RESUMO
Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 µM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.
Assuntos
Ácido Aspártico Proteases , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Aspergillus/metabolismo , Concentração de Íons de Hidrogênio , Pepstatinas/metabolismo , Peptídeo HidrolasesRESUMO
Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase.
Assuntos
Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/metabolismo , Trichosporon/enzimologia , Brasil , Catepsina D/metabolismo , DNA Fúngico , Gelatina , HIV-1/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G , Peso Molecular , Mucinas , Pepstatinas/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Inibidores de Proteases , Albumina Sérica , Pele/microbiologia , Trichosporon/crescimento & desenvolvimento , Trichosporon/isolamento & purificação , Trichosporon/patogenicidadeRESUMO
The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 A resolution and 15.8% (R(free)=19.2%) at 1.85 A resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Proteínas Fúngicas/química , Pepstatinas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Trichoderma/enzimologia , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Análise por Conglomerados , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Pepstatinas/genética , Pepstatinas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação ProteicaRESUMO
During larva to adult transition, the larval fat body of the Medfly (Ceratitis capitata) progressively disintegrates to be replaced by the adult one, after imago ecdysis. Here we show that a temporal correlation exists among the microscopy images of fat body progressive disintegration, the activation of fat body lysosomes (as judged by acid phosphatase activity), and the activity of a novel fat body aspartyl proteinase. The enzyme was purified and partially characterized. This proteinase exhibited a wide range of acid isoforms with isoelectric points from 5.6 to 7.3, an optimum pH of 3.0 for hemoglobin digestion, and was completely inhibited by pepstatin A. The apparent molecular weight was estimated (42 +/- 1 kDa) and the protein was characterized as N-glycosylated, judging from affinity to Concanavalin A. From the biochemical characteristics, the enzyme that we called "Early Metamorphosis Aspartyl Proteinase" (EMAP) appears to be similar to mammalian Cathepsin D. However, the N-terminal sequence of EMAP showed no similarity with any known animal Cathepsins and exhibited an important instability to neutral and alkaline pH. This feature seems to be a peculiar characteristic of insect aspartyl proteinases. The temporal activity profile of EMAP during metamorphosis correlated well with the microscopy images of fat body cell autolytic death. Our data support the notion that EMAP is a metamorphosis-specific lysosomal proteinase, mostly expressed during larval fat body histolysis.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Ceratitis capitata/enzimologia , Corpo Adiposo/metabolismo , Metamorfose Biológica/fisiologia , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Ceratitis capitata/fisiologia , Cromatografia de Afinidade , Concanavalina A , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Corpo Adiposo/citologia , Técnicas Histológicas , Concentração de Íons de Hidrogênio , Isoenzimas , Lisossomos/metabolismo , Pepstatinas/metabolismo , Análise de Sequência de ProteínaRESUMO
The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 A. Crystallographic refinement led to an R factor of 0.161 at 2.45 A resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.