Aspartic Proteinase Activity Research Articles

Differential proteolytic activity in Anisakis simplex s.s. and Anisakis pegreffii, two sibling species from the complex Anisakis simplex s.l., major etiological agents of anisakiasis

Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen–host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p < 0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p < 0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although, in L3-0h, the activity of these proteases was significantly higher (p < 0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites. This may be related to the significantly higher activity (p ≤ 0.05) of serine protease in all the larval stages of A. simplex studied at pH 6.0. Thus, there are interspecific differences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

Characterization of the \u03b3-secretase subunit interactome in Arabidopsis thaliana

Gamma secretase is a multi-subunit complex with aspartic intramembrane protease activity that is involved in the pathogenesis of Alzheimer’s disease in humans. In Arabidopsis thaliana, γ-secretase subunits are localized to endomembrane system compartments and interact with each other in a similar manner to their human counterparts. Here, we identified the protein partners of two plant γ-secretase subunits, presenilin 2 and PEN-2, by tandem affinity purification and co-immunoprecipitation, respectively. Integral membrane proteins were found to interact with presenilin 2, whereas secreted proteins were found to interact with PEN-2. Microscopy screening revealed that the reticulon family protein, RTNLB1, and two single transmembrane domain proteins, TIR-X and the phytolongin PHYL1.1, interact with presenilins. Finally, we show that RTNLB1 interacts with TIR-X. These results represent a step toward elucidating the functions of γ-secretase subunits in plant cells.

Imidazolidinones and Imidazolidine-2,4-diones as Antiviral Agents.

Imidazolidinones and imidazolidine-2,4-diones are important classes of heterocyclic compounds that possess potent activities against several viruses such as dengue virus, enterovirus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV). The first imidazolidinone derivative as an anti-HIV agent was reported in 1996. Imidazolidinones inhibit HIV aspartic protease activity, and also act as CCR5 co-receptor antagonists. Significant effort has been devoted to the design of various imidazolidinone analogues that are active against drug-resistant HIV strains, with fewer side effects. Different scaffolds have been designed through both rational drug design strategies and computer-aided drug design. Imidazolidinones have been found to be potent against HIV, and preclinical studies are currently in progress. There are some reports of imidazolidinones as having both anti-HCV and anti-dengue virus activity, and more research has yet to be done along these lines. These compounds inhibit NS3 serine protease of HCV, and NS2B-NS3 protease of dengue virus. Pyridyl-imidazolidinones possess very specific and potent activity against human enterovirus 71 (EV71) by targeting the EV71 capsid protein VP1, and inhibiting viral adsorption and/or viral RNA uncoating.

Cariogenicity of Candida albicans of distinct genotypes among 3-5-year-old Uygur children in Kashgar, China- a case-control study

BackgroundIn recent years, the opportunistic fungus C.albicans has been linked to ECC.It is important to investigate the relationship between the distribution of C.albicans and early childhood caries (ECC) in 3–5-year-old Uygur and Han children in Kashgar, and the role of C.albicans of distinct genotypes in caries.MethodsTwo hundred fifty-six Uygur and 141 Han children were enrolled in the study. The identified C.albicans isolates were genotyped based on 25S rDNA Polymerase Chain Reaction(PCR) amplification, and their acidogenicity, aciduricity, and adhesiveness were examined. Moreover, secreted aspartic protease (Sap) activity and SAP1–5 transcriptional levels were compared in the ECC and caries-free (CF) groups of Uygur children.ResultsC.albicans detection rate was significantly higher in Uygur children than in Han children (44. 5% vs. 31. 2%; χ2= 6.739, P = 0.009);the A genotype was dominant in Uygur and Han children with ECC, the C genotype was dominant in Uygur CF children(P = 0.022). C.albicans acidogenicity and growth were more pronounced in the Uygur ECC group than in CF group, especially at pH 4.0 and 4. 5(pH 4.0, P = 0.012; pH 4. 5, P = 0.029); the average ratios of glass-wall adhesion and the Sap activity was higher in ECC group than in CF group(P < 0.01), and SAP2(P < 0.001) and SAP5(P = 0.001) were expressed more robustly in ECC group.ConclusionsThe strong acidogenicity and aciduricity, Sap activity, and high SAP2 and SAP5 expression might be closely associated with ECC. C.albicans potentially plays a key role in the progression of caries, which most readily affects genotype A carriers and could be attributed to person-to-person environmental variation.

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

Growth and protease secretion of Scedosporium aurantiacum under conditions of hypoxia

One of the micro-environmental stresses that fungal pathogens, such as Scedosporium aurantiacum, colonising human lungs encounter in vivo is hypoxia, or deficiency of oxygen. In this work, we studied the impacts of a hypoxic micro-environment (oxygen levels ≤1%) on the growth of a clinical S. aurantiacum isolate (WM 06.482; CBS 136046) and an environmental strain (S. aurantiacum WM 10.136; CBS 136049) on mucin-containing synthetic cystic fibrosis sputum medium. Additionally, profiles of secreted proteases were compared between the two isolates and protease activity was assessed using class-specific substrates and inhibitors. Overall, both isolates grew slower and produced less biomass under hypoxia compared to normoxic conditions. The pH of the medium decreased to 4.0 over the cultivation time, indicating that S. aurantiacum released acidic compounds into the medium. Accordingly, secreted proteases of the two isolates were dominated by acidic proteases, including aspartic and cysteine proteases, with optimal protease activity at pH 4.0 and 6.0 respectively. The clinical isolate produced higher aspartic and cysteine protease activities. Conversely, all serine proteases, including elastase-like, trypsin-like, chymotrypsin-like and subtilisin-like proteases had higher activities in the environmental isolate. Sequence similarities to 13 secreted proteases were identified by mass spectrometry (MS) by searching against other fungal proteases in the NCBI database. Results from MS analysis were consistent with those from activity assays. The clinical highly-virulent, and environmental low-virulence S. aurantiacum isolates responded differently to hypoxia in terms of the type of proteases secreted, which may reflect their different virulence properties.

A coupled process of same- and opposite-sex mating generates polyploidy and genetic diversity in Candida tropicalis.

Sexual reproduction is a universal mechanism for generating genetic diversity in eukaryotes. Fungi exhibit diverse strategies for sexual reproduction both in nature and in the laboratory. In this study, we report the discovery of same-sex (homothallic) mating in the human fungal pathogen Candida tropicalis. We show that same-sex mating occurs between two cells carrying the same mating type (MTLa/a or α/α) and requires the presence of pheromone from the opposite mating type as well as the receptor for this pheromone. In ménage à trois mating mixes (i.e., “a x a + α helper” or “α x α + a helper” mixes), pheromone secreted by helper strains promotes diploid C. tropicalis cells to undergo same-sex mating and form tetraploid products. Surprisingly, however, the tetraploid mating products can then efficiently mate with cells of the opposite mating type to generate hexaploid products. The unstable hexaploid progeny generated from this coupled process of same- and opposite-sex mating undergo rapid chromosome loss and generate extensive genetic variation. Phenotypic analysis demonstrated that the mating progeny-derived strains exhibit diverse morphologies and phenotypes, including differences in secreted aspartic proteinase (Sap) activity and susceptibility to the antifungal drugs. Thus, the coupling of same- and opposite-sex mating represents a novel mode to generate polyploidy and genetic diversity, which may facilitate the evolution of new traits in C. tropicalis and adaptation to changing environments.

Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines

SummaryThe protease inhibitor component ofHeterodera glycinescyst contents was explored using a battery of peptide substrates andH. glycinesandMeloidogyne incognitasecond-stage juveniles as enzyme sources. Protease inhibitors were prepared by heat-denaturingH. glycinescyst-egg extract (hHglCE), which was used in all inhibition exploration. Eight substrates targeting four endoprotease groups (aspartic, cysteine, metallo- and serine proteases) revealed that protease inhibition by hHglCE varied significantly betweenH. glycinesandM. incognitawith seven of the eight substrates. Only cysteine protease activity was inhibited equally betweenH. glycinesandM. incognita. Aspartic protease activity was inhibited more strongly inH. glycinesand serine protease activity was inhibited more strongly inM. incognita. Digestion of five matrix metalloprotease (MMP) substrates was inhibited more strongly inH. glycines(two substrates) andM. incognita(three substrates). These variations were particularly intriguing given the potential association of MMP proteases with developing embryos. Inhibition of digestion of nematode FMRFamide-like peptides (FLPs) showed less variation between nematode species than the targeted substrates, but inhibition did vary significantly across substrates within each species. Digestion of FLP-6 was the least affected by hHglCE but was inhibited significantly more inM. incognitathan inH. glycines. Residue differences between two FLP-14 sequences significantly affected inhibition of FLP-14 digestion in bothH. glycinesandM. incognita. RP-HPLC fractionation of hHglCE clearly demonstrated the presence of high (Fr No.5) and low (Fr No.14) polarity inhibitor components. Potency of inhibition ofM. incognitaserine protease activity, based upon IC50values (1.68 and 2.78 hHglCEeq reaction−1for Fr No.5 and Fr No.14, respectively), was reduced significantly from unfractionated hHglCE (IC50 = 0.61), suggesting inhibitor dilution, loss of component synergy, or both, due to fractionation.

Antifungal mechanism of the combination of Cinnamomum verum and Pelargonium graveolens essential oils with fluconazole against pathogenic Candida strains.

The present study aimed to investigate the anti-Candida activity of ten essential oils (EOs) and to evaluate their potential synergism with conventional drugs. The effect on secreted aspartic protease (SAP) activity and the mechanism of action were also explored. The antifungal properties of essential oils were investigated using standard micro-broth dilution assay. Only Cinnamomum verum, Thymus capitatus, Syzygium aromaticum, and Pelargonium graveolens exhibited a broad spectrum of activity against a variety of pathogenic Candida strains. Chemical composition of active essential oils was performed by gas chromatography-mass spectrometry (GC-MS). Synergistic effect was observed with the combinations C. verum/fluconazole and P. graveolens/fluconazole, with FIC value 0.37. Investigation of the mechanism of action revealed that C. verum EO reduced the quantity of ergosterol to 83%. A total inhibition was observed for the combination C. verum/fluconazole. However, P. graveolens EO may disturb the permeability barrier of the fungal cell wall. An increase of MIC values of P. graveolens EO and the combination with fluconazole was observed with osmoprotectants (sorbitol and PEG6000). Furthermore, the combination with fluconazole may affect ergosterol biosynthesis and disturb fatty acid homeostasis in C. albicans cells as the quantity of ergosterol and oleic acid was reduced to 52.33 and 72%, respectively. The combination of P. graveolens and C. verum EOs with fluconazole inhibited 78.31 and 64.72% SAP activity, respectively. To our knowledge, this is the first report underlying the mechanism of action and the inhibitory effect of SAP activity of essential oils in synergy with fluconazole. Naturally occurring phytochemicals C. verum and P. graveolens could be effective candidate to enhance the efficacy of fluconazole-based therapy of C. albicans infections.

Toxic effect of Canavalia ensiformis seed coat on larval development of Callosobruchus maculatus

The outer-covering of seeds is known as the seed coat, and this is the seed's first protective barrier against the penetration of insects. In this sense, seed coat toxicity can affect insect development. In this work, we study the influence of the Canavalia ensiformis seed coat on embryonic and larval development of Callosobruchus maculatus. Oviposition of C. maculatus was negatively affected by the seed coat, while embryonic development occurred without any morphological or biochemical alterations. During larval development, physiological changes comprising delay in body weight, the inability of larvae to penetrate the C. ensiformis seed coat, and decreases in biochemical parameters, such as contents of glucose, proteins, and triglycerides, were recorded. Although larvae survived until 17 days after oviposition, they could not penetrate the C. ensiformis seed coat. A dose-dependent effect was observed on female oviposition, mass, and survival of larvae of C. maculatus. Cysteine protease, α-amylase and α-glucosidase activities decreased while aspartic protease activity increased with increases in ingestion of seed coat flour. Larval post-embryonic development was affected by C. ensiformis seed and this interference is related with a toxic seed coat which reduced activities of several key digestive enzymes, resulting in severe weight loss.

Detection of Aspartic Proteinase Activities Using Gel Zymography.

Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.

Exogenous enzymes in dairy technology: acidic proteases from processing discards of shrimp Pleoticus muelleri and their use as milk‐clotting enzymes for cheese manufacture

SummaryArgentina is the seventh largest world producer of cheese. Owing to the profitability of this industry, more than a thousand of dairy companies and farms exclusively produce cheeses, and, thus, this activity utilises 42% of total raw milk production of the country. Due to the increase in cheese demand, and because calf rennet is relatively expensive, this has motivated the study of new sources of proteases due to the potential use in biotechnological processes. Previously, crustacean proteases have been considered as good rennet substitutes for milk coagulation during the cheese making process. The aim of this study was to evaluate and characterise acidic proteases obtained from Pleoticus muelleri discards and their proteolytic capability as well as their potential use as a milk‐clotting agent. The activity of aspartic proteases and the satisfactory acceptation by the taste panel for cheese made with shrimp's enzymes suggest that P. muelleri proteins offer a high potential for use in dairy biotechnological processes with potential use as milk‐clotting agents for cheese making.

Different classes of hydrolytic enzymes produced by multidrug-resistant yeasts comprising the Candida haemulonii complex.

The production of enzymes in clinical isolates of Candida haemulonii (Ch, n = 5), Candida duobushaemulonii (Cd, n = 4) and Candida haemulonii var. vulnera (Chv, n = 3) was identified by agar plate. Aspartic protease, phytase, caseinolytic and hemolytic activities were detected in all the isolates. A distinct scenario was evidenced regarding the production of lipases. In this way, 80%, 50% and 100% of Ch, Cd and Chv, respectively, were phospholipase producers. Regarding esterase activity, 100%, 50% and 66.7% of Ch, Cd and Chv, respectively, were positive isolates. Esterase activity was significantly higher in isolates recovered from cutaneous candidiasis compared with those recovered from body fluids.

Zymographic Detection of Aspartic Proteinase Activities in Porcine Ovarian Extracts

Zymographic Detection of Aspartic Proteinase Activities in Porcine Ovarian Extracts

Effects of the aspartic protease inhibitor from Lupinus bogotensis seeds on the growth and development of Hypothenemus hampei: An inhibitor showing high homology with storage proteins

The coffee berry borer Hypothenemus hampei is a pest that causes great economic damage to coffee grains worldwide. Because the proteins consumed are digested by aspartic proteases in the insect's midgut, the inhibition of these proteases by transferring a gene encoding an aspartic protease inhibitor from Lupinus bogotensis Benth. to coffee plants could provide a promising strategy to control this pest. Five aspartic protease inhibitors from L. bogotensis (LbAPI) were accordingly purified and characterized. The gene encoding the L. bogotensis aspartic protease inhibitor (LbAPI), with the highest inhibitory activity against H. hampei, was expressed in Escherichia coli and the purified recombinant protein (rLbAPI), with a molecular mass of 15 kDa, was subsequently assessed for its ability to inhibit the aspartic protease activity present in the H. hampei midgut in vitro, as well as its effects on the growth and development of H. hampei in vivo. The in vitro experiments showed that rLbAPI was highly effective against aspartic proteases from H. hampei guts, with a half maximal inhibitory concentration (IC50) of 2.9 μg. The in vivo experiments showed that the concentration of rLbAPI (w/w) in the artificial diet necessary to cause 50% mortality (LD50) of the larvae was 0.91%. The amino acid sequence of LbAPI had high homology (52-80%) to the seed storage proteins, vicilin and β-conglutin, suggesting that this protein was generated by evolutionary events from a β-conglutin precursor. Based on these results, LbAPI may have a dual function as storage protein, and as defense protein against H. hampei. These results provide a promising alternative to obtain a coffee plant resistant to H. hampei.

An Aspartic Protease of the Scabies Mite Sarcoptes scabiei Is Involved in the Digestion of Host Skin and Blood Macromolecules

BackgroundScabies is a disease of worldwide significance, causing considerable morbidity in both humans and other animals. The scabies mite Sarcoptes scabiei burrows into the skin of its host, obtaining nutrition from host skin and blood. Aspartic proteases mediate a range of diverse and essential physiological functions such as tissue invasion and migration, digestion, moulting and reproduction in a number of parasitic organisms. We investigated whether aspartic proteases may play role in scabies mite digestive processes.Methodology/Principle FindingsWe demonstrated the presence of aspartic protease activity in whole scabies mite extract. We then identified a scabies mite aspartic protease gene sequence and produced recombinant active enzyme. The recombinant scabies mite aspartic protease was capable of digesting human haemoglobin, serum albumin, fibrinogen and fibronectin, but not collagen III or laminin. This is consistent with the location of the scabies mites in the upper epidermis of human skin.Conclusions/SignificanceThe development of novel therapeutics for scabies is of increasing importance given the evidence of emerging resistance to current treatments. We have shown that a scabies mite aspartic protease plays a role in the digestion of host skin and serum molecules, raising the possibility that interference with the function of the enzyme may impact on mite survival.

Antifungal properties of the anti-hypertensive drug: Aliskiren

ObjectivesAliskiren, the first in a new class of orally active renin inhibitors, is licensed for the treatment of hypertension. It inhibits plasma renin activity directly, thereby reducing generation of angiotensin II. In this study, we have explored the anti-Candida properties of aliskiren. MethodsCandida albicans was cultured in the presence or absence of aliskiren for various time periods. Subsequently, inhibition of growth, germtube formation, adhesion, early/matured biofilm development and secreted aspartic protease (SAP) activity were studied. ResultsWhen cultured in the presence of aliskiren, Candida showed significant reduction in the activity of aspartic proteases. Aliskiren impaired in vitro growth of C. albicans. It also affected two other virulence factors, germtube and adhesion. There is reduction in early and matured biofilm when treated with aliskiren. ConclusionsAliskiren could be considered as a candidate for antifungal drug development.

Storage Stability of Wheat Bran Proteases for Producing Angiotensin I-converting Enzyme Inhibitory Peptides

The stability of wheat bran proteases for producing angiotensin I-converting enzyme (ACE) inhibitory peptides during storage was investigated. The aspartic proteinase activity upon storage at 15, 20, and 30°C was > 90% of initial levels after 30 days, while storage at 40 and 50°C resulted in levels of 87.3 and 63.1%, respectively. The carboxypeptidase activity remained constant at all temperatures during the storage period. The yields of ACE inhibitory peptides from wheat bran at 15, 20, 30, 40, and 50°C after 30 days were 99.0, 98.0, 97.1, 97.1, and 92.2% of the initial level, respectively. The ACE inhibitory activity of all fractions was > 95% of the initial level. These results indicated that the storage of wheat bran at 15 to 40°C results in maintenance of the ACE inhibitory peptide-producing activity, for at least 30 days.

A Peptide Derived from the Highly Conserved Protein GAPDH Is Involved in Tissue Protection by Different Antifungal Strategies and Epithelial Immunomodulation

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis but also in non-metabolic processes, including transcription activation and apoptosis. We report the isolation of an hGAPDH (2-32) fragment peptide from human placental tissue exhibiting antimicrobial activity. The peptide was internalized by cells of the pathogenic yeast Candida albicans and initiated a rapid apoptotic mechanism, leading to killing of the fungus. Killing was dose-dependent, with 10 µg/ml (3.1 µM) and 100 µg/ml hGAPDH (2-32) depolarizing 45% and 90% of the fungal cells in a population, respectively. Experimental C. albicans infection induced epithelial hGAPDH (2-32) expression. Addition of the peptide significantly reduced the tissue damage as compared to untreated experimental infection. Secreted aspartic proteinases (Saps) activity of C. albicans was inhibited by the fragment at higher concentrations with an ED50 of 160 mg/l (50 μM) for Sap1p and 200 mg/l (63 μM) for Sap2p while Sap3 was not inhibited at all. Interestingly, hGAPDH (2-32) induced significant epithelial IL-8 and GM-CSF secretion and stimulated TLR4 expression at low concentrations independently of the presence of C. albicans without any toxic mucosal effects.In the future, the combination of different antifungal strategies, e.g. a conventional fungicidal with immunomodulatory effects and the inhibition of fungal virulence factors might be a promising treatment option.

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 103 and 6.52 × 103 M−1. The binding reactions were enthalpy driven with ΔHb values between −50.99 and −46.07 kJ mol−1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.