Antihypertensive treatment of end-stage renal disease patients on hemodialysis does not alter circulating ACE and ACE2 activity and angiotensin peptides.

Cardiovascular diseases (CVD) are the main causes of death in hemodialysis patients, representing a public health challenge. We investigated the effect of different antihypertensive treatments on circulating levels of renin-angiotensin system (RAS) components in end-stage renal disease (ESRD) patients on hemodialysis. ESRD patients were grouped following the prescribed antihypertensive drugs: ß-blocker, ß-blocker+ACEi and ß-blocker+AT1R blocker. ESDR patients under no antihypertensive drug treatment were used as controls. Blood samples were collected before hemodialysis sessions. Enzymatic activities of the angiotensin-converting enzymes ACE and ACE2 were measured through fluorescence assays and plasma concentrations of the peptides Angiotensin II (Ang II) and Angiotensin-(1-7) [Ang-(1-7)] were quantified using mass spectrometry (LC-MS/MS). ACE activity was decreased only in the ß-blocker+ACEi group compared to the ß-blocker+AT1R, while ACE2 activity did not change according to the antihypertensive treatment. Both Ang II and Ang-(1-7) levels also did not change according to the antihypertensive treatment. We concluded that the treatment of ESRD patients on hemodialysis with different antihypertensive drugs do not alter the circulating levels of RAS components.

Pediatric dyslipidemia is associated with increased urinary ACE activity, blood pressure values, and carotidal-femoral pulse wave velocity.

This study aimed to evaluate the enzymatic activity of the angiotensin-converting enzyme (ACE) in children and adolescents to investigate their relationship with dyslipidemia and other cardiometabolic alterations. Anthropometric measurements, blood pressure (BP), and fasting lipid concentrations were taken from 360 subjects. Categorization was done according to the levels of each lipoprotein (total cholesterol, triglycerides (TG), LDL-C, HDL-C, and non-HDL-C) into three groups: normolipidemic (NL), borderline (BL), and dyslipidemic (DL). Enzymatic activity in urine was measured using the substrates Z-FHL-OH and hippuryl-HL-OH (h-HL-OH) and the ACE activity ratio (Z-FHL-OH/h-HL-OH) was calculated. Dyslipidemic levels of HDL-C, TG, and LDL-C were observed in 23%, 9%, and 3% of the participants, respectively, and were more frequent in obese children (Chi-square, p < 0.001). ACE activity ratio was augmented in BL(HDL-C) when compared to NL(HDL-C) (5.06 vs. 2.39, p < 0.01), in DL(LDL-C) in comparison to BL(LDL-C) and NL(LDL-C) (8.7 vs. 1.8 vs. 3.0, p < 0.01), and in DL(non-HDL-C) than in BL(non-HDL-C) and in NL(non-HDL-C) (6.3 vs. 2.1 vs. 2.9, p = 0.02). The groups with impaired HDL-C and TG levels presented an increased diastolic BP percentile, and a higher systolic BP percentile was observed in BL(TG) and DL(TG). The carotidal-femoral pulse wave velocity (cfPWV) was higher in the groups with DL levels of TG and LDL-C than in NL groups. Hypertriglyceridemia was associated with higher cfPWV. No direct impact of the ACE activity on BP values was observed in this cohort, however, there was an association between hyperlipidemia and ACE upregulation which can trigger mechanisms driving to early onset of hypertension and cardiovascular disease. Graphical abstract exemplifying the cohort, categorization of subjects into the groups NL normolipidemic, BL borderline, DL dyslipidemic, methods, and main findings. Pediatric dyslipidemia was consistent with dyslipidemia secondary to obesity (DSO), associated with higher urinary angiotensin-converting enzyme (ACE) activity ratio, BP blood pressure values, and carotidal-femoral pulse wave velocity (cfPWV).

Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus.

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM-1.s-1), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM-1.s-1), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM-1.s-1). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk.

Biochemical Properties and Catalytic Specificity of a Novel Neutral Serine Peptidase Secreted by Fungus Pyrenochaetopsis sp.

The fungal genus Pyrenochaetopsis has received particular attention because of its different lifestyles, such as numerous plant pathogenic, saprophytic, and endophytic species. Its ability to infect plant cells relies heavily upon secreted peptidases. Here, we investigated the biochemical properties and catalytic specificity of a new serine peptidase secreted by the filamentous fungus Pyrenochaetopsis sp. We found that while this neutral serine peptidase displayed optimal activity at a pH of 7.0 and temperature of 45 °C, it tolerated a wide range of pH conditions and temperatures lower than 45 °C. Its peptidase activity was depressed by some metallic ions (such as aluminum, cobalt, and copper (II) chloride) and enhanced by others (such as sodium, lithium, magnesium, potassium, calcium, and manganese). Lastly, the enzyme showed the greatest specificity for non-polar amino acids, particularly leucine and isoleucine, and moderate specificity for basic and neutral polar amino acids. It displayed the least specificity for acidic residues.

Design and characterization of crotamine-functionalized gold nanoparticles.

This paper describes the development of a facile and environmentally friendly strategy for supporting crotamine on gold nanoparticles (GNPs). Our approach was based on the covalent binding interaction between the cell penetrating peptide crotamine, which is a snake venom polypeptide with preference to penetrate dividing cells, and a polyethylene glycol (PEG) ligand, which is a nontoxic, water-soluble and easily obtainable commercial polymer. Crotamine was derivatized with ortho-pyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide (OPSS-PEG-SVA) cross-linker to produce OPSS-PEG-crotamine as the surface modifier of GNP. OPSS-PEG-SVA can serve not only as a surface modifier, but also as a stabilizing agent for GNPs. The successful PEGylation of the nanoparticles was demonstrated using different physicochemical techniques, while the grafting densities of the PEG ligands and crotamine on the surface of the nanoparticles were estimated using a combination of electron microscopy and mass spectrometry analysis. In vitro assays confirmed the internalization of these GNPs, into living HeLa cells. The results described herein suggest that our approach may serve as a simple platform for the synthesis of GNPs decorated with crotamine with well-defined morphologies and uniform dispersion, opening new roads for crotamine biomedical applications.

Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium.

For a long time, proteolytic enzymes have been employed as key tools of industrial processes, especially in the dairy industry. In the present work, we used Phanerochaete chrysosporium for biochemical characterization and analysis of catalytic specificity of an aspartic peptidase. Our results revealed an aspartic peptidase with molecular mass ∼38kDa, maximal activity at pH 4.5 and 50°C, and stability above 80% in the pH range of 3-8 and temperature up to 55°C for 1h. In a milk-clotting assay, this peptidase showed maximal milk clotting activity at 60-65°C and maintenance of enzymatic activity above 80% in the presence of 20mM CaCl2. In a specificity assay, we observed stronger restriction of catalysis at the S1 subsite, with a preference for lysine, arginine, leucine, tyrosine, and phenylalanine residues. The restricted proteolysis and milk-clotting potential are attractive properties for the use in cheese production.

Activity of a peptidase secreted by Phanerochaete chrysosporium depends on lysine to subsite S'1.

Peptidases are enzymes that catalyze the rupture of peptide bonds. Catalytic specificity studies of these enzymes have illuminated their modes of action and preferred hydrolysis targets. We describe the biochemical characteristics and catalytic specificity of a lysine-dependent peptidase secreted by the basidiomycete fungus Phanerochaete chrysosporium. We attained 5.7-fold purification of a ∼23-kDa neutral peptidase using size-exclusion (Sephadex G-50 resin) and ion-exchange (Source 15S resin) chromatography. Using the Fluorescence Resonance Energy Transfer substrate Abz-KLRSSKQ-EDDnp, we detected maximal activity at pH 7.0 and 45-55°C. The peptidase retained ∼80% of its enzymatic activity for a wide range of conditions (pH 4-9; temperatures up to 50°C for 1h). The peptidase activity was lowered by the ionic surfactants, sodium dodecyl sulfate and cetyltrimethylammonium bromide; the reducing agent, dithiothreitol; the chaotrope, guanidine; copper (II) ion; and the cysteine peptidase-specific inhibitors, iodoacetic acid and N-ethylmaleimide. The peptidase preferred the basic amino acids K and R and high selectivity on S'1 subsite, exhibiting a condition of lysine-dependence to catalysis on anchoring of this subsite.

Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.

Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus.

The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.

The identification and biochemical properties of the catalytic specificity of a serine peptidase secreted by Aspergillus fumigatus Fresenius.

Aspergillus fumigatus is a saprophytic fungus as well as a so-called opportunist pathogen. Its biochemical potential and enzyme production justify intensive studies about biomolecules secreted by this microorganism. We describe the alkaline serine peptidase production, with optimum activity at 50°C and a pH of 7.5 and a reduction in proteolytic activity in the presence of the Al(+3) ions. When using intramolecularly quenched fluorogenic substrates, the highest catalytic efficiency was observed with the amino acid leucine on subsite S'(3) (60,000 mM(-1)s(-1)) and preference to non-polar amino acids on subsite S(3). In general, however, the peptidase shows non-specificity on other subsites studied. According to the biochemical characteristics, this peptidase may be an important biocatalyst for the hydrolysis of an enormous variety of proteins and can constitute an essential molecule for the saprophytic lifestyle or invasive action of the opportunistic pathogen. The peptidase described herein exhibits an estimated molecular mass of 33 kDa. Mass spectrometry analysis identified the sequence GAPWGLGSISHK displaying similarities to that of serine peptidase from Aspergillus fumigatus. These data may lead to a greater understanding of the advantageous biochemical potential, biotechnological interest, and trends of this fungus in spite of being an opportunist pathogen.

Purification, characterization, and specificity determination of a new serine protease secreted by Penicillium waksmanii.

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl(2), KCl, and BaCl, and partially inhibited by CuCl(2), CoCl(2) and totally inhibited by AlCl(3) and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k (cat)/K (m) of 10,666 mM(-1) s(-1), followed by the peptide Abz-GLRSSKQ-EDDnp with a k (cat)/K (m) of 7,500 mM(-1) s(-1). Basic and acidic side chain-containing amino acids performed best at subsite S(1). Subsites S(2), S(3), S(') (2), and S(') (1), S(') (3) showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k (cat)/K (m) were observed for the subsites S(2), S(3), and S(') (2.) The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.