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Introduction: Dermatophytes are pathogenic fungi that cause cutaneousinfection of human and animal and grow exclusively on the stratumcorneum, nail and hair. Methods: The present study was conducted on 160 samples from clinicallydiagnosed onycomycosis patients, further subjected to culture from nailsamples of patients attending dermatology OPD of a tertiary care hospital,Delhi from January 2016 to December 2018 Results: In a soy protein culture, a substantial proteolytic activity was seen, which was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55–75% inhibited by O-phenanthroline, which authenticate that metalloproteases were secreted by all three species. A consensus probe was constructed on previously characterized genes, which encodes metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis. From genomic libraries of T. rubrum, T. mentagrophytes and M. canis, T. rubrum & T. Mentagrophytesa five-member MEP family was isolated and also secretes MCP A & MCPB, MEP 3, MEP4, IL, CS, MS AND DPP V of the M14 family accordingto the MEROPS proteolytic enzyme database. Conclusion: This study shows that Metalloprotease, Metallocarboxypeptidase, Isocitrate Lyase, Citrate Synthase, Malate Synthase and Dipeptidylpeptidase V are expressed in Trichophyton Rubrum Isolates from Dermatophytosis Patients.

Skin infections by keratinophilic fungi are commonly referred to as dermatophytosis and represent a major health burden worldwide. Although patient numbers are on the rise, data on virulence factors, their function and kinetics are scarce. We employed an ex vivo infection model based on guinea pig skin explants (GPSE) for the zoonotic dermatophyte Trichophyton (T.) benhamiae to investigate kinetics of the virulence factors subtilisin (sub) 3, sub 6, metallocarboxypeptidase A (mcpA) and isocitrate lyase (isol) at gene level for ten days. Fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to detect and quantify the transcripts, respectively. Kingdom-spanning, species-specific and virulence factor-specific probes were successfully applied to isolated fungal elements showing inhomogeneous fluorescence signals along hyphae. Staining results for inoculated GPSE remained inconsistent despite thorough optimization. qPCR revealed a significant increase of sub 3- and mcpA-transcripts toward the end of culture, sub 6 and isol remained at a low level throughout the entire culture period. Sub 3 is tightly connected to the de novo formation of conidia during culture. Since sub 6 is considered an in vivo disease marker. However, the presented findings urgently call for further research on the role of certain virulence factors during infection and disease.

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.

Background: Carboxypeptidases are exopeptidases that catalyze the release of amino acids from the C-terminus of peptides or proteins. The peptides consisting of hydrophobic amino acids taste bitter. Therefore, the hydrolytic capability of carboxypeptidase toward hydrophobic amino acids at the C-terminus of peptides is useful for the degradation of bitter peptides. Results: Using the genome data of Streptomyces cinnamoneus TH-2, we expressed and characterized a novel metallocarboxypeptidase (TH2-CP) in Streptomyces lividans. TH2-CP had a molecular mass of 37.7 kDa. As TH2-CP possesses a zinc-binding consensus motif (HXXE......H) and N-terminal prosegment residues, we suggest that TH2-CP could be classified into the M14A subfamily. In the presence of Z-Gly-Leu as the substrate, TH2-CP showed optimum activity at pHs 7 and 8 in potassium phosphate and Tris-HCl buffers, respectively. The optimum temperature for activity was 51 °C. Furthermore, 50 % activity was conserved after incubation at 38 °C for 30 min. TH2-CP showed broad substrate specificity, with a preference for hydrophobic amino acids, as demonstrated by casein hydrolysate breakdown. Conclusions: A novel metallocarboxypeptidase, TH2-CP, from S. cinnamoneus TH-2 was characterized. TH2-CP preferred substrates with hydrophobic amino acids at the C-terminal position for casein peptides. This property indicates that TH2-CP can be used to decrease the bitterness of peptides in food industries. [ABSTRACT FROM AUTHOR]

Digestion and absorption of old cuticles during insect molting are necessary for new cuticle formation, during which complicated enzyme catalysis is essential. To date, a few carboxypeptidases, aminopeptidases and serine proteases (mostly trypsins) connected with cuticle digestion, zymogen activation and histological differentiation during the ecdysis of lepidopteran, dipteran and hymenopteran insects have been identified. However, little is known about these proteins in hemimetabolous insects. In this study, we identified 33 candidate trypsin and trypsin-like homologs, 14 metallocarboxypeptidase and 32 aminopeptidase genes in the brown planthopper Nilaparvata lugens , a hemipteran rice pest. Among the proteins encoded by these genes , 9 trypsin-like proteases, 3 metallocarboxypeptidases and 1 aminopeptidase were selected as potential procuticle hydrolases by bioinformatics analysis and in vivo validation. RNA interference targeting these genes demonstrated that 3 trypsin-like proteases (NlTrypsin-8, NlTrypsin-29 and NlTrypsin-32) genes and 1 metallocarboxypeptidase (NlCpB) gene were found to be essential for ecdysis in N. lugens ; specifically, gene silencing led to incomplete cuticle degradation and arrested ecdysis, causing lethal morphological phenotype acquisition. Spatiotemporal expression profiling by quantitative PCR and western blotting revealed their specific expression in the integument and their periodic expression during each stadium, with a peak before ecdysis and eclosion. Transmission electron microscopy demonstrated corresponding ultrastructural defects after RNAi targeting, with NlCpB -silenced specimens having the most undigested old procuticles. Immunohistochemical staining revealed that NlTrypsin-8, NlTrypsin-29 and NlCpB were predominantly located in the exuvial space. This research further adds to our understanding of proteases and its potential role in insect ecdysis. [Display omitted] • 33 trypsin, 14 metallocarboxypeptidase and 32 aminopeptidase genes were identified in the planthopper. • NlTrypsin-8 , NlTrypsin-29 , NlTrypsin-32 and NlCpB are integument-specific and periodic expression during each stadium. • Knockdown of NlTrypsin-8 , NlTrypsin-29 , NlTrypsin-32 and NlCpB caused lethal phenotype during ecdysis and eclosion. • NlTrypsin-8, NlTrypsin-29 and NlCpB located in between the new emerging cuticle and the old procuticle. [ABSTRACT FROM AUTHOR]

[Display omitted] • CpeNeo/Neo mice do not express carboxypeptidase E, an important neuropeptide processing enzyme. • Homozygous CpeNeo/Neo mice have greatly reduced levels of most neuropeptides and are overweight, subfertile, anxious, and depressed. • Heterozygous CpeNeo/+ mice have ∼ 50% reduced expression of CPE mRNA and greatly reduced enzyme activity. • Heterozygous CpeNeo/+ mice are identical to wild-type mice in terms of neuropeptide levels, physiology and behavior, indicating that CPE is not a rate-limiting enzyme. Carboxypeptidase E (CPE) is an essential enzyme that contributes to the biosynthesis of the vast majority of neuropeptides and peptide hormones. There are several reports claiming that small decreases in CPE activity cause physiological changes in animals and/or cultured cells, but these studies did not provide evidence that neuropeptide levels were affected by decreased CPE activity. In the present study, we tested if CPE is a rate-limiting enzyme in neuropeptide production using CpeNeo mice, which contain a neomycin cassette within the Cpe gene that eliminates enzyme expression. Homozygous CpeNeo/Neo mice show defects found in Cpefat/fat and/or Cpe global knockout (KO) mice, including greatly decreased levels of most neuropeptides, severely impaired fertility, depressive-like behavior, adult-onset obesity, and anxiety-like behavior. Removal of the neomycin cassette with Flp recombinase under a germline promoter restored expression of CPE activity and resulted in normal behavioral and physiological properties, including levels of neuropeptides. Mice heterozygous for the CpeNeo allele have greatly reduced levels of Cpe mRNA and CPE-like enzymatic activity. Despite the decreased levels of Cpe expression, heterozygous CpeNeo mice are behaviorally and physiologically identical to wild-type mice, with normal levels of most neuropeptides. These results indicate that CPE is not a rate-limiting enzyme in the production of most neuropeptides, casting doubt upon studies claiming small decreases in CPE activity contribute to obesity or other physiological effects. [ABSTRACT FROM AUTHOR]

Background: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. Results: Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. Conclusions: We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol. [ABSTRACT FROM AUTHOR]

Abstract: The carboxypeptidase gene from Geobacillus SBS-4S was cloned and sequenced. The sequence analysis displayed the gene consists of an open reading frame of 1503 nucleotides encoding a protein of 500 amino acids (CBPSBS). The amino acid sequence comparison revealed that CBPSBS exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBPSBS contained an active site motif 265HEXXH269 which is conserved in family-M32 of carboxypeptidases. The gene was expressed with His-Tag utilizing Escherichia coli expression system and purified to apparent homogeneity. The purified CBPSBS showed highest activity at pH 7.5 and 70°C. The enzyme activity was metal ion dependent. Among metal ions highest activity was found in the presence of Co2+. Thermostability studies of CBPSBS by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77°C. The enzyme exhibited K m and V max values of 14mM and 10526μmol min−1 mg−1 when carbobenzoxy–alanine–arginine was used as substrate. k cat and k cat /K m valves were 10175s−1 and 726mM−1 s−1. To our knowledge this is the highest ever reported enzyme activity of a metallocarboxypeptidase and the first characterization of a metallocarboxypeptidase from genus Geobacillus. [Copyright &y& Elsevier]

Abstract: In what corresponds to a life span, metallocarboxypeptidases (MCPs) have jumped from being mere contaminants in animal pancreas powders (in depression year 1929) to be key players in cellular and molecular processes (in yet-another-depression years 2009–2010). MCPs are unique zinc-dependent enzymes that catalyze the breakdown of the amide bond at the C-terminus of peptide and protein substrates and participate in the recovery of dietary amino acids, tissue organogenesis, neurohormone and cytokine maturation and other important physiological processes. More than 26 genes code for MCPs in the human genome, many of them still waiting to be fully understood in terms of physiological function. A variety of MCPs have been linked to diseases in man: acute pancreatitis and pancreas cancer, type 2 diabetes, Alzheimer’s Disease, various types of cancer, and fibrinolysis and inflammation. Many of these discoveries have been made possible thanks to recent advances, as exemplified by plasma carboxypeptidases N and B, known for fifty and twenty years, respectively, which have had their structures released only very recently. Plasma carboxypeptidase B is a biological target for therapy because of its involvement in the coagulation/fibrinolysis processes. Besides, the widespread use of carboxypeptidase A as a benchmark metalloprotease since the early days of Biochemistry has allowed the identification and design of an increasingly vast repertory of small molecular weight inhibitors. With these two examples we wish to emphasize that MCPs have become part of the drug discovery portfolio of pharmaceutical companies and academic research laboratories. This paper will review key developments in the discovery and design of MCP small molecular weight inhibitors, with an emphasis on the discovery of chemically diverse entities. Although encouraging advances have been achieved in the last few years, the specificity and oral bioavailability of the new chemotherapeutic agents seem to pose a challenge to medicinal chemists. [Copyright &y& Elsevier]

Abstract: Drosophila melanogaster silver gene is the ortholog of the coding gene of mammalian carboxypeptidase D (CPD). The silver gene gives rise to eight different splicing variants of differing length that can contain up to three homologous repeats. Among the protein variants encoded, the short form 1B alias DmCPD1Bs (D . melanogaster CPD variant 1B short) is necessary and sufficient for viability of the fruit fly. It has one single repeat, it is active against standard peptide substrates, and it is localized to the secretory pathway. In this work, the enzyme was found as a monomer in solution and as a homodimer in the crystal structure, which features a protomer with an N-terminal 311-residue catalytic domain of α/β-hydrolase fold and a C-terminal 84-residue all-β transthyretin-like domain. Overall, DmCPD1Bs conforms to the structure of N/E-type funnelins/M14B metallopeptidases, but it has two unique structural elements potentially involved in regulation of its activity: (i) two contiguous surface cysteines that may become palmitoylated and target the enzyme to membranes, thus providing control through localization, and (ii) a surface hot spot targetable by peptidases that would provide a regulatory mechanism through proteolytic inactivation. Given that the fruit fly possesses orthologs of only two out of the five proteolytically competent N/E-type funnelins found in higher vertebrates, DmCPD1Bs may represent a functional analog of at least one of the missing mammalian CPs. [ABSTRACT FROM AUTHOR]

metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0–7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity). [ABSTRACT FROM AUTHOR]

Abstract: A structure-based virtual screening survey was used to identify potential inhibitors of the human M14 family of metallocarboxypeptidases. A good correlation between docking energy scores and measured K i values was observed, indicating an efficient performance of the screening procedure. Among various compounds displaying K i values in the low micromolar range, N-(3-chlorophenyl)-4-((5-(3-methoxybenzylthio)-1,3,4-oxadiazol-2-yl)methyl)thiazol-2-amine emerged as the most powerful inhibitor for human carboxypeptidase B (CPB). According to molecular docking, this compound fits into CPB active site cleft through coordination of the catalytic zinc ion with the 1,3,4-oxadiazole moiety. This represents a novel five-membered heterocyclic type of inhibitor for disease-linked metallocarboxypeptidases and an interesting lead for further development. [Copyright &y& Elsevier]

Abstract: Latexin, the endogenous protein inhibitor of the A/B subfamily of metallocarboxypeptidases, is expressed in small nociceptive neurons in sensory ganglia and in a subset of neurons in the telencephalon. In this study, we generated latexin-deficient mice that exhibited increased tail-flick latency compared to wild-type animals upon noxious heat stimulation. The reduced pain sensitivity in the mutants was rescued by the systemic administration of a plant carboxypeptidase inhibitor that inhibits the A/B subfamily of metallocarboxypeptidases. These findings suggest that latexin is involved in the transmission of pain. [Copyright &y& Elsevier]

Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn ear-worm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A/B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase α/β-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1' substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the β8-α9 loop, and α7 together with the α7-α8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors. [ABSTRACT FROM AUTHOR]

Treatment of advanced stages of prostate carcinoma with histone-deacetylase inhibitors entails expression of human procarboxypeptidase-A4 (hPCPA4). The three-dimensional structure of hPCPA4 has been solved and shows the features of related metallocarboxypeptidase zymogens, with a preformed α/β/-hydrolase active-enzyme moiety (hCPA4) and an inhibiting pro-domain (PD). The protease moiety recalls a sphere, out of which a spherical cone has been cut. This results in a funnel-like structure, at the bottom of which the active-site cleft resides. The border of this funnel is shaped by loops, which are responsible for the interaction with the PD, characterised by a large interface area and relatively few contacts. Such an inhibitory mode is evocative of the recently reported structure of the human inhibitor latexin in its complex with hCPA4. The main contacting structure of latexin is similar to the one employed for PD inhibition. In both cases, active-site blocking relies mainly on a loop provided by the central part of a β sheet. [ABSTRACT FROM AUTHOR]

Abstract: Carboxypeptidase A-6 (CPA6) was recently discovered in the human genome. To gain information regarding the potential function of this novel protein, the mouse homolog of CPA6 was identified using a combination of bioinformatics and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, homologs in rat, chicken, and frog were identified using a bioinformatics approach. The distribution of CPA6 mRNA in mouse tissues was examined using RT-PCR and in situ hybridization. A strong RT-PCR signal is detectable in olfactory bulb, and much lower levels are present in other regions such as the cerebral cortex, hippocampus, hypothalamus, striatum, and medulla. In peripheral tissues, a moderate RT-PCR signal is present in epididymis, and low levels are detectable in colon and spleen. The high level of CPA6 in adult mouse brain olfactory bulb was confirmed by in situ hybridization. Lower levels of CPA6 mRNA were found to be present in the cingulate cortex, lateral septum, pontine nucleus, and inferior olivary nucleus of the hindbrain. Within the olfactory bulb, CPA6 mRNA is enriched in the mitral and granular layer. A lower level of CPA6 mRNA is present in the internal and external plexiform layers, and no signal is detectable in the olfactory nerve layer. The distribution was also examined in whole embryos at embryonic day 14.5 and CPA6 mRNA was found to be enriched in eye, ear, osteoblasts, stomach, skin, dorsal root ganglia, and throughout the CNS. The presence of CPA6 mRNA in the rectus muscle layer of the eye at embryonic day 14.5 is consistent with the observation that the CPA6 gene is disrupted in a patient with Duane syndrome, a congenital eye defect. Taken together, the distribution of CPA6 suggests a specific role in a limited number of tissues, and it is possible that this role involves an aspect of cell migration. [Copyright &y& Elsevier]

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine-specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C-terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a K i in the pm range. [ABSTRACT FROM AUTHOR]

Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this “traditional” pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named “CPA-5,” has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites. [Copyright &y& Elsevier]

Human carboxypeptidase D (CPD) is a 180-kDa type I membrane protein with three tandem active site domains. CPD is a B-type (or kininase I-type) carboxypeptidase that cleaves C-terminal basic residues from proteins and peptides, such as Arg9 from bradykinin. The human carboxypeptidase D (CPD) gene was found to encompass ∼88.3 kb of genomic sequence, containing 21 exons ranging in size from 65 to 1813 bp, and 21 introns ranging in size from 112 bp to 35.6 kb. Although CPD and CPM belong to the same metallocarboxypeptidase subfamily, their intron/exon structures differ significantly. Multiple transcription start sites were found in the CPD gene within a GC-rich sequence lacking the typical TATA box, but containing three GC boxes. Luciferase reporter assays with various size constructs containing the promoter region upstream of the start sites showed that it was active in three different cell lines, especially in the human hepatoma cell line HepG2 and the human monocytic cell line THP-1, which have high constitutive expression of CPD. [Copyright &y& Elsevier]

ProSAAS (encoded by mouse gene Pcsk1n) is a recently described neuroendocrine secretory pathway protein that is cleaved into smaller peptides that may function in cell–cell signalling. ProSAAS and its processing intermediates are also potent inhibitors of prohormone convertase 1 (PC1), which is encoded by mouse gene Pcsk1. In order to gain insight into the function of proSAAS, we have examined the distribution of several proSAAS-derived peptides and PC1 by immunohistochemistry throughout mouse development. The distribution patterns of both SAAS and PC1 are broad from E9 to E11, with some enrichment in neural tube-derived tissues. By E15, the expression of SAAS is largely restricted to neuroendocrine tissues known to produce bioactive peptides. In general, the expression pattern of PC1 overlaps with that of SAAS and other proSAAS-derived peptides, consistent with the hypothesis that proSAAS functions as an endogenous PC1 inhibitor. [Copyright &y& Elsevier]

Carboxypeptidases perform many diverse functions in the body. The well-studied pancreatic enzymes (carboxypeptidases A1, A2 and B) are involved in the digestion of food, whereas a related enzyme (mast-cell carboxypeptidase A) functions in the degradation of other proteins. Several members of the metallocarboxypeptidase gene family (carboxypeptidases D, E, M and N) are more selective enzymes and are thought to play a role in the processing of intercellular peptide messengers. Three other members of the metallocarboxypeptidase gene family do not appear to encode active enzymes; these members have been designated CPX-1, CPX-2 and AEBP1/ACLP. In this review, we focus on the recently discovered carboxypeptidase Z (CPZ). This enzyme removes C-terminal Arg residues from synthetic substrates, as do many of the other members of the gene family. However, CPZ differs from the other enzymes in that CPZ is enriched in the extracellular matrix and is broadly distributed during early embryogenesis. In addition to containing a metallocarboxypeptidase domain, CPZ also contains a Cys-rich domain that has homology to Wnt-binding proteins; Wnts are important signaling molecules during development. Although the exact function of CPZ is not yet known, it is likely that this protein plays a role in development by one of several possible mechanisms. [ABSTRACT FROM AUTHOR]

Crude enzyme extract was obtained from a low-pH soil from a tea field by shaking with 0.1 M PO4 buffer (pH 7.0). Hydrolytic activity toward benzyloxycarbonyl- L-Phe- L-Leu (Z- L-Phe- L-Leu) and Z- L-Phe- L-Tyr- L-Leu showed two pH optima, at about pH 5 and 9, suggesting that the soil contained at least two protease components. The acid-type protease in the extract was assumed to be Ser-carboxypeptidase because phenylmethanesulphonyl fluoride and diisopropylphosphoro fluoridate inhibited its activity. Peptide bonds in the C-terminal residues of Leu-enkephalin and angiotensin I were split more by protease than those in the N-terminal residue. The apparent molecular weight of the acid-type protease was estimated to be 75 kDa by Sephadex G-100 gel filtration and the isoelectric point 4.4 by isoelectric focusing. A neutral-type protease in the extract was assumed to be a metallocarboxypeptidase because only o-phenanthrorine inhibited its activity. Peptide bonds in the C-terminal residues of Leu-enkephalin and angiotensin I were hydrolyzed to a greater extent than those in the N-terminal residues. The apparent molecular weight of the neutral-type protease was estimated to be 37 kDa and the isoelectric point 5.8, 8.0 and 9.4. The isoelectric point 9.4 fraction showed the highest relative activity. [ABSTRACT FROM AUTHOR]3– buffer (pH 7.0). Hydrolytic activity toward benzyloxycarbonyl- L-Phe- L-Leu (Z- L-Phe- L-Leu) and Z- L-Phe- L-Tyr- L-Leu showed two pH optima, at about pH 5 and 9, suggesting that the soil contained at least two protease components. The acid-type protease in the extract was assumed to be Ser-carboxypeptidase because phenylmethanesulphonyl fluoride and diisopropylphosphoro fluoridate inhibited its activity. Peptide bonds in the C-terminal residues of Leu-enkephalin and angiotensin I were split more by protease than those in the N-terminal residue. The apparent molecular weight of the acid-type protease was estimated to be 75 kDa by Sephadex G-100 gel filtration and the isoelectric point 4.4 by isoelectric focusing. A neutral-type protease in the extract was assumed to be a metallocarboxypeptidase because only o-phenanthrorine inhibited its activity. Peptide bonds in the C-terminal residues of Leu-enkephalin and angiotensin I were hydrolyzed to a greater extent than those in the N-terminal residues. The apparent molecular weight of the neutral-type protease was estimated to be 37 kDa and the isoelectric point 5.8, 8.0 and 9.4. The isoelectric point 9.4 fraction showed the highest relative activity. [ABSTRACT FROM AUTHOR]

Skin infections by keratinophilic fungi are commonly referred to as dermatophytosis and represent a major health burden worldwide. Although patient numbers are on the rise, data on virulence factors, their function and kinetics are scarce. We employed an ex vivo infection model based on guinea pig skin explants (GPSE) for the zoonotic dermatophyte Trichophyton (T.) benhamiae to investigate kinetics of the virulence factors subtilisin (sub) 3, sub 6, metallocarboxypeptidase A (mcpA) and isocitrate lyase (isol) at gene level for ten days. Fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to detect and quantify the transcripts, respectively. Kingdom-spanning, species-specific and virulence factor-specific probes were successfully applied to isolated fungal elements showing inhomogeneous fluorescence signals along hyphae. Staining results for inoculated GPSE remained inconsistent despite thorough optimization. qPCR revealed a significant increase of sub 3- and mcpA-transcripts toward the end of culture, sub 6 and isol remained at a low level throughout the entire culture period. Sub 3 is tightly connected to the de novo formation of conidia during culture. Since sub 6 is considered an in vivo disease marker. However, the presented findings urgently call for further research on the role of certain virulence factors during infection and disease. [ABSTRACT FROM AUTHOR]

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications. [ABSTRACT FROM AUTHOR]