RESUMEN
Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.
Asunto(s)
Proteínas de Transporte de Membrana/fisiología , Percepción de Quorum/fisiología , Trypanosoma/metabolismo , Diferenciación Celular , Secuencia Conservada/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Transporte de Membrana/genética , Oligopéptidos/genética , Oligopéptidos/fisiología , Filogenia , Proteínas Protozoarias/metabolismo , Percepción de Quorum/genética , Transducción de Señal , Trypanosoma/fisiología , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/parasitología , Virulencia/fisiologíaRESUMEN
The emergence of multidrug-resistant bacterial pathogens is a growing threat to global public health. Here, we report the development and characterization of a panel of nine-amino acid residue synthetic peptides that display potent antibacterial activity and the ability to disrupt preestablished microbial biofilms. The lead peptide (Peptide K6) showed bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus in culture and in monocultures and mixed biofilms in vitro. Biophysical analysis revealed that Peptide K6 self-assembled into nanostructured micelles that correlated with its strong antibiofilm activity. When surface displayed on the outer membrane protein LamB, two copies of the Peptide K6 were highly bactericidal to Escherichia coli. Peptide K6 rapidly increased the permeability of bacterial cells, and resistance to this toxic peptide occurred less quickly than that to the potent antibiotic gentamicin. Furthermore, we found that Peptide K6 was safe and effective in clearing mixed P. aeruginosa-S. aureus biofilms in a mouse model of persistent infection. Taken together, the properties of Peptide K6 suggest that it is a promising antibiotic candidate and that design of additional short peptides that form micelles represents a worthwhile approach for the development of antimicrobial agents.
Asunto(s)
Antibacterianos , Coinfección , Animales , Ratones , Antibacterianos/farmacología , Micelas , Staphylococcus aureus , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Biopelículas , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosaRESUMEN
Macropinocytosis mediates non-selective bulk uptake of extracellular fluid. It is the major route by which axenic Dictyostelium cells obtain nutrients and has emerged as a nutrient-scavenging pathway in mammalian cells. How environmental and cellular nutrient status modulates macropinocytic activity is not well understood. By developing a high-content imaging-based genetic screen in Dictyostelium discoideum we identified Slc15A, an oligopeptide transporter located at the plasma membrane and early macropinosome, as a novel macropinocytosis regulator. We show that deletion of slc15A but not two other related slc15 genes, leads to reduced macropinocytosis, reduced cell growth and aberrantly increased autophagy in cells grown in nutrient-rich medium. Expression of Slc15A protein or supplying cells with free amino acids rescues these defects. In contrast, expression of transport-defective Slc15A or supplying cells with amino acids in their di-peptide forms fails to rescue these defects. Therefore, Slc15A modulates the level of macropinocytosis by maintaining the intracellular availability of key amino acids through extraction of oligopeptides from the early macropinocytic pathway. We propose that Slc15A constitutes part of a positive feedback mechanism coupling cellular nutrient status and macropinocytosis. This article has an associated First Person interview with the first authors of the paper.
Asunto(s)
Dictyostelium , Animales , Dictyostelium/genética , Endosomas , Humanos , Mamíferos , Nutrientes , Oligopéptidos , PinocitosisRESUMEN
Iron (Fe) needs to be delivered to different organs and tissues of above-ground parts for playing its multiple physiological functions once it is taken up by the roots. However, the mechanisms underlying Fe distribution are poorly understood. We functionally characterized OsOPT7, a member of oligo peptide transporter family in terms of expression patterns, localization, transport activity and phenotypic analysis of knockdown lines. OsOPT7 was highly expressed in the nodes, especially in the uppermost node I, and its expression was upregulated by Fe-deficiency. OsOPT7 transports ferrous iron into the cells coupled with proton. Immunostaining revealed that OsOPT7 is mainly localized in the xylem parenchyma cells of the enlarged vascular bundles in the nodes and vascular tissues in the leaves. Knockdown of OsOPT7 did not affect the Fe uptake, but altered Fe distribution; less Fe was distributed to the new leaf, upper nodes and developing panicle, but more Fe was distributed to the old leaves. Furthermore, knockdown of OsOPT7 also resulted in less Fe distribution to the leaf sheath, but more Fe to the leaf blade. Taken together, OsOPT7 is involved in the xylem unloading of Fe for both long-distance distribution to the developing organs and local distribution within the leaf in rice.
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Hierro , Oryza , Proteínas de Plantas , Xilema , Transporte Biológico , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Oryza/genética , Oryza/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Xilema/metabolismoRESUMEN
Bisacridinyl-bisarginyl porphyrin (BABAP) is a trisintercalating derivative of a tricationic porphyrin, formerly designed and synthesized in order to selectively target and photosensitize the ten-base pair palindromic sequence d(CGGGCGCCCG)2 . We resorted to the previously derived (Far etâ al., 2004) lowest energy-minimized (EM) structure of the BABAP complex with this sequence as a starting point. We performed polarizable molecular dynamics (MD) on this complex. It showed, over a 150â ns duration, the persistent binding of the Arg side-chain on each BABAP arm to the two G bases upstream from the central porphyrin intercalation site. We subsequently performed progressive shortenings of the connector chain linking the Arg-Gly backbone to the acridine, from n=6 methylenes to 4, followed by removal of the Gly backbone and further connector shortenings, from n=4 to n=1. These resulted into progressive deformations ('kinks') of the DNA backbone. In its most accented kinked structure, the DNA backbone was found to have a close overlap with that of DNA bound to Cre recombinase, with, at the level of one acridine intercalation site, negative roll and positive tilt values consistent with those experimentally found for this DNA at its own kinked dinucleotide sequence. Thus, in addition to their photosensitizing properties, some BABAP derivatives could induce sequence-selective, controlled DNA deformations, which are targets for cleavage by endonucleases or for repair enzymes.
Asunto(s)
Simulación de Dinámica Molecular , Porfirinas , Porfirinas/química , ADN/química , Oligopéptidos , AcridinasRESUMEN
Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.
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Acetilcisteína , Sobrecarga de Hierro , Oligopéptidos , Animales , Masculino , Ratas , Acetilcisteína/farmacología , Acetilcisteína/metabolismo , Caspasas/metabolismo , Claudinas/genética , Giro Dentado/metabolismo , Giro Dentado/patología , Dextranos/metabolismo , Dextranos/farmacología , Regulación hacia Abajo , Glutatión/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Hierro/metabolismo , Hierro/farmacología , Sobrecarga de Hierro/complicaciones , Sobrecarga de Hierro/tratamiento farmacológico , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/farmacología , Nestina/genética , Nestina/metabolismo , Nestina/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Regulación hacia Arriba , Oligopéptidos/farmacología , Hemo-Oxigenasa 1/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismoRESUMEN
This study reports the formation of self-assembled nanostructures with homo-oligopeptides consisting of amino acids (i.e., alanine, threonine, valine, and tyrosine), the resulting morphologies (i.e., spherical shape, layered structure, and wire structure) in aqueous solution, and their potential as ice growth inhibitors. Among the homo-oligopeptides investigated, an alanine homo-oligopeptide (n = 5) with a spherical nanostructure showed the highest ice recrystallization inhibition (IRI) activity without showing a burst ice growth property and with low ice nucleation activity. The presence of nanoscale self-assembled structures in the solution showed superior IRI activity compared to an amino acid monomer because of the higher binding affinity of structures on the growing ice crystal plane. Simulation results revealed that the presence of nanostructures induced a significant inhibition of ice growth and increased lifetime of hydrogen bonding compared with unassembled homo-oligopeptide. These results envision extraordinary performance for self-assembled nanostructures as a desirable and potent ice growth inhibitor.
Asunto(s)
Proteínas Anticongelantes , Hielo , Proteínas Anticongelantes/química , Cristalización , Aminoácidos , Alanina , OligopéptidosRESUMEN
Cutibacterium acnes is abundant and commonly exists as a superficial bacteria on human skin. Recently, the resistance of C. acnes to antimicrobial agents has become a serious concern, necessitating the development of alternative pharmaceutical products with antimicrobial activity against C. acnes. To address this need, we evaluated the antimicrobial activity of CKR-13-a mutant oligopeptide of FK-13 with increased net charge and theoretical α-helical content-against C. acnes in modified Gifu Anaerobic Medium broth by determining the minimum inhibitory concentration (MIC). CKR-13 exerted greater antimicrobial activity against C. acnes than FK-13 in the broth at pH 7.0. The antimicrobial activity of CKR-13 with RXM against C. albicans was pH-dependent. The ionization of CKR-13 and pH-dependent growth delay of C. albicans was suggested to be associated with the increase in CKR-13 antimicrobial activity.
Asunto(s)
Candida albicans , Pruebas de Sensibilidad Microbiana , Oligopéptidos , Oligopéptidos/química , Oligopéptidos/farmacología , Candida albicans/efectos de los fármacos , Conformación Proteica en Hélice alfa , Propionibacteriaceae/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Antiinfecciosos/farmacología , Antiinfecciosos/química , Antibacterianos/farmacología , Antibacterianos/químicaRESUMEN
Candida tropicalis is often reported as the second or third most common pathogen causing fungal infections. Antimicrobial peptides (AMPs) have attracted increasing attention for their broad-spectrum antimicrobial properties and low cytotoxicity. Our previous studies have shown that CGA-N9, a non-membrane-rupturing AMP, crosses the cell membrane to exert anticandidal activity. We speculate that there are some related transporters that assist in the transmembrane transport of CGA-N9. In this study, the relationship between CGA-N9 lethality kinetics and its real-time transmembrane amount in C. tropicalis cells was investigated. The results demonstrated that there was a positive correlation between its candicidal activity and transmembrane amount. A total of 12 oligopeptide transporter (OPT) coding sequences (CDSs) were cloned from C. tropicalis by using the conservative OPT gene sequences of Candida spp. to design primers and were named C. tropicalis OPTs (CtOPTs). The results of RTâqPCR demonstrated that the expression levels of CtOPT1, CtOPT9 and CtOPT12 were correlated with the CGA-N9 transmembrane amount in a time-dependent manner. The results of molecular docking demonstrated that CtOPT1, CtOPT9 and CtOPT12 interact strongly with CGA-N9. Therefore, CtOPT1, CtOPT9 and CtOPT12 were predicted to assist in the transmembrane transport of the AMP CGA-N9.
Asunto(s)
Péptidos Antimicrobianos , Candida tropicalis , Candida tropicalis/genética , Candida tropicalis/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Oligopéptidos/metabolismo , Antifúngicos/farmacología , Antifúngicos/metabolismoRESUMEN
IMPORTANCE: Limited nitrogen supply can prevent the completion of alcoholic fermentation. Supplementation through peptides as an alternative, natural source of nitrogen for yeast offers an interesting solution for this issue. In this work, the S. cerevisiae peptide transporters of the Opt and Fot families were studied. We demonstrated that Fot and Opt2 have a broader peptide length preference than previously reported, enabling yeasts to acquire sufficient nitrogen from peptides without requiring additional ammonia or amino acids to complete fermentation. On the contrary, Opt1 was unable to consume any peptide in the given conditions, whereas it has been described elsewhere as the main peptide transporter for peptides longer than three amino acid residues in experiments in laboratory conditions. This controversy signifies the need in applied sciences for approaching experimental conditions to those prevalent in the industry for its more accurate characterization. Altogether, this work provides further evidence of the importance of peptides as a nitrogen source for yeast and their consequent positive impact on fermentation kinetics.
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Saccharomyces cerevisiae , Vino , Humanos , Saccharomyces cerevisiae/metabolismo , Nitrógeno/metabolismo , Transporte Biológico , Oligopéptidos/metabolismo , FermentaciónRESUMEN
We have been interested in the development of rubisco-based biomimetic systems for reversible CO2 capture from air. Our design of the chemical CO2 capture and release (CCR) system is informed by the understanding of the binding of the activator CO2 (A CO2 ) in rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). The active site consists of the tetrapeptide sequence Lys-Asp-Asp-Glu (or KDDE) and the Lys sidechain amine is responsible for the CO2 capture reaction. We are studying the structural chemistry and the thermodynamics of CO2 capture based on the tetrapeptide CH3 CO-KDDE-NH2 ("KDDE") in aqueous solution to develop rubisco mimetic CCR systems. Here, we report the results of 1 H NMR and 13 C NMR analyses of CO2 capture by butylamine and by KDDE. The carbamylation of butylamine was studied to develop the NMR method and with the protocol established, we were able to quantify the oligopeptide carbamylation at much lower concentration. We performed a pH profile in the multi equilibrium system and measured amine species and carbamic acid/carbamate species by the integration of 1 H NMR signals as a function of pH in the range 8≤pH≤11. The determination of ΔG1 (R) for the reaction R-NH2 +CO2 â â ${ \mathbin{{\stackrel{\textstyle\rightarrow} { {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss}}}} } }} }$ R-NH-COOH requires the solution of a multi-equilibrium equation system, which accounts for the dissociation constants K2 and K3 controlling carbonate and bicarbonate concentrations, the acid dissociation constant K4 of the conjugated acid of the amine, and the acid dissociation constant K5 of the alkylcarbamic acid. We show how the multi-equilibrium equation system can be solved with the measurements of the daughter/parent ratio X, the knowledge of the pH values, and the initial concentrations [HCO3 - ]0 and [R-NH2 ]0 . For the reaction energies of the carbamylations of butylamine and KDDE, our best values are ΔG1 (Bu)=-1.57â kcal/mol and ΔG1 (KDDE)=-1.17â kcal/mol. Both CO2 capture reactions are modestly exergonic and thereby ensure reversibility in an energy-efficient manner. These results validate the hypothesis that KDDE-type oligopeptides may serve as reversible CCR systems in aqueous solution and guide designs for their improvement.
RESUMEN
This mini-review describes the role of the solute carrier (SLC)15 family of proton-coupled oligopeptide transporters (POTs) and particularly Pept2 (Slc15A2) and PhT1 (Slc15A4) in the brain. That family transports endogenous di- and tripeptides and peptidomimetics but also a number of drugs. The review focuses on the pioneering work of David E. Smith in the field in identifying the impact of PepT2 at the choroid plexus (the blood-CSF barrier) as well as PepT2 and PhT1 in brain parenchymal cells. It also discusses recent findings and future directions in relation to brain POTs including cellular and subcellular localization, regulatory pathways, transporter structure, species differences and disease states.
Asunto(s)
Simportadores , Simportadores/metabolismo , Protones , Transporte Biológico , Proteínas de Transporte de Membrana/metabolismo , Oligopéptidos/metabolismo , Encéfalo/metabolismoRESUMEN
To investigate the effects of bonito oligopeptide SEP-3 on the repair of liver damage and regulation of liver biorhythm in sleep-deprived mice (SDM), C57BL/6 male mice were subjected to sleep deprivation by modified multi-platform water environment method, and were given different doses of bonito oligopeptide SEP-3 in groups. To determine the liver organ index, liver tissue-related apoptotic protein levels, Wnt/ß-Catenin pathway-related protein expression levels, serum alanine transaminase (ALT), glutamicum transaminase (AST), glucocorticoid (GC), and adrenocorticotropin (ACTH) content in each group of mice, four time points were selected to examine the mRNA expression levels of circadian clock-related genes in mouse liver tissue. The results showed that low, medium, and high doses of SEP-3 significantly increased SDM, ALT, and AST (p < 0.05), and medium and high doses of SEP-3 significantly reduced SDM liver index and GC and ACTH. As SEP-3 increased the apoptotic protein and Wnt/ß-Catenin pathway, mRNA expression gradually tended to normal (p < 0.05). This suggests that sleep deprivation can cause excessive oxidative stress in mice, which can lead to liver damage. Additionally, oligopeptide SEP-3 achieves the repair of liver damage by inhibiting SDM hepatocyte apoptosis, activating liver Wnt/ß-Catenin pathway, and promoting hepatocyte proliferation and migration, and suggests that oligopeptide SEP-3 is closely related to repair of liver damage by regulating the biological rhythm of SDM disorder.
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Privación de Sueño , beta Catenina , Ratones , Masculino , Animales , beta Catenina/metabolismo , Privación de Sueño/complicaciones , Privación de Sueño/metabolismo , Ratones Endogámicos C57BL , Hígado , Apoptosis , Estrés Oxidativo , Oligopéptidos/farmacología , Oligopéptidos/metabolismo , ARN Mensajero/metabolismoRESUMEN
The oxidative state of intestinal tracts of healthy animals were investigated after short-term intake of half-fin anchovy hydrolysates (HAHp) and their thermal or Maillard reaction products (MRPs). After one month of continuous oral gavage of HAHp, HAHp-heated products (HAHp-H), the MRPs of HAHp with 3% of glucose (HAHp-3%G MRPs), and the MRPs of HAHp with 3% of fructose (HAHp-3%F MRPs) at a dose of 1.0 g/kg of body weight per day into healthy ICR male mice, the concentrations of serum low-density and high-density lipoprotein cholesterol did not significantly change compared to the control group (CK, gavage with saline). Similar results were found for the interleukin-6 concentrations of all groups. By comparison, HAHp-H, HAHp-3%G MRPs, and HAHp-3%F MRPs administration decreased serum tumor necrosis factor-α concentration as compared to the CK group (p < 0.05). No histological damage was observed in the jejunum, ileum, and colonic tissues of all groups. However, HAHp-H treatment induced higher upregulation of Kelch-like ECH-associated protein 1, transcription factors Nrf-2, associated protective phase-II enzymes of NAD(P)H: quinine oxidoreductase-1, and hemoxygenase-1 in colon tissue, as well as higher upregulation of endogenous antioxidant enzymes, including copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase 2 than other groups (p < 0.05). Additionally, increases in Nε-carboxymethyllysine expression in the colonic tissues of all groups were consistent with their increased oligopeptide transporter 1 expressions. Our results suggest that the thermal products of HAHp might have a broad application prospect in improving antioxidant defense in vivo in healthy animals.
Asunto(s)
Antioxidantes , Reacción de Maillard , Ratones , Animales , Masculino , Antioxidantes/farmacología , Ratones Endogámicos ICR , Peces/metabolismo , Productos Finales de Glicación AvanzadaRESUMEN
In this project, we combine two areas of research, experimental characterization and molecular docking studies of the interaction of positively charged oligopeptides with crucial blood plasma proteins. The investigated peptides are rich in NH2 groups of amino acid side chains from Dap, Orn, Lys, and Arg residues, which are relevant in protein interaction. The peptides are 9- and 11-mer with the following sequences: (Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt), (Lys-Dab-Ala-Gly-Orn-Pro-His-Lys-Arg), and (Lys-Dab-Dab-Gly-Orn-Pro-Phe(2-F)-Lys-Arg). The net charge of the compound strongly depends on the pH environment and it is an important aspect of protein binding. The studied oligopeptides exhibit therapeutic properties: anti-inflammatory activity and the capacity to diminish reactive oxygen species (ROS). Therefore, the mechanism of potential binding with blood plasma components is the next challenge. The binding interaction has been investigated under pseudo-physiological conditions with the main blood plasma proteins: albumin (BSA), α1-acid glycoprotein (AAG), and γ-globulin fraction (GGF). The biomolecular quenching constant (kq) and binding constant (Kb) were obtained by fluorescence spectroscopy at various temperatures. Simultaneously, the changes in the secondary structure of proteins were monitored by circular dichroism (CD) and infrared spectroscopy (IR) by quantity analysis. Moreover, molecular docking studies were conducted to estimate the binding affinity, the binding domain, and the chemical nature of these interactions. The results show that the investigated oligopeptides could be mainly transported by albumin, and the binding domain I is the most favored cavity. The BSA and GGF are able to form stable complexes with the studied compounds as opposed to AAG. The binding reactions are spontaneous processes. The highest binding constants were determined for Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt peptide, in which the values of the binding constants Kb to BSA and GGF were 10.1 × 104 dm3mol-1 and 3.39 × 103 dm3mol-1, respectively. The positively charged surface of peptides participated in salt bridge interaction with proteins; however, hydrogen bonds were also formed. The secondary structure of BSA and GGF after contact with peptides was changed. A reduction in the α-helix structure was observed with an increase in the ß-sheet and ß-turn and random coil structures.
Asunto(s)
Oligopéptidos , Péptidos , Secuencia de Aminoácidos , Simulación del Acoplamiento Molecular , Péptidos/química , Oligopéptidos/química , Lisina/química , Proteínas Sanguíneas , AlbúminasRESUMEN
Antibodies recognizing RBD and the S-protein have been previously demonstrated to be formed in humans after SARS-CoV-2 infection and vaccination with the Sputnik V adenovirus vaccine. These antibodies were found to be active when hydrolyzing FITC-labeled oligopeptides corresponding to linear epitopes of the S-protein. The thin-layer chromatography method allows the relative accumulation of the reaction product to be estimated but cannot identify hydrolysis sites. This study used the MALDI-TOF MS method to establish oligopeptide hydrolysis sites. Using the MALDI-TOF MS method in combination with the analysis of known hydrolysis sites characteristic of canonical proteases allowed us to establish the unique hydrolysis sites inherent only to catalytically active antibodies. We have discovered two 12-mer oligopeptides to have six hydrolysis sites equally distributed throughout the oligopeptide. The other three oligopeptides were found to have two to three closely spaced hydrolysis sites. In contrast to trypsin and chymotrypsin proteases, the catalytically active antibodies of COVID-19 patients have their peptide bond hydrolyzed mainly after proline, threonine, glycine, or serine residues. Here, we propose a new high-throughput experimental method for analyzing the proteolytic activity of natural antibodies produced in viral pathology.
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COVID-19 , SARS-CoV-2 , Humanos , Hidrólisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Anticuerpos , Oligopéptidos , Péptido Hidrolasas , Anticuerpos AntiviralesRESUMEN
Poly(amidoamine) (PAMAM) dendrimers have attracted considerable attention in the field of gene therapy due to their flexibility in introducing different functional moieties and reduced toxicity at low generations. However, their transfection efficiency remains a limitation. Therefore, an essential approach for improving their transfection efficiency as gene carriers involves modifying the structure of PAMAM by conjugating functional groups around their surface. In this study, we successfully conjugated an RRHRH oligopeptide to the surface of PAMAM generation 2 (PAMAM G2) to create RRHRH-PAMAM G2. This construction aims to condense plasmid DNA (pDNA) and facilitate its penetration into cell membranes, leading to its promising potential for gene therapy. RRHRH-PAMAM G2/pDNA complexes were smaller than 100 nm and positively charged. Nano-polyplexes can enter the cell and show a high transfection efficiency after 24 h of transfection. The RRHRH-PAMAM G2 was non-toxic to HeLa, NIH3T3, A549, and MDA-MB-231 cell lines. These results strongly suggest that RRHRH-PAMAM G2 holds promise as a gene carrier for gene therapy owing to its biocompatibility and ability to deliver genes to the cell.
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Dendrímeros , Ratones , Animales , Humanos , Dendrímeros/química , Células 3T3 NIH , ADN/química , Plásmidos/genética , Transfección , Oligopéptidos/químicaRESUMEN
BACKGROUND: sh-oligopeptide-1 is a cosmetic that is considered to be the bioequivalent of the Epidermal Growth Factor (EGF). OBJECTIVE: The systematic search and selection of the information available on sh-oligopeptide-1, and field research on laboratories manufacturing cosmetics and the legal regulation of their products. METHODS: Specialised search in Medline and Google Scholar (March 2023). A total of 22 references were obtained and 12 articles were selected for analysis, 7 of which were included in Medline. 9 articles correspond to clinical trials that use sh-oligopeptide-1 as equivalent to a functional recombinant human EGF (hr-EGF). The manufacturers are investigated and they are requested to provide scientific information. RESULTS: Using an inactive ingredient, clinical trials are published with favourable results in order to treat diabetics and cancer patients. These trials do not follow medical standards. Active EGF is not a cosmetic, rather it is a potent unauthorised drug. Likewise, sh-oligopeptide-1 is not a functional EGF, and it is not authorised for medical treatments. Topical hr-EGF is not authorised as a medication in any concentration, except for clinical trials. However, sh-EGF (sh-oligopeptide-1) is authorised as a cosmetic in several different concentrations for generalised use, with unknown long-term risks. CONCLUSION: Clinical studies on sh-oligopeptide-1 have no scientific basis, as the preclinical bioactivity of this molecule has not been proven.
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Cosméticos , Factor de Crecimiento Epidérmico , Humanos , Oligopéptidos , FraudeRESUMEN
Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.
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Lipoproteínas , Thermococcus , Proteínas Bacterianas/química , Proteínas Portadoras/química , Lipoproteínas/química , Proteínas de Transporte de Membrana/metabolismo , Oligopéptidos/química , Péptidos/metabolismoRESUMEN
The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.