RESUMO
Experimental inhibition of the (pro)renin receptor [(P)RR] is a promising therapeutic strategy in different disease models ranging from cardiorenal to oncological entities. Here, we briefly review the direct protein-protein interaction partners of the (P)RR and the plethora of distinct diseases in which the (P)RR is involved. The first structural work on the (P)RR using AlphaFold, which was recently published by Ebihara et al., is the center of this mini-review since it can mechanistically link the protein-protein interaction level with the pathophysiological level. More detailed insights into the 3D structure of the (P)RR and its interaction domains might guide drug discovery on this novel target. Finally, antibody- and small molecule-based approaches to inhibit the (P)RR are shortly discussed.
Assuntos
Receptor de Pró-Renina , Receptores de Superfície Celular , Humanos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Renina/química , Renina/metabolismo , Comunicação CelularRESUMO
Past several decades, therapeutic investigations lead to the discovery of numerous antihypertensive drugs. Although it has been proved for their potency, altered efficacy is common norms in several conditions due to genetic variations. Cytochrome P450 plays a crucial role in drug metabolism and responsible for the pharmacokinetic and pharmacodynamic properties of the drug molecules. Here, we report the deleterious point mutations in the genes associated with the altered response of antihypertensive drug molecules and their metabolizers. Missense variants were filtered as potential nonsynonymous single nucleotide polymorphisms among the available data for the target genes (REN, CYP2D6, CYP3A4). The key objective of the work is to identify the deleterious single nucleotide polymorphisms (SNPs) responsible for the drug response and metabolism for the application of personalized medication. The molecular docking studies revealed that Aliskiren and other clinically approved drug molecules have a high binding affinity with both wild and mutant structures of renin, CYP2D6, and CYP3A4 proteins. The docking (Glide XP) score was observed to have in the range of -8.896 to -11.693 kcal/mol. The molecular dynamics simulation studies were employed to perceive the structural changes and conformational deviation through various analyses. Each studied SNPs was observed to have disparate scoring in the binding affinity to the specific drug molecules. As a prospective plan, we assume this study might be applied to identify the risky SNPs associated with hypertension from the patients to recommend the suitable drug for personalized hypertensive treatment. Further, extensive clinical pharmacogenomics studies are required to support the findings.
Assuntos
Anti-Hipertensivos/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Renina/metabolismo , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Sítios de Ligação , Biologia Computacional , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Farmacogenética , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Renina/química , Renina/genéticaRESUMO
Pepsin, the archetypal pepsin-like aspartic protease, is irreversibly denatured when exposed to neutral pH conditions whereas renin, a structural homologue of pepsin, is fully stable and optimally active in the same conditions despite sharing highly similar enzyme architecture. To gain insight into the structural determinants of differential aspartic protease pH stability, the present study used comparative bioinformatic and structural analyses. In pepsin, an abundance of polar and aspartic acid residues were identified, a common trait with other acid-stable enzymes. Conversely, renin was shown to have increased levels of basic amino acids. In both pepsin and renin, the solvent exposure of these charged groups was high. Having similar overall acidic residue content, the solvent-exposed basic residues may allow for extensive salt bridge formation in renin, whereas in pepsin, these residues are protonated and serve to form stabilizing hydrogen bonds at low pH. Relative differences in structure and sequence in the turn and joint regions of the ß-barrel and ψ-loop in both the N- and C-terminal lobes were identified as regions of interest in defining divergent pH stability. Compared to the structural rigidity of renin, pepsin has more instability associated with the N-terminus, specifically the B/C connector. By contrast, renin exhibits greater C-terminal instability in turn and connector regions. Overall, flexibility differences in connector regions, and amino acid composition, particularly in turn and joint regions of the ß-barrel and ψ-loops, likely play defining roles in determining pH stability for renin and pepsin.
Assuntos
Pepsina A/química , Renina/química , Sequência de Aminoácidos , Aminoácidos , Animais , Biologia Computacional , Estabilidade Enzimática , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Estrutura Terciária de Proteína , Desdobramento de Proteína , Alinhamento de Sequência , Solventes/químicaRESUMO
RATIONALE: Human prorenin, representing the precursor of mature renin, has been discussed as a potential biomarker, e.g. in diagnosing primary hyperaldosteronism or diabetes-induced nephropathy. Currently, only immunoassays are available for prorenin quantification. As the similarity of prorenin to active renin impedes its accurate determination by immunoassay, mass spectrometry appears as an accurate alternative for differentiation of that protein. METHODS: Immunoaffinity purification plus a mixed-solvent-triggered digestion was combined with liquid chromatography/mass spectrometry (LC/MS) to enable a fast, sensitive, and less laboratory-intensive approach to the quantification of prorenin. Statistical experimental planning, which is known as Design of Experiments (DOE), was used to identify the optimal conditions for the generation of the signature peptides within a manageable number of experiments. The efficiency of the mixed-solvent-triggered digestion by trypsin was investigated using four different organic solvents: acetonitrile, acetone, tetrahydrofuran and methanol. RESULTS: By utilizing a D-optimal design, we found that the optimal mixed-solvent type for the generation of both signature peptides was acetonitrile at a concentration of 84% and an incubation temperature of 16°C. Using the mixed-solvent-triggered digestion, the procedure time allowed a fast analysis of active renin and prorenin with a short digestion time of 98 min. This optimized mixed-solvent-triggered digestion procedure was applied to detect renin and prorenin successfully in human plasma by the newly developed hybrid approach. CONCLUSIONS: The identification of unique surrogates for human prorenin enabled the mass spectrometric differentiation between the two similar proteins. The novel hybrid approach successfully proved its ability to purify, detect and distinguish between prorenin and active renin in human plasma.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Renina , Humanos , Imunoensaio , Limite de Detecção , Masculino , Renina/sangue , Renina/química , Renina/isolamento & purificação , Projetos de Pesquisa , Solventes/químicaRESUMO
Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin's prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.
Assuntos
Endocitose/fisiologia , Precursores Enzimáticos/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Renina/metabolismo , Amidas/farmacologia , Animais , Linhagem Celular Transformada , Células Epiteliais/metabolismo , Fumaratos/farmacologia , Humanos , Túbulos Renais Proximais/citologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley , Receptor IGF Tipo 2/antagonistas & inibidores , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Renina/química , Renina/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/farmacologia , Especificidade por Substrato , Temperatura , Tripsina/metabolismo , Saco Vitelino/citologia , Receptor de Pró-ReninaRESUMO
Renin is the rate-limiting step within the renin-angiotensin-aldosterone system, but the reliable quantification of human endogenous renin levels by liquid chromatography coupled with mass spectrometry remains challenging. The complex sample matrix triggering ion suppression and the detection of the low-abundance as well as the proteolytical-resistant renin make a hybrid approach using immunocapture coupled with LC-HRMS a promising method for investigation. Therefore, in-silico digestion and BLAST® experiments were conducted in order to identify the unique amino acid sequence for mass spectrometric detection. To enhance mass spectrometric response, impacting parameters within the denaturation, alkylation, and digestion experiments were identified and optimized by a multistep Design of Experiments process. The optimal denaturation buffer consisted of RapiGest® and urea, leading to a signature peptide intensity increase of 56% at 20⯰C, whereas the optimal reducing agent improved intensity by 27%. The most effective generation of signature peptide I was achieved using a high trypsin concentration and a low incubation temperature enhancing digestion by 75%. The applicability of this hybrid approach was confirmed in human matrix and allowed for a fivefold reduction in total assay procedure time without limiting the reliable quantification if compared to a conventional digestion approach.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Renina , Simulação por Computador , Humanos , Renina/sangue , Renina/química , Renina/metabolismo , Projetos de Pesquisa , Extração em Fase Sólida , Temperatura , Tripsina/metabolismoRESUMO
Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous renal disorder leading to progressive loss of renal function. ADTKD-REN is due to rare mutations in renin, all localized in the protein leader peptide and affecting its co-translational insertion in the endoplasmic reticulum (ER). Through exome sequencing in an adult-onset ADTKD family we identified a new renin variant, p.L381P, mapping in the mature protein. To assess its pathogenicity, we combined genetic data, computational and predictive analysis and functional studies. The L381P substitution affects an evolutionary conserved residue, co-segregates with renal disease, is not found in population databases and is predicted to be deleterious by in silico tools and by structural modelling. Expression of the L381P variant leads to its ER retention and induction of the Unfolded Protein Response in cell models and to defective pronephros development in zebrafish. Our work shows that REN mutations outside of renin leader peptide can cause ADTKD and delineates an adult form of ADTKD-REN, a condition which has usually its onset in childhood. This has implications for the molecular diagnosis and the estimated prevalence of the disease and points at ER homeostasis as a common pathway affected in ADTKD-REN, and possibly more generally in ADTKD.
Assuntos
Genes Dominantes , Mutação , Nefrite Intersticial/genética , Renina/genética , Adulto , Idade de Início , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Humanos , Masculino , Nefrite Intersticial/metabolismo , Nefrite Intersticial/patologia , Linhagem , Renina/químicaRESUMO
The objective of this was to determine the impact of enzymatic hydrolysis on the multifunctionality of tomato seed protein hydrolysates (TSPH) and their physicochemical properties. The enzymatic hydrolysis was performed using alcalase and two factors response surface methodology. The best conditions were 131.4 min and 3% enzyme/substrate (E/S) for antioxidant activity; 174.5 min and 2.93% E/S for angiotensin-converting enzyme (ACE) inhibition; and 66.79 min and 2.27% E/S for the calcium binding. Antioxidant and ACE hydrolysates were characterized by higher solubility, zeta potential, and thermal stability while properties of the calcium binding hydrolysate were only minimally affected by the enzymatic hydrolysis. Gel electrophoresis showed that molecular weights of polypeptides in the calcium binding TSPH were higher compared to those in ACE and antioxidant TSPHs. This was due to the low degree of hydrolysis of the calcium binding hydrolysate. PRACTICAL APPLICATIONS: Nowadays, different protein sources are used to produce protein hydrolysates containing bioactive peptides that can help alleviate oxidation of foods, oxidative stress, and chronic conditions (e.g., hypertension, diabetes, cardiovascular disorder). Hydrolyzed proteins also have the potential to increase mineral absorption through the formation of mineral-binding complexes. Biological activities of proteins and peptides from tomato processing byproduct (i.e., pomace) have received until now little attention. The determination of physicochemical properties and biological activities of the hydrolyzed proteins has application in the formulation of value-added food products for the reduction of oxidative stress and risks of developing chronic diseases. In addition, there will be a reduction of pomace waste generated by the tomato processing industry.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Antioxidantes/química , Extratos Vegetais/química , Proteínas de Plantas/química , Sementes/química , Solanum lycopersicum/química , Cálcio/química , Hidrólise , Peptídeos/química , Renina/química , Subtilisinas/químicaRESUMO
The renin-angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket.
Assuntos
Angiotensinogênio/química , Renina/química , Regulação Alostérica , Angiotensina I , Angiotensinogênio/genética , Angiotensinogênio/metabolismo , Cristalografia por Raios X , Humanos , Domínios Proteicos , Renina/genética , Renina/metabolismoRESUMO
Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides dose-dependently inhibited ACE and various synthetic X-Hyp-Gly-type tripeptides showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 µM, respectively. Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type tripeptides highlight their potential for hypertension control.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Colágeno/química , Peptídeos/química , Animais , Hidroxilação , Hidroxiprolina/química , Cinética , Camundongos , Simulação de Acoplamento Molecular , Peptídeos/sangue , Renina/química , Renina/metabolismoRESUMO
Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.
Assuntos
Amidas/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/química , Fumaratos/farmacologia , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/terapia , Renina/química , Amidas/uso terapêutico , Quimiotaxia/efeitos dos fármacos , Criança , Complemento C3/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C4/química , Complemento C5a/química , Complemento C5a/metabolismo , Complemento C5b/química , Complemento C5b/metabolismo , Fator B do Complemento/química , Fator D do Complemento/química , Feminino , Fumaratos/uso terapêutico , Membrana Basal Glomerular/patologia , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Mastócitos/fisiologia , Renina/antagonistas & inibidores , Renina/metabolismoRESUMO
Protein flexibility poses a major challenge to docking of potential ligands in that the binding site can adopt different shapes. Docking algorithms usually keep the protein rigid and only allow the ligand to be treated as flexible. However, a wrong assessment of the shape of the binding pocket can prevent a ligand from adapting a correct pose. Ensemble docking is a simple yet promising method to solve this problem: Ligands are docked into multiple structures, and the results are subsequently merged. Selection of protein structures is a significant factor for this approach. In this work we perform a comprehensive and comparative study evaluating the impact of structure selection on ensemble docking. We perform ensemble docking with several crystal structures and with structures derived from molecular dynamics simulations of renin, an attractive target for antihypertensive drugs. Here, 500 ns of MD simulations revealed binding site shapes not found in any available crystal structure. We evaluate the importance of structure selection for ensemble docking by comparing binding pose prediction, ability to rank actives above nonactives (screening utility), and scoring accuracy. As a result, for ensemble definition k-means clustering appears to be better suited than hierarchical clustering with average linkage. The best performing ensemble consists of four crystal structures and is able to reproduce the native ligand poses better than any individual crystal structure. Moreover this ensemble outperforms 88% of all individual crystal structures in terms of screening utility as well as scoring accuracy. Similarly, ensembles of MD-derived structures perform on average better than 75% of any individual crystal structure in terms of scoring accuracy at all inspected ensembles sizes.
Assuntos
Simulação de Acoplamento Molecular , Renina/química , Renina/metabolismo , Algoritmos , Sítios de Ligação , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Herein, the ability of ginger glycoproteins to inhibit the angiotensin-converting enzyme (ACE) is characterized. The activity is monitored via HPLC, and then the crude glycoproteins are enriched with lectin microarrays and magnetic microspheres. The N-linked glycans released from the enriched glycoproteins by PNGase F are identified by MALDI-TOF-MS. The results suggest that the crude ginger glycoproteins are active against ACE with an IC50 value of 0.83 ± 0.09 mg mL-1. The ginger glycoproteins are enriched by concanavalin A (Con A) and solanum tuberosum (Potato) lectin (STL), and the structures of the N-glycans released from the ginger glycoproteins include high-mannose type glycans, fucosylated-type glycans, and hybrid-type glycans, as analyzed by MALDI-TOF-MS. The results of this study are expected to provide a reference for the glycan structure of ginger glycoproteins with ACE-inhibitory activity.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Glicoproteínas/química , Proteínas de Plantas/química , Zingiber officinale/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Glicoproteínas/isolamento & purificação , Glicosilação , Proteínas de Plantas/isolamento & purificação , Polissacarídeos/química , Renina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
One way to control hypertension is inactivation of the Renin- Angiotensin- Aldosterone System (RAAS). Inhibition of renin as a rate-limiting step of this system is an effective way to stop up RAAS. It has been proved that soyasaponin I, an herbal compound obtained from soybeans, has anti-hypertensive effect via renin inhibition, so it has the potential of being an anti-hypertensive drug. Herein, some theoretical approaches such as Docking Simulation, Molecular Dynamics (MD) Simulation and MMPBSA analysis have been used to study how soyasaponin I inhibits renin at the structural level. The results of docking simulation and hydrogen bond pattern show that this ligand is able to bind to the active site of renin and a region near the active site. Results of MD simulation for renin - soyasaponin I complexes confirm that soyasaponin I binds to the active site of renin and has inhibition effect on it via competing with the substrate. Besides, according to MMPBSA analysis, the binding free energy for renin - soyasaponin I complex is -42.61 kcal/mol when it binds to the active site. Comparing to the peptide obtained from angiotensinogen, ΔG = -74.96 kcal/mol, it may inferred that although binding of soyasaponin I to the active site of renin does not have a complete competition with the substrate, it might attenuate the formation of renin - angiotensinogen complex and have partial non-competitive effect. The results of this survey might be helpful to design partial non - competitive renin inhibitors with pharmaceutical capability.
Assuntos
Hipertensão/prevenção & controle , Ácido Oleanólico/análogos & derivados , Sistema Renina-Angiotensina/efeitos dos fármacos , Renina/antagonistas & inibidores , Saponinas/farmacologia , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Domínio Catalítico , Humanos , Hipertensão/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacologia , Ligação Proteica , Renina/química , Renina/metabolismo , Saponinas/química , Saponinas/metabolismo , TermodinâmicaRESUMO
Body fluid volume regulation is a complex process involving the interaction of various afferent (sensory) and neurohumoral efferent (effector) mechanisms. Historically, most studies focused on the body fluid dynamics in heart failure (HF) status through control of the balance of sodium, potassium, and water in the body, and maintaining arterial circulatory integrity is central to a unifying hypothesis of body fluid regulation in HF pathophysiology. The pathophysiologic background of the biochemical determinants of vascular volume in HF status, however, has not been known. I recently demonstrated that changes in vascular and red blood cell volumes are independently associated with the serum chloride concentration, but not the serum sodium concentration, during worsening HF and its recovery. Based on these observations and the established central role of chloride in the renin-angiotensin-aldosterone system, I propose a unifying hypothesis of the "chloride theory" for HF pathophysiology, which states that changes in the serum chloride concentration are the primary determinant of changes in plasma volume and the renin-angiotensin-aldosterone system under worsening HF and therapeutic resolution of worsening HF.
Assuntos
Líquidos Corporais/metabolismo , Cloretos/química , Insuficiência Cardíaca/fisiopatologia , Rim/metabolismo , Volume Sanguíneo , Eletrólitos , Hemodinâmica , Humanos , Modelos Teóricos , Renina/química , Sistema Renina-Angiotensina , Sódio/químicaRESUMO
In this work, the inhibitory effects of potato patatin-derived peptides Trp-Gly (WG) and Pro-Arg-Tyr (PRY) on angiotensin I-converting enzyme (ACE) and renin activities were investigated using kinetics, intrinsic fluorescence and molecular docking. The results indicated that PRY was a more potent ACE- and renin-inhibitory peptide than WG. Enzyme inhibition kinetics showed that WG and PRY inhibited ACE activity through mixed-type and competitive modes, respectively. The inhibitory mechanism of WG and PRY towards renin was determined to be mixed-type. PRY exhibited stronger affinity towards ACE and renin molecules, when compared to WG as determined by intrinsic fluorescence intensity. Molecular docking data confirmed that the higher inhibitory potency of PRY might be attributed to formation of more hydrogen bonds with the enzyme's active site or non-active sites that distorted the configuration necessary for catalysis.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Hidrolases de Éster Carboxílico/química , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Proteínas de Plantas/química , Renina/antagonistas & inibidores , Renina/metabolismo , Domínio Catalítico , Humanos , Cinética , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/química , Renina/químicaRESUMO
BACKGROUND: An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities. RESULTS: Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTSâ¢+ to a three-fold greater extent compared to the isolate. CONCLUSION: BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Fabaceae/química , Sequestradores de Radicais Livres/química , Proteínas de Plantas/química , Radicais Livres/química , Cinética , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/química , Renina/químicaRESUMO
BACKGROUND: The function of prorenin, the inactive precursor of renin, remains unclear after many decades of research. The discovery of a (pro)renin receptor suggested that prorenin, by binding to this receptor, would become active, that is, obtain an 'open' conformation. However, the receptor only interacted with prorenin at levels that were many orders of magnitude above its normal levels, making such interaction in-vivo unlikely. Prorenin occurs in two conformations, an open, active form, and a closed, inactive form. Under physiological conditions (pH 7.4, 37â°C), virtually all prorenin occurs in the closed conformation. This study investigated to what degree prorenin-synthesizing cells release prorenin in an open conformation. METHODS AND RESULTS: Renin plus prorenin-synthesizing human mast cells, and prorenin-synthesizing HEK293 cells (transfected with the mammalian expression vector pRhR1100, containing human prorenin) and human decidua cells were incubated with the renin inhibitor VTP-27999. This inhibitor will trap open prorenin, as after VTP-27999 binding, prorenin can no longer return to its closed conformation, thus allowing its detection in a renin immunoradiometric assay. No evidence for the release of open prorenin was found. Moreover, incubating decidua cells with angiotensinogen yielded low angiotensin levels, corresponding with the activity of ≈1% of prorenin in the medium, that is, the amount of open prorenin expected based upon the equilibrium between open and closed prorenin under physiological conditions. CONCLUSION: Our study does not reveal evidence for the release of open, active prorenin by prorenin-synthesizing cells, at least under cell culture conditions. This argues against prorenin activity at the site of its release.
Assuntos
Renina/biossíntese , Renina/química , Adulto , Angiotensinogênio/farmacologia , Angiotensinas/metabolismo , Anti-Hipertensivos , Carbamatos/farmacologia , Decídua/citologia , Decídua/efeitos dos fármacos , Decídua/metabolismo , Feminino , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Pessoa de Meia-Idade , Piperidinas/farmacologia , Renina/antagonistas & inibidoresRESUMO
BACKGROUND: Angiotensinogen (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. RESULTS: When 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 K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG. CONCLUSIONS: Recombinant 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.
Assuntos
Angiotensinogênio/metabolismo , Escherichia coli/genética , Proteínas Recombinantes/metabolismo , Renina/metabolismo , Angiotensinogênio/química , Angiotensinogênio/genética , Angiotensinogênio/isolamento & purificação , Animais , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Renina/química , OvinosRESUMO
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-ß-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 ß-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.