Renin Substrate Research Articles

Hypothyroid on cardiopulmonary bypass usage in children with congenital heart disease: A literature review

Congenital heart disease (CHD) is a birth defect that affects the structure or function of the heart. Cardiopulmonary bypass (CPB) is frequently used to treat CHD, which raises the risk of hypothyroidism. The disorder can cause circulatory problems, edema, and pleural effusion, all of which indicate heart failure. Hypothyroid conditions can cause decreased cardiac output due to poor relaxation of cardiac smooth muscle cells and reduced endothelial nitric oxide accessibility. This causes a chain reaction of increasing arterial stiffness, which raises systemic vascular resistance (SVR). Thyroid hormones also have an effect on the renin-angiotensin-aldosterone pathway, promoting the synthesis of renin substrates in the liver. This causes higher diastolic blood pressure, narrow pulse pressure, and decrease in renin levels in hypothyroid patients. Thyroid hormones also regulate the transcription of pacemaker genes, hence hypothyroid patients have a slower heartbeat. Hypothyroidism raises the likelihood of atrial fibrillation.

Abstract 11373: Suppression of Plasma Angiotensin II by GLP-1 in Humans is Independent of Changes in Plasma Angiotensinogen

Introduction: In humans, the natriuretic effect of GLP-1 depends on GLP-1 receptor interaction, is independent of changes in renal blood flow, renin, ANP, and aldosterone but is accompanied by suppression of angiotensin II (ANGII). The mechanism by which GLP-1 reduces ANGII independently of changes in renin is not clarified. Hypothesis: It was hypothesized that GLP-1 suppresses renin substrate, angiotensinogen, in plasma. Methods: To test this proposal, we measured arterial angiotensinogen concentration in biobanked plasma samples from healthy males during a 3-hour GLP-1 infusion that induced natriuresis (1). Samples were from a study with fixed sodium intake where eight healthy men were examined twice in random order during a 3-hour infusion of either GLP-1(1.5 pmol kg -1 min -1 ) or vehicle together with an intravenous infusion of 0.9% NaCl (750 mL/hour). Arterial plasma angiotensinogen was measured in plasma by dilution (1:200) and addition of excess exogenous human renin (Inst. Of Biological Standards UK). After prolonged incubation, ANGI was quantified by radioimmunoassay. Intraassay variation was 9-12%. Results: Arterial plasma angiotensinogen and renin levels decreased similarly and modestly (~15 and ~30%, respectively) after NaCl infusion during both studies whereas ANGII levels decreased significantly (~25%) only during GLP-1 infusion. Conclusions: Suppression of plasma angiotensinogen is less likely to account for GLP-1-mediated ANGII suppression in healthy young males. In perspective, GLP-1 could enhance pathways of ANGII degradation rather than inhibit its formation. 1. Asmar A et al. Extracellular fluid volume expansion uncovers a natriuretic action of GLP-1: a functional GLP-1-renal axis in man. J Clin Endocrinol Metab. 2019 Jul 1;104(7):2509-2519.

1652-P: Suppression of Plasma Angiotensin II by GLP-1 in Humans Is Independent of Changes in Plasma Angiotensinogen

In humans, the natriuretic effect of GLP-1 depends on GLP-1 receptor interaction, is independent of changes in renal blood flow, renin, ANP, and aldosterone but is accompanied by suppression of angiotensin II (ANGII). The mechanism by which GLP-1 reduces ANGII independently of changes in renin is not clarified. It was hypothesized that GLP-1 suppresses renin substrate, angiotensinogen, in plasma. To test this proposal, we measured arterial angiotensinogen concentration in biobanked plasma samples from healthy males during a 3-hour GLP-1 infusion that induced natriuresis (1). Samples were from a study with fixed sodium intake where eight healthy men were examined twice in random order during a 3-hour infusion of either GLP-1(1.5 pmol kg-1 min-1) or vehicle together with an intravenous infusion of 0.9% NaCl (750 mL/hour). Arterial plasma angiotensinogen was measured in plasma by dilution (1:200) and addition of excess exogenous human renin (Inst. of Biological Standards UK). After prolonged incubation, ANGI was quantified by radioimmunoassay. Intraassay variation was 9-12%. Arterial plasma angiotensinogen and renin levels decreased similarly and modestly (~15 and ~30%, respectively) after NaCl infusion during both studies whereas ANGII levels decreased significantly (~25%) only during GLP-1 infusion. Suppression of plasma angiotensinogen is less likely to account for GLP-1-mediated ANGII suppression in healthy young males. In perspective, GLP-1 could enhance pathways of ANGII degradation rather than inhibit its formation. 1. Asmar A et al. Extracellular fluid volume expansion uncovers a natriuretic action of GLP-1: a functional GLP-1-renal axis in man. J Clin Endocrinol Metab. 2019 Jul 1;104(7):2509-2519. Disclosure B.L.Jensen: None. A.Asmar: Research Support; Novo Nordisk A/S.

Fast algorithm for time decay estimation with applications to electrostatic ion beam traps

The ability of peptide trapping in an electrostatic ion beam trap (EIBT) is used for the measurement of renin substrate lifetime dependence from the pressure. The time decay estimation is traditionally obtained by optimization of nonlinear curve-fitting in the least-squares sense. This paper presents a novel algorithm to address this problem, using a numerical differentiation method as the basis for lifetime estimation. Simulations results show that the proposed method provides results similar to those obtained with the classical approach, but is faster by about two orders of magnitude. An experimental result is detailed, which shows the adequacy of this algorithm for the real-life monitoring of decay measurements, not only for EIBT, but also for other processes such as luminescence where exponential decay is involved.

Renin-Angiotensin-Aldosterone System in Women Using Combined Oral Contraceptive: A Systematic Review.

To describe the effects of combined oral contraceptives (COC) on the renin-angiotensin-aldosterone system (RAAS). This is a systematic review according to the criteria of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), registered in PROSPERO under the ID: CRD42020200019. Searches were performed between August 2020 and December 2021, in the following databases: Medline via Pubmed, Cochrane Central Library, Scientific Electronic Library Online, and Latin American and Caribbean Literature in Health Sciences via Virtual Health Library. The effects of the combined oral contraceptive on plasma renin activity values, plasma renin values, angiotensinogen values- also known as plasma renin substrate- angiotensin, and/or aldosterone values. A total of 877 studies were selected and, of these, 10 articles met the eligibility criteria and were included in this review. Data were combined through qualitative synthesis and included in a spreadsheet previously prepared by the authors. The collected samples ranged from 18 to 137 participants, totaling 501 women aged between 18 and 49 years throughout all studies. The studies showed increased activity of plasma renin, plasma renin substrate, angiotensin II, and aldosterone in this population. The findings of this study suggest that the COC promotes greater activation of the RAAS. Supporting the idea that its use is related to an increased risk of cardiovascular events, including systemic arterial hypertension.

Resemblance in Physiological Systems during Phylogeny and Ontogeny

Although it is not certain whether some characteristics of the mammalian fetus/embryo may repeat the adult stage of ancestral vertebrates, comparison of these two time‐dependent processes should provide new insight into the molecular and functional evolution and its significance. Here we discuss two important biological models: 1) Renin‐angiotensin system (RAS) and 2) Urine concentration mechanism.Renin is a hormone and substrate‐dependent enzyme secreted mostly by the juxtaglomerular (JG) cells of the afferent arteriole located at the vascular pole of the glomerulus. Renin acts on renin substrates and forms angiotensin (Ang) I which is subsequently converted to Ang II by Ang converting enzyme (ACE). Renin containing cells evolved early both in phylogeny and ontogeny, whereas, the macula densa (tubule component), and extracellular mesangium appeared at later stages.Renin containing cells are distributed more widely intrarenally and/or extrarenally in early stages during phylogeny and ontogeny. Granulated cells and renin expression become progressively restricted to JG areas with phylogenic and ontogenic advancement. Kidney renin activities tend to be higher in primitive fishes than in more advanced, while the relative level of extrarenal renin expression to that of the kidney is higher in embryos than in more mature chicken and mice. Extra renal renin is noted in primitive vertebrates. Also, in both the mammalian fetus and nonmammalian vertebrates, the vasopressor and vascular action of Ang II evolved early. A single application of ACE inhibitors decreases the basal level of blood pressure and increase plasma renin activity in conscious adult teleost fish. Ang II stimulates, whereas ACE inhibitors inhibit, arteriolar branching in zebrafish. Likewise, the RAS is important during early ontogeny of mammals in vascular growth and development.Distal tubules of freshwater trout show secondary active NaCl cotransport mechanism without accompaniment of water and act as the so‐called “diluting segment.” Teleost fish cannot concentrate urine because they lack structural and functional countercurrent mechanisms. Only birds and mammals can form urine clearly hyperosmotic to plasma and conserve body water. The structure and function of adult quail kidneys have similarities to those of developing rodents. Both have a loop of Henle, consisting of descending limb and thick ascending limb, but they lack the thin ascending limb. Countercurrent urine concentration of adult quail and developing rodents heavily depends on NaCl recycling without participation of urea transport. Moreover, in both adult quail and developing rodent kidneys, aquaporin 2 gene expression and protein are present in the apical membrane of the collecting tubules, but the water conservation effect of neurohypophysial hormones is smaller than that of adult rodent kidneys. Hence, comparing these two time‐dependent processes may provide new insights into the interpretation of existing findings and provide perspective for future investigations.

Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris.

Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature (Tm ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.

SUN-365 Characteristics of Primary Aldosteronism without Suppression of Plasma Renin Activity

Introduction: Primary aldosteronism (PA) is a common cause of secondary hypertension. It is characterized by excess production of aldosterone from the adrenal glands resulting in suppressed plasma renin activity (PRA) by a negative feedback regulation. Thus, if a patient with hypertension had an unsuppressed PRA, the possibility of PA might be excluded. However, we have observed some PA patients with unsuppressed PRA in our hospital. Here, we report clinical characters of PA patients with unsuppressed PRA. Methods: We searched for PA patients with unsuppressed PRA (Escaped PRA group) from 969 patients who underwent segmental adrenal venous sampling (S-ATS) in our hospital between April 2009 and March 2018. Escaped PRA group were those with PRA level of >1.0 ng/mL/hr in any two or more blood samples obtained in resting position, and had aldosterone producing adenomas (APAs) in which CYP11B2 expression was confirmed histopathologically. We also enrolled patients with APAs with suppressed PRA as a control group (34 females, 35 females ). All patients in the control group had PRA level of < 1.0 ng/mL/hr in any two or more blood samples obtained in resting position and had macroadenomas in which CYP11B2 expression was confirmed. We compared clinical characteristics between Escaped PRA group and Control group. Results: We found that 7 cases (3 males, 4 females) were in the Escaped PRA group (PRA; 2.3±0.8 ng/mL/hr). One of them showed a gap between PRA and Active Renin Concentration (ARC). Compared to the control group (PRA; 0.3±0.2 ng/mL/hr), the escaped PRA group had no significant difference in body mass Index, blood pressure, urinary aldosterone, and serum potassium. The escaped PRA group tended to be younger compared to control group (53.0±4.4 years-old vs 47.4±10.8 years-old, P = 0.12) and have decreased eGFR Escaped (74.4±20.0 mL/min/1.73 m2 vs 86.3±19.3 mL/min/1.73 m2, P = 0.088). Only serum cortisol level after 1 mg dexamethasone suppression test showed a significant difference (Escaped PRA group; 1.9±0.5 mg/dL, Control group 1.4±3.0 mg/dL, P = 0.011). Conclusion: Excess cortisol has been reported to increase PRA through increasing renin substrate and suppressing ACTH-dependent aldosterone secretion. We should not exclude the possibility of PA in patients without suppressed plasma renin activity, especially who had a cortisol excess.

Establishing a novel assay system for measuring renin concentration using cost effective recombinant ovine angiotensinogen

BackgroundPlasma renin can predict future cardiovascular events as well as the prevalence of chronic renal disease in hypertensive subjects. Ovine angiotensinogen (oANG) is a better substrate for measuring renin concentration through activity assay. Recombinant oANG expressed in Escherichia coli cells can be utilized as the substrate while measuring plasma renin. We aim to establish an immunoassay for measuring renin concentration at picomolar level using recombinant oANG. Material and methodsRecombinant oANG was expressed in E. coli cells and purified to homogeneity. Various concentrations (0–1.5 pM) of recombinant human renin standard were prepared and incubated with recombinant oANG. Renin activity was determined by angiotensin-I specific enzyme-linked immunosorbent assay. ResultsAbout 4.5 mg of purified recombinant oANG was obtained from 0.5 L of E. coli culture. The Michaelis constant and turnover number of human renin with recombinant oANG were 0.16 μM and 0.51 s−1, respectively. A linear relationship was obtained when renin activity was plotted as a function of renin concentration using recombinant oANG as the renin substrate. Picomolar amounts of renin can be measured from known renin activity using this method. ConclusionThis study established a novel assay system for measuring renin at picomolar level using cost effective recombinant oANG.

The Higher Frequency of G Allele Found in the -152 Site of Agt Gene But was Not Aligned with the Angiotensinogen Levels

Pathogenesis of essential hypertension involves an interaction between genetic and environmental factors. Genetic variant G-152A of angiotensinogen (AGT) promoter is thought to affect AGT gene transcription and angiotensinogen levels. Angiotensinogen is an important substrate for renin in the RAS that is finally converted into angiotensin II that plays a key role in the control of blood pressure. However, the studies that discuss the single nucleotide polymorphism (SNP) G-152A of AGT gene are still limited, especially in Indonesian hypertensive patients. Therefore, this study was designed to detect the SNP G-152A of AGT and reported its serum angiotensinogen levels. The variants were identified in 62 patients in Malang, Indonesia with essential hypertension by PCR and further identified by automated sequencing. Serum samples were collected to analyze the angiotensinogen levels by a sandwich ELISA. The data showed that the AGT promoter in our patients had genetic variants -152G (G allele/GG genotype) and -152A (A allele/AG genotype) with a frequency of 0.92 : 0.08, respectively. There were no patients with AA genotype. In the analysis of serum angiotensinogen levels (mean ± SD), it was found that A allele had an angiotensinogen level of 371.30 ± 69.92 (ng/mL) and G allele was 343.53 ± 74.95 (ng/mL). The A allele had slightly higher angiotensinogen level than G allele with no significant difference (P=0.437). Thus, in our study is found a genetic variant G-152A of AGT gene represented the GG, AG genotypes in the (-152) site and it has higher frequency of G allele. Further research is still needed to determine that G/A allele directly affects on the occurrence of essential hypertension.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala-Thr) and the other to the non-pathogenic Aβ48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Arterial wall hypertrophy is ameliorated by α2-adrenergic receptor antagonist or aliskiren in kidneys of angiotensinogen-knockout mice.

Arterial hypertrophy and interstitial fibrosis are important characteristics in kidneys of angiotensinogen-knockout (Atg -/-) mice. In these mice, which exhibit polyuria and hypotension, sympathetic nerve signaling is estimated to be compensatorily hyperactive. Furthermore, transforming growth factor (TGF)-β1 is overexpressed in mice kidneys. To determine whether sympathetic nerve signaling and TGF-β1 exacerbate arterial hypertrophy and interstitial fibrosis, intervention studies of such signaling are required. We performed renal denervation and administered the α2-adrenergic receptor (AR) antagonist, atipamezole, to Atg -/- mice. A renin inhibitor, aliskiren, which was preliminarily confirmed to reduce TGF-β1 gene expression in kidneys of the mice, was additionally administered to assess the effect on the arterial hypertrophy and interstitial fibrosis. Norepinephrine content in kidneys of Atg -/- mice was three times higher than in kidneys of wild-type mice. Interventions by renal denervation and atipamezole resulted in amelioration of the histological findings. Overexpression of TGF-β1 gene in kidneys of Atg -/- mice was altered in a manner linked to the histological findings. Surprisingly, aliskiren reduced α2-AR gene expression, interstitial fibrosis, and arterial hypertrophy in kidneys of Atg -/- mice, which lack renin substrate. Alpha2-AR signaling is one of the causes of persistent renal arterial hypertrophy in Atg -/- mice. Aliskiren also angiotensinogen-independently reduces the extent of renal arterial hypertrophy, partly thorough downregulation of α2-ARs. Although renal arterial hypertrophy in Atg -/- mice appears to be of multifactorial origin, TGF-β1 may play a key role in the persistence of such hypertrophy.

Renin-angiotensin system in vertebrates: phylogenetic view of structure and function.

Renin substrate, biological renin activity, and/or renin-secreting cells in kidneys evolved at an early stage of vertebrate phylogeny. Angiotensin (Ang) I and II molecules have been identified biochemically in representative species of all vertebrate classes, although variation occurs in amino acids at positions 1, 5, and 9 of Ang I. Variations have also evolved in amino acid positions 3 and 4 in some cartilaginous fish. Angiotensin receptors, AT1 and AT2 homologues, have been identified molecularly or characterized pharmacologically in nonmammalian vertebrates. Also, various forms of angiotensins that bypass the traditional renin-angiotensin system (RAS) cascades or those from large peptide substrates, particularly in tissues, are present. Nonetheless, the phylogenetically important functions of RAS are to maintain blood pressure/blood volume homeostasis and ion-fluid balance via the kidney and central mechanisms. Stimulation of cell growth and vascularization, possibly via paracrine action of angiotensins, and the molecular biology of RAS and its receptors have been intensive research foci. This review provides an overview of: (1) the phylogenetic appearance, structure, and biochemistry of the RAS cascade; (2) the properties of angiotensin receptors from comparative viewpoints; and (3) the functions and regulation of the RAS in nonmammalian vertebrates. Discussions focus on the most fundamental functions of the RAS that have been conserved throughout phylogenetic advancement, as well as on their physiological implications and significance. Examining the biological history of RAS will help us analyze the complex RAS systems of mammals. Furthermore, suitable models for answering specific questions are often found in more primitive animals.

Quantification of intact plasma AGT consisting of oxidized and reduced conformations using a modified ELISA.

The pleiotropic actions of the renin-angiotensin system (RAS) depend on the availability of angiotensinogen (AGT) which generates angiotensin I (ANG I) when cleaved by renin. Thus, quantification of the intact AGT (iAGT) concentrations is important to evaluate the actual renin substrate available. The iAGT conformation exists as oxidized AGT (oxi-AGT) and reduced AGT (red-AGT) in a disulfide bond, and oxi-AGT has a higher affinity for renin, which may exacerbate RAS-associated diseases. Accordingly, we determined iAGT, oxi-AGT, and red-AGT levels in plasma from rats and mice. Blood samples were obtained by cardiac puncture and then immediately mixed with an inhibitor solution containing a renin inhibitor. Total AGT (tAGT) levels were measured by tAGT ELISA which detects both cleaved and iAGT. iAGT levels were determined by iAGT ELISA which was found to only detect red-AGT. Thus, it was necessary to treat samples with dithiothreitol, a reducing agent, to quantify total iAGT concentration. tAGT levels in rat and mouse plasma were 1,839 ± 139 and 1,082 ± 77 ng/ml, respectively. iAGT levels were 53% of tAGT in rat plasma but only 22% in mouse plasma, probably reflecting the greater plasma renin activity in mice. The ratios of oxi-AGT and red-AGT were ∼4:1 (rat) and 16:1 (mouse). Plasma iAGT consists of oxi-AGT and red-AGT, suggesting that oxidative stress can influence ANG I generation by the AGT conformation switch. Furthermore, the lower availability of plasma iAGT in mice suggests that it may serve as a limiting factor in ANG I formation in this species.

3-Hydroxy-4-nitrobenzoic Acid as a MALDI Matrix for In-Source Decay

In-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) is a rapid sequencing method for peptides. 1,5-Diaminonaphthalene (1,5-DAN) is a most frequently used matrix for ISD. However, using 1,5-DAN generates mainly c- and z-series ions by N-Cα bond cleavage, which makes it difficult to distinguish leucine (Leu) and isoleucine (Ile), and frequently lacks c(n-1)-series ions owing to proline (Pro) at residues n. Several oxidizing matrices generating a- and x-series ions accompanied by d-series ions by Cα-C bond cleavage have been reported, but an issue remained concerning their sensitivity. 3-Hydroxy-4-nitrobenzoic acid (3H4NBA) has been reported as a matrix for 2-nitrobenzenesulfenyl-labeled peptides by Matsuo et al. (Proteomics 2006, 6, 2042-2049). Here, we used 3H4NBA as an oxidizing matrix for ISD. As a result, numerous a- and d-series ions for amyloid β 1-40 were generated with high sensitivity using 3H4NBA. Each of the two Leu and two Ile was identified by the d-series ions. The sensitivity of the a-series ions using 3H4NBA was a little lower than that of c-series ions using 1,5-DAN. The same tendency was observed for N-acetyl renin substrate and ACTH 18-39. The a-series ions were detected, even at the left side of Pro. The sensitivity of the a-series ions using 3H4NBA was higher than with other existing oxidizing matrices, such as 5-nitrosalicylic acid and 5-formyl salycilic acid. The ions were detected over the entire area of the matrix-analyte spot using 3H4NBA. 3H4NBA was confirmed to be a useful oxidizing matrix for ISD, leading to higher sequence coverage of peptides.

Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.

BackgroundAngiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system.ResultsWhen recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum of E. coli-expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. The Km values of both oANG preparations were similar; the kcat value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG.ConclusionsRecombinant oANG expressed in E. coli functions as a human renin substrate. This study presents an E. coli-based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0265-x) contains supplementary material, which is available to authorized users.

Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry

The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one μs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 μs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 μs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 μs is obtained for the formation of a β-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 μs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 μs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 μs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.

Novel renin inhibitors containing derivatives of N-alkylleucyl-β-hydroxy-γ-amino acids.

In search for new drugs lowering arterial blood pressure, which could be applied in anti-hypertensive therapy, research concerning agents blocking of renin-angiotensin-aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8-13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N-methylleucyl-β-hydroxy-γ-amino acids at the P2-P1' position: 4-[N-(N-methylleucyl)-amino]-3-hydroxy-7-(3-nitroguanidino)-heptanoic acid (AHGHA), 4-[N-(N-methylleucyl)-amino]-3-hydroxy-5-phenyl-pentanoic acid (AHPPA) or 4-[N-(N-methylleucyl)-amino]-8-benzyloxycarbonylamino-3-hydroxyoctanoic acid (AAHOA). The previously listed synthetic β-hydroxy-γ-amino acids constitute pseudodipeptidic units that correspond to the P1-P1' position of the inhibitor molecule. An unnatural amino acid, 4-methoxyphenylalanin (Phe(4-OMe)), was introduced at the P3 position of the obtained compounds. Three of these compounds contain isoamylamide of 6-aminohexanoic acid (ε-Ahx-Iaa) at the P2'-P3' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc-Phe(4-OMe)-MeLeu-AHGHA-OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10(-6)-10(-9) M. The compound Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa proved to be the most active (IC50 = 1.05 × 10(-9) M). The compounds Boc-Phe(4-OMe)-MeLeu-AHGHA-Ahx-Iaa and Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa are resistant to chymotrypsin.

Urinary Angiotensinogen is Increased in Human Heart Failure

Angiotensin II (AII) is an activator of the AT1 receptor and mediates deleterious actions in heart failure (HF) especially in the kidney. A major unmet need is a biomarker to assess intrarenal AII activity to optimize anti-AII therapies as well as serve as a possible biomarker for HF severity. Angiotensinogen (AGT) is the only known substrate of renin. Human AGT is converted to Angiotensin I by renin which is further converted by Angiotensin-converting enzyme to AII. Recent studies have established that AGT is present in proximal tubules and secreted into the tubular lumen and excreted in the urine. Increases in urinary AGT parallel increases in AII production in the kidney in renal disease and may be the key regulator of the renin angiotensin system (RAS) in the kidney. To date, urinary AGT in human HF is undefined. Urinary AGT is increased in patients with HF compared to normal human subjects. Our study recruited healthy subjects (Age 18-85) with no prescription medication use, and no history of HF or hypertension (n= 5) and hospitalized patients (Age 20 - 95) with acute decompensated HF (n= 5). Blood samples (EDTA) and 24 h urine samples were collected from normal and HF subjects. Twenty four hour urine samples were collected on ice and preserved with acetic acid. Blood was collected in EDTA vacutainers on ice. All aliquots were stored at -80°C until the time of assay. Angiotensinogen (AGT) was measured in EDTA-plasma and urine (24h) samples using the Human Total Angiotensinogen Assay Kit (IBL, Japan). Plasma AGT was expressed as mg/mL. Urinary AGT was normalized to respective urinary creatinine levels of each subject and expressed as AGT (ng/mg Ucre). Comparisons between two groups were performed using Student t-test. All values are expressed as Mean ±SD. P<0.05 was considered significant. Plasma AGT was not different between HF and normal subjects (56.0 ± 11.9 mg/mL vs 49.8 ±10.6 mg/mL). Importantly, urinary angiotensinogen in HF patients was increased compared to normal subjects (9.5 ±6.2 ng/mg Ucre vs 0.7 ± 0.3 ng/mg Ucre, P<0.05) and approximately 10 fold greater in HF than in normals. Urinary angiotensinogen is increased in HF patients in this proof of concept study when compared to normal subjects which may indicate increased intrarenal RAS activity. Further studies are needed to validate our findings and determine if use of urinary AGT may facilitate optimization of anti-AII therapies as well as serve as a renal biomarker for adverse outcomes in human HF.

Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.