptide/small molecular inhibitor: Mechanism for drug resistance

As one potent plant protease inhibitor, potato carboxypeptidase inhibitor (PCI) can competitively inhibit insect digestive metallocarboxypeptidases (MCPs) through interfering with its digestive system that causes amino acid deficiencies and leading to serious developmental delay and mortality. However, this effective biological pest control is significantly impaired by the PCI-resistant insect MCPs. Therefore, deep understanding of the resistant mechanism of insect MCPs is particularly necessary for designing new durable pest control regimen and developing effective pesticides. In this study, the binding of PCI and small molecular inhibitor THI to insect PCI-sensitive/-resistant MCPs and human MCP was investigated by docking, molecular dynamics (MD) simulations and thermodynamic analysis. The structural analysis from MD simulations indicates that the PCI-resistant mechanism of CPBHz is mainly dominated by the Trp277A, which changes the conformation of β8-α9 loop and therefore narrow the access to the active site of CPBHz, prohibiting the entrance of the C termini tail of PCI. Additionally, the insertion of Gly247A weakens the stabilization of CPBHz and PCI through disrupting the hydrogen bond formation with its surrounding residues. Furthermore, the predicted binding free energies gives explanation of structure affinity relationship of PCI and THI with MCPs and suggest that the electrostatic energy is the main contribution term affecting the difference in binding affinities. Finally, the decomposition analysis of binding free energies infers that the key residues Glu72, Arg127, Ile247/Leu247 and Glu270 are critical for the binding of PCI/THI to MCPs.

Effect of fibrinolysis on bleeding phenotype in moderate and severe von Willebrand disease

Patients with von Willebrand disease (VWD), the most common inherited bleeding disorder, display large variation in bleeding tendency, which is not completely related to VWF levels. The cause of variability in clinical expression is largely unknown. The effect of plasma fibrinolytic capacity on bleeding tendency in VWD patients has not been investigated. We hypothesized that enhanced fibrinolysis may result in a more severe bleeding phenotype. Therefore, we measured the fibrinolytic potential in patients with moderate or severe VWD to investigate the contribution of fibrinolysis to the bleeding tendency. Fibrinolytic potential was measured as plasma clot lysis time (CLT) with and without addition of potato carboxypeptidase inhibitor (PCI) in 638 patients with moderate or severe VWD who participated in a nationwide multicentre cross-sectional study. Bleeding severity was measured using the Bleeding Score (BS).The CLTs were significantly longer, indicative of hypofibrinolysis, in men compared to women with VWD [106.2 (IQR 95.7-118.1) vs. 101.9 (IQR 92.8-114.0) min]. The CLTs prolonged with increasing age. No association was found between VWF or FVIII levels and CLT, or between VWF or FVIII levels and CLT(+PCI) . No association was observed for BS in a model with 10log-transformed CLT, adjusted for age, gender, VWF:Act and FVIII [b = 6.5 (95%CI -0.3 to 13.4)]. Our study showed that the plasma fibrinolytic potential does not influence bleeding tendency in VWD patients and therefore does not explain the variability in bleeding phenotype in VWD.

Contribution of exopeptidases to formation of nonprotein nitrogen during ensiling of alfalfa

The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main exopeptidases contributing to the formation of NH3-N. Among the 5 exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role in the formation of NH3-N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.

Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests

Environmental impacts and resistance to insecticides pose serious challenges to stored-product insect and other types of pest control. Insect-resistant transgenic grain is a potential alternative to fumigants, but candidate control proteins are needed, especially for coleopterans. Therefore, we evaluated the efficacy of a coleopteran-active toxin, Bacillus thuringiensis Cry3Aa, with or without protease inhibitors, in laboratory feeding assays against coleopteran storage pests. In a comparison of the toxicity of Cry3Aa protoxin towards three species of coleopteran storage pests, Tenebrio molitor L. was found to be most sensitive, Tribolium castaneum (Herbst.) was most refractory and Rhyzopertha dominica F. displayed an intermediate response. For R. dominica, Cry3Aa combined with 3500 mg potato carboxypeptidase inhibitor or 5000 mg aprotinin kg(-1) diet resulted in both delayed development and increased mortality. Potato carboxypeptidase inhibitor and bovine aprotinin reduced the LC(50) of Cry3Aa for R. dominica two- and threefold respectively. Cry3Aa treatment resulted in fewer progeny from R. dominica, and progeny was further reduced when the protoxin was combined with potato carboxypeptidase inhibitor. These data support the hypothesis that a combination of Cry3Aa protoxin and protease inhibitors, particularly a potato carboxypeptidase inhibitor, may have applications in control strategies for preventing damage to stored products and grains by coleopteran pests.

Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h −1, 1.4 g PCI L −1 were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h −1, and 2.0-fold compared to μ = 0.25 h −1. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.

Inactivation of the complement anaphylatoxin C5a by secreted products of parasitic nematodes

Given the importance of the complement anaphylatoxins in cellular recruitment during infection, the ability of secreted products from larval stages of Brugia malayi and Trichinella spiralis to influence C5a-mediated chemotaxis of human peripheral blood granulocytes in vitro was examined. Secreted products from B. malayi microfilariae almost completely abolished chemotaxis. This inhibition was blocked by phenylmethylsulphonyl fluoride, indicating the presence of a serine protease, which was subsequently shown to cleave C5a. In contrast, secreted products from T. spiralis infective larvae showed modest inhibition of C5a-mediated granulocyte chemotaxis, and this was blocked by potato carboxypeptidase inhibitor, an inhibitor of several metallocarboxypeptidases. Adult and larval stages of both parasites were demonstrated to secrete carboxypeptidases which cleaved hippuryl-l-lysine and hippuryl-l-arginine, and the T. spiralis enzyme was partially characterised. The data are discussed with reference to inflammation in parasitic nematode infection.

A novel metallocarboxypeptidase‐like enzyme from the marine annelid Sabellastarte magnifica– a step into the invertebrate world of proteases

After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N-terminal and internal sequence homologies with M14 metallocarboxypeptidase-like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca2+ and is drastically inhibited by the metal chelator 1,10-phenanthroline, as well as by excess Zn2+ or Cu2+, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase-A synthetic substrates, such as those with hydrophobic residues at the C-terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O-like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic-like subfamily) with a wider specificity that has not been described previously.