Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola / Produção de protease e caracterização molecular de um gene de protease - DPAP (dipeptidil-aminopeptidase) de diferentes cepas de Sordaria fimicola

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

A pesquisa atual foi projetada para alcançar o potencial de produção de protease extracelular em diferentes cepas de Sordaria fimicola que foram previamente obtidas do Dr. Lamb (Imperial College, Londres) de North Facing Slope e South Facing Slope de Evolution Canyon. Após a triagem inicial e secundária, duas cepas hiperprodutoras S2 e N6 foram selecionadas para fermentação submersa e condições culturais, incluindo temperatura, pH, período de incubação, tamanho do inóculo, concentração de substrato, e diferentes fontes de carbono e nitrogênio foram otimizadas para produção de enzima. A cepa S2 apresentou produção máxima de protease de 3,291 U/mL após 14 dias de incubação a 30 °C com pH 7, concentração de substrato de 1% e inóculo de 1 mL, enquanto a cepa N6 apresentou produção máxima de protease de 1,929 U/mL em condições otimizadas de fermentação. Outro objetivo da presente pesquisa foi sustentar a biodiversidade da genética e modificações pós-tradicionais (PTMs) da protease DPAP (peptidil-aminopeptidase) em Sordaria fimicola. Cinco sítios polimórficos foram observados na sequência de aminoácidos de cepas de S. fimicola com referência a Neurospora crassa. A previsão de PTMs a partir de ferramentas de bioinformática previu 38 locais de fosforilação em resíduos de serina para protease peptidil-aminopeptidase na cepa S1 de S. fimicola, enquanto 45 locais de fosforilação em serina na cepa N7 e 47 modificações de fosforilação de serina foram previstas em N. crassa. A pesquisa atual deu uma ideia de que a mudança na composição genética afetou os PTMs que, em última análise, afetaram a produção da enzima protease em diferentes cepas do mesmo organismo (S. fimicola). A produção e os dados moleculares da pesquisa revelaram que o estresse ambiental tem fortes efeitos sobre genes específicos por meio de mutações que podem causar diversidade genética. S. fimicola é um fungo não patogênico e tem um ciclo de vida curto. Esse fungo pode ser escolhido para produzir enzima protease em escala comercial.

A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Crosslinking of membrane CD13 in human neutrophils mediates phagocytosis and production of reactive oxygen species, neutrophil extracellular traps and proinflammatory cytokines.

Aminopeptidase N, or CD13, is a cell membrane ectopeptidase highly expressed in myeloid cells. Through its enzymatic activity, CD13 regulates the activity of several bioactive peptides, such as endorphins and enkephalins, chemotactic peptides like MCP-1 and IL-8, angiotensin III, bradikinin, etc. In recent years, it has been appreciated that independently of its peptidase activity, CD13 can activate signal transduction pathways and mediate effector functions such as phagocytosis and cytokine secretion in monocytes and macrophages. Although neutrophils are known to express CD13 on its membrane, it is currently unknown if CD13 can mediate effector functions in these cells. Here, we show that in human neutrophils CD13 can mediate phagocytosis, which is dependent on a signaling pathway that involves Syk, and PI3-K. Phagocytosis mediated by CD13 is associated with production of reactive oxygen species (ROS). The level of phagocytosis and ROS production mediated by CD13 are similar to those through FcγRIII (CD16b), a widely studied receptor of human neutrophils. Also, CD13 ligation induces the release of neutrophil extracellular traps (NETs) as well as cytokine secretion from neutrophils. These results support the hypothesis that CD13 is a membrane receptor able to activate effector functions in human neutrophils.

Overexpression of Plasmodium falciparum M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development.

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in P. falciparum.

KBE009: A Bestatin-Like Inhibitor of the Trypanosoma cruzi Acidic M17 Aminopeptidase with In Vitro Anti-Trypanosomal Activity.

Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, is a human tropical illness mainly present in Latin America. The therapies available against this disease are far from ideal. Proteases from pathogenic protozoan have been considered as good drug target candidates. T. cruzi acidic M17 leucyl-aminopeptidase (TcLAP) mediates the major parasite's leucyl-aminopeptidase activity and is expressed in all parasite stages. Here, we report the inhibition of TcLAP (IC50 = 66.0 ± 13.5 µM) by the bestatin-like peptidomimetic KBE009. This molecule also inhibited the proliferation of T. cruzi epimastigotes in vitro (EC50 = 28.1 ± 1.9 µM) and showed selectivity for the parasite over human dermal fibroblasts (selectivity index: 4.9). Further insight into the specific effect of KBE009 on T. cruzi was provided by docking simulation using the crystal structure of TcLAP and a modeled human orthologous, hLAP3. The TcLAP-KBE009 complex is more stable than its hLAP3 counterpart. KBE009 adopted a better geometrical shape to fit into the active site of TcLAP than that of hLAP3. The drug-likeness and lead-likeness in silico parameters of KBE009 are satisfactory. Altogether, our results provide an initial insight into KBE009 as a promising starting point compound for the rational design of drugs through further optimization.

Bacitracin is a non-competitive inhibitor of porcine M1 family neutral and glutamyl aminopeptidases.

Membrane alanyl and glutamyl aminopeptidases (APN and APA, respectively) are established targets for the development of biomedical tools in human pathologies. APN overexpression correlates with the progression of tumours, including melanoma. Bacitracin, widely used as a topical antibiotic, inhibits subtilisin-like serine peptidases and disulphide isomerases. In the present contribution, we demonstrate that bacitracin is a non-competitive α = 1 and α < 1 inhibitor of porcine kidney APN and APA, respectively, with Ki values in the micromolar range. To test a potential application of this result, we assayed the effect of bacitracin on murine melanoma MB16F10 cell line viability. We demonstrated the cell line expresses an APN-like activity inhibited by bacitracin and bestatin. Additionally, we identified a cytotoxic effect of bacitracin. Further experiments are required to understand in depth the mechanisms of action of bacitracin on melanoma cells. They will clarify the therapeutic potential of bacitracin for melanoma treatment.

Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline.

Metallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate's neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.

Angiotensin-Converting Enzyme Inhibitor Protects Against Cisplatin Nephrotoxicity by Modulating Kinin B1 Receptor Expression and Aminopeptidase P Activity in Mice.

Cisplatin is a highly effective chemotherapeutic agent. However, its use is limited by nephrotoxicity. Enalapril is an angiotensin I-converting enzyme inhibitor used for the treatment of hypertension, mainly through the reduction of angiotensin II formation, but also through the increase of kinins half-life. Kinin B1 receptor is associated with inflammation and migration of immune cells into the injured tissue. We have previously shown that the deletion or blockage of kinin B1 and B2 receptors can attenuate cisplatin nephrotoxicity. In this study, we tested enalapril treatment as a tool to prevent cisplatin nephrotoxicity. Male C57Bl/6 mice were divided into 3 groups: control group; cisplatin (20 mg/kg i.p) group; and enalapril (1.5 mg;kg i.p) + cisplatin group. The animals were treated with a single dose of cisplatin and euthanized after 96 h. Enalapril was able to attenuate cisplatin-induced increase in creatinine and urea, and to reduce tubular injury and upregulation of apoptosis-related genes, as well as inflammatory cytokines in circulation and kidney. The upregulation of B1 receptor was blocked in enalapril + cisplatin group. Carboxypeptidase M expression, which generates B1 receptor agonists, is blunted by cisplatin + enalapril treatment. The activity of aminopeptidase P, a secondary key enzyme able to degrade kinins, is restored by enalapril treatment. These findings were confirmed in mouse renal epithelial tubular cells, in which enalaprilat (5 µM) was capable of decreasing tubular injury and inflammatory markers. We treated mouse renal epithelial tubular cells with cisplatin (100 µM), cisplatin+enalaprilat and cisplatin+enalaprilat+apstatin (10 µM). The results showed that cisplatin alone decreases cell viability, cisplatin plus enalaprilat is able to restore cell viability, and cisplatin plus enalaprilat and apstatin decreases cell viability. In the present study, we demonstrated that enalapril prevents cisplatin nephrotoxicity mainly by preventing the upregulation of B1 receptor and carboxypeptidase M and the increased concentrations of kinin peptides through aminopeptidase activity restoration.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

Inhibitors of tumor necrosis factor synthesis as a new approach for the treatment of rheumatoid arthritis

Objective: The treatment of rheumatoid arthritis (RA) is based on the inhibition of TNF. Here we evaluated whether drugs that might inhibit TNF, such as pentoxifylline (PTX), rupatadine (RUP), rolipram (ROL) and thalidomide (THA), could be an alternative for RA treatment. Methods: In wistar male rats the changes in paw thickness, plasma TNF and by the activity of basic aminopeptidase (APB) in soluble fraction of synovial tissue and peripheral blood mononuclear cells (PBMC) evaluated after daily injection for 30 days were taken as anti-inflammatory outputs, while hepatotoxicity was assessed by measuring plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity. The content of IL1-β, IL-6 in serum and synovial fluid and the histology of the injured tissue were determined only for ROL, THA and ROL+THA. Prednisolone was used as a standard drug. Results: Collagen treatment induced paw thickness, histological changes in the tibiotarsal joint, increase in synovial fluid of both cytokines and synovial tissue of APB activity. Furthermore, the APB activity in PBMC was reduced and ALT and AST activity were enhanced. The most effective drug schedule in reducing arthritis induced changes described above, as well as recovering from control levels TNF, IL1-β, APB in synovial tissue and AST activities were THA and the association of ROL and THA. However, only THA alone reduced the levels of ALT. Conclusion: The synthesis of TNF in RA models can be blocked by drugs acting at different targets. We show that THA and THA+ROL emerges as simple and effective therapeutic alternatives for RA.

Inhibition of Porcine Aminopeptidase M (pAMP) by the Pentapeptide Microginins.

Aminopeptidase M (AMP) inhibition is of interest for several diseases, such as highly vascularized cancer types. AMP can be inhibited by linear pentapeptides isolated from Microcystis aeruginosa LTPNA08 (MG7XX). Porcine AMP inhibition-a model for human AMP-activity was spectrophotometrically measured by the formation of p-nitroanilide from L-leucine-p-nitroanilide substrate by AMP. AMP inhibition by MG770 exhibited comparable inhibition levels to amastatin (IC50 values: 1.20 ± 0.1 µM and 0.98 ± 0.1 µM, respectively), while MG756 was slightly less potent (with IC50 values of 3.26 ± 0.5 µM). Molecular modelling suggests a potential binding mode, based on the interaction with the Zn2+ cofactor, where MG770's extra methyl group contributes to the disturbance of the Zn2+ cofactor complex and highlights the importance of hydrophobicity for the site.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV.

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin.

Bacillus thuringiensis Cry1Ca is toxic to different Spodoptera species. The aims of this work were to identify the Cry1Ca-binding proteins in S. frugiperda, to provide evidence on their participation in toxicity, and to identify the Cry1Ca amino acid residues involved in receptor binding. Pulldown assays using Spodoptera frugiperda brush border membrane vesicles (BBMV) identified aminopeptidase N (APN), APN1, and APN2 isoforms as Cry1Ca-binding proteins. Cry1Ca alanine substitutions in all residues of domain III ß16 were characterized. Two ß16 nontoxic mutants (V505A and S506A) showed a correlative defect on binding to the recombinant S. frugiperda APN1 (SfAPN1). Finally, silencing the expression of APN1 transcript, by double-stranded RNA (dsRNA) feeding, showed that silenced larvae are more tolerant of the Cry1Ca toxin, which induced less than 40% mortality in silenced larvae whereas nonsilenced larvae had 100% mortality. Overall, our results show that Cry1Ca relies on APN1 binding through domain III ß16 to impart toxicity to S. frugiperdaIMPORTANCEBacillus thuringiensis Cry toxins rely on receptor binding to exert toxicity. Cry1Ca is toxic to different populations of S. frugiperda, a major corn pest in America. Nevertheless, the S. frugiperda midgut proteins that are involved in Cry1Ca toxicity have not been identified. Here we identified aminopeptidase N1 (APN1) as a functional receptor of Cry1Ca. Moreover, we showed that Cry1Ca domain III ß16 is involved in APN1 binding. These results give insights on potential target sites for improving Cry1Ca toxicity to S. frugiperda.

Trichomonas vaginalis metalloproteinase TvMP50 is a monomeric Aminopeptidase P-like enzyme.

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.

Periplasmic form of dipeptidyl aminopeptidase IV from Pseudoxanthomonas mexicana WO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis.

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2'…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Šresolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84 Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

A chromatographic approach to distinguish Gram-positive from Gram-negative bacteria using exogenous volatile organic compound metabolites.

This paper utilized L-alanine aminopeptidase activity as a useful approach to distinguish between Gram-negative and Gram-positive bacteria. This was done using two enzyme substrates, specifically 2-amino-N-phenylpropanamide and 2-amino-N-(4-methylphenyl)propanamide which liberated the volatile compounds aniline and p-toluidine, respectively. Two complementary analytical techniques have been used to identify and quantify the VOCs, specifically static headspace multicapillary column gas chromatography ion mobility spectrometry (SHS-MCC-GC-IMS) and headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS). Superior limits of detection were obtained using HS-SPME-GC-MS, typically by a factor of x6 such that the LOD for aniline was 0.02µg/mL and 0.01µg/mL for p-toluidine. In addition, it was also possible to determine indole interference-free by HS-SPME-GC-MS at an LOD of 0.01µg/mL. The approach was applied to a range of selected bacteria: 15 Gram-negative and 7 Gram-positive bacteria. Use of pattern recognition, in the form of Principal Component Analysis, confirmed that it is possible to differentiate between Gram-positive and Gram-negative bacteria using the enzyme generated VOCs, aniline and p-toluidine. The exception was Stenotrophomonas maltophilia which showed negligible VOC concentrations for both aniline and p-toluidine, irrespective of the analytical techniques used and hence was not characteristic of the other Gram-negative bacteria investigated. The developed methodology has the potential to be applied for clinical and food applications.