RESUMEN
Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.
Asunto(s)
Unión Proteica , Conformación Proteica , ARN Bicatenario , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/química , Dominios Proteicos , Humanos , Conformación de Ácido Nucleico , Modelos MolecularesRESUMEN
Fabrication of gold nanoparticles (GNPs) with phytochemicals is an emerging green nanotechnology approach with therapeutic implications. Garlic, known for its culinary and medicinal properties, has been extensively investigated for its anticancer properties. Here, we report a method to substantially enhance the antiproliferative potency of garlic by functionalizing its phytochemicals to GNPs and demonstrate a possible mechanism of action of these nanoparticles in the triple-negative breast cancer cell line, MDA-MB-231. Garlic gold nanoparticles (As-GNPs) were synthesized using garlic extract (As-EX) and gold chloride and characterized using a variety of spectroscopy techniques, and transmission electron microscopy (TEM). Compared to As-EX, which has a negligible effect on the viability of the cells, As-GNPs inhibited cell viability with an IC50of 0.310 ± 0.04 mg ml-1and strongly inhibited the clonogenic and migratory propensities of these cells. As indicated by TEM, the As-GNPs entered the cells via endocytosis and dispersed in the cellular milieu. Since tubulin, the protein involved in cell division, is a verified target for several antiproliferative drugs, we next examined whether the As-GNPs interact with this protein. The As-GNPs showed concentration-dependent binding to purified tubulin, slightly but consistently perturbing its secondary helical integritywithout grossly damaging the tertiary structure of the protein or the net polymer mass of the microtubules, as indicated by a tryptophan-quenching assay, far UV-circular dichroism spectroscopy, anilinonaphthalene sulfonate-binding assay, and polymer mass analysis, respectively. In cells, As-GNPs killed the cancer cells without cell cycle arrest, as evidenced by flow cytometry.
Asunto(s)
Proliferación Celular , Supervivencia Celular , Ajo , Oro , Nanopartículas del Metal , Neoplasias de la Mama Triple Negativas , Humanos , Ajo/química , Oro/química , Nanopartículas del Metal/química , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fitoquímicos/farmacología , Fitoquímicos/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Femenino , Antineoplásicos/farmacología , Antineoplásicos/química , Tubulina (Proteína)/metabolismo , Movimiento Celular/efectos de los fármacos , Microscopía Electrónica de TransmisiónRESUMEN
The current trend in the biopharmaceutical market has boosted the development and production of biological drugs with high efficacy and fidelity for receptor binding. While high-resolution structural insights into binding epitopes of the receptor are indispensable for better therapeutic design, it is tedious and costly. In this work, we develop a protocol by integrating two well-known NMR-based solution-state methods. Saturation transfer double-difference with methyl-TROSY (STDD-Methyl TROSY NMR) was used to probe methyl binding epitopes of the ligand in a label-free environment. This study was carried out with Human insulin as a model peptide drug, with the insulin growth factor receptor (IGFR), which is an off-target receptor for insulin. Methyl epitopes identified from STDD-Methyl TROSY NMR spectroscopy were validated through the HADDOCK platform to generate a drug-receptor model. Since this method can be applied at natural abundance, it has the potential to screen a large set of peptide-drug interactions for optimum receptor binding. Thus, we propose STDD-Methyl TROSY NMR spectroscopy as a technique for rapid screening of biologics for the development of optimized biopharmaceutics.
Asunto(s)
Insulinas , Péptidos , Humanos , Epítopos , Espectroscopía de Resonancia Magnética/métodos , Ligandos , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Barbituric acid is believed to be a proto-RNA nucleobase that was used for biological information transfer on prebiotic earth before DNA and RNA in their present forms evolved. Nucleobases have various tautomeric forms and the relative stability of these forms is critical to their biological function. It has been shown that barbituric acid has a tri-keto form in the gas phase and an enol form in the solid state. However, its dominant tautomeric form in aqueous medium that is most relevant for biology has been investigated only to a limited extent and the findings are inconclusive. We have used multiple approaches, namely, molecular dynamics, quantum chemistry, NMR, and IR spectroscopy to determine the most stable tautomer of barbituric acid in solution. We find a delicate balance in the stability of the two tautomers, tri-keto and enol, which is tipped toward the enol as the extent of solvation by water increases.
Asunto(s)
Prebióticos , Agua , ADN/química , Simulación de Dinámica Molecular , ARN , Agua/químicaRESUMEN
Many double-stranded RNA-binding domains (dsRBDs) interact with topologically distinct dsRNAs in biological pathways pivotal to viral replication, cancer causation, neurodegeneration, and so on. We hypothesized that the adaptability of dsRBDs is essential to target different dsRNA substrates. A model dsRBD and a few dsRNAs, slightly different in shape from each other, were used to test the systematic shape dependence of RNA on the dsRBD-binding using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. NMR-based titrations showed a distinct binding pattern for the dsRBD with the topologically distinct dsRNAs. The line broadening upon RNA binding was observed to cluster in the residues lying in close proximity, thereby suggesting an RNA-induced conformational exchange in the dsRBD. Further, while the intrinsic microsecond dynamics observed in the apo-dsRBD were found to quench upon binding with the dsRNA, the microsecond dynamics got induced at residues spatially proximal to quench sites upon binding with the dsRNA. This apparent relay of conformational exchange suggests the significance of intrinsic dynamics to help adapt the dsRBD to target various dsRNA-shapes. The conformational pool visualized in MD simulations for the apo-dsRBD reported here has also been observed to sample the conformations seen previously for various dsRBDs in apo- and in dsRNA-bound state structures, further suggesting the conformational adaptability of the dsRBDs. These investigations provide a dynamic basis for the substrate promiscuity for dsRBD proteins.
Asunto(s)
ARN Bicatenario , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
The self-assembly reactions of tetratopic metal acceptors with the flexible bidentate ligands are known to yield self-assembled molecular squares of the type [M4L8], triangles of composition [M3L6], or a mixture of these two. In this work, we demonstrate the preferential formation of a trimeric cage assembly of the formula [Pd3(L1)6·(BF4)6] (1a) over the tetrameric cage [Pd4(L1)8·(BF4)8] (1b) by employing a flexible dipodal phosphoramide ligand, [PhPO(NH(3-Py))2] (L1; 3-Py = 3-aminopyridine), in a reaction with [Pd(CH3CN)4·(BF4)2]. The entropically favored trimeric self-assembly of 1a is the predominant species in the solution [dimethyl sulfoxide (DMSO)-d6] at room temperature. In fact, at higher temperatures, 1a was found to be the only product, as observed from the disappearance of the peak due to 1b in the 31P NMR spectrum. However, in a 1:1 mixture of acetonitrile (MeCN)-d3 and DMSO-d6, the tetrameric species 1b is the preferred species, as revealed by the 31P NMR and electrospray ionization mass spectral analyses. The structure of the molecular trimer 1a has been established in the solid state by using single-crystal X-ray diffraction analysis. Interestingly, treatment of an another flexible ligand, [MePO(NH(3-Py))2] (L2), with the same Pd(II) acceptor resulted in exclusive formation of the trimeric cage [Pd3(L2)6·(BF4)6] (2).
RESUMEN
We report two pyridyl-linked benzimidazolyl hydrazones as HCl cotransporters that are 5 and 2 times superior to prodigiosin, a natural product whose transport efficiency has never been routed by synthetic molecules. These hydrazones provide a suitable HCl binding site through a cooperative protonation and chloride ion recognition. HCl transport by the most active compound induces lysosome deacidification. Viability assays confirmed that the compounds induce cytotoxicity toward human breast cancer MCF-7 cells but are relatively nontoxic toward noncancerous HEK293T cells.
RESUMEN
The aggregation of α-synuclein is linked to neurological disorders, and of these, Parkinson's disease (PD) is among the most widely studied. In this background, we have investigated here the effects of three α, ß-unsaturated carbonyl based plant metabolites, daidzein, fisetin and scopoletin on α-Syn aggregation. The ThT and light scattering kinetics studies establish that these compounds have ability to inhibit α-Syn fibrillation to different extents; this is confirmed by TEM studies. It is pertinent to note here that daidzein and scopoletin have been predicted to be able to cross the blood brain barrier. ANS binding assays demonstrate that the compounds interfere in the hydrophobic interactions. The tyrosine quenching, molecular docking and MD simulation studies showed that the compounds bind with α-Syn and provide structural rigidity which delays onset of structural transitions, which is confirmed by CD spectroscopy. The results obtained here throw light on the mechanisms underlying inhibition of α-Syn fibrillation by these compounds. Thus, the current work has significant therapeutic implications for identifying plant based potent therapeutic molecules for PD and other synucleinopathies, an area which needs extensive exploration.
Asunto(s)
Flavonoles/farmacología , Isoflavonas/farmacología , Metaboloma , Agregado de Proteínas/efectos de los fármacos , Escopoletina/farmacología , alfa-Sinucleína/metabolismo , Productos Biológicos/química , Productos Biológicos/farmacología , Línea Celular , Flavonoles/química , Fluorescencia , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoflavonas/química , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Conformación Proteica , Escopoletina/química , Tirosina/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/ultraestructuraRESUMEN
The prebiotic soup of a putative 'RNA World' would have been replete with a plethora of molecules resulting from complex chemical syntheses and exogeneous delivery. The presence of background molecules could lead to molecular crowding, potentially affecting the course of the reactions facilitated therein. Using NMR spectroscopy, we have analyzed the effect of crowding on the stacking ability of RNA monomers. Our findings corroborate that the purines stack more efficiently than the pyrimidine ribonucleotides. This competence is further enhanced in the presence of a crowding agent. This enhanced stacking could result in greater sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic reactions into question. Thus, this study demonstrates the need for systematic characterization of molecular crowding in the context of prebiotically pertinent processes. Unraveling such phenomena is essential for our understanding of the transition from abiotic to biotic, during the origin of life.
RESUMEN
Charge-neutral chiral hosts are attractive due to their ability to recognize a wide range of guest functionalities and support enantioselective processes. However, reports on such charge-neutral cages are very scarce in the literature. Here, we report an enantiomeric pair of tetrahedral Pd(ii) cages built from chiral tris(imido)phosphate trianions and oxalate linkers, which exhibit enantioselective separation capabilities for epichlorohydrin, ß-butyrolactone, and 3-methyl- and 3-ethyl cyclopentanone.
RESUMEN
TAR RNA binding protein (TRBP) is a double-stranded RNA binding protein involved in various biological processes like cell growth, development, death, etc. The protein exists as two isoforms TRBP2 and TRBP1. TRBP2 contains additional 21 amino acids at its N-terminus, which are proposed to be involved in its membrane localization, when compared to TRBP1. The resonance assignment (19-228) of the double-stranded RNA binding domains (dsRBD 1 and 2) of TRBP2 has been reported earlier. Here, we report 1H, 13C and 15N resonance assignment for dsRBD1 of TRBP2 (1-105) containing the additional N-terminal residues. This assignment will provide deeper insights to understand the effect of these residues on the structure and dynamics of TRBP2 and would therefore help in further elucidating the differences in the role of these isoforms.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Proteínas de Unión al ARN/química , Humanos , Modelos Moleculares , Dominios ProteicosRESUMEN
A charge-neutral tetrahedral [(Pd3X)4L6] cage assembly built from a trinuclear polyhedral building unit (PBU), [Pd3X](3+), cis-blocked with an imido P(V) ligand, [(N(i)Pr)3PO](3-) (X(3-)), and oxalate dianions (L(2-)) is reported. Use of benzoate or ferrocene dicarboxylate anions, which do not offer wide-angle chelation as that of oxalate dianions, leads to smaller prismatic clusters instead of polyhedral cage assemblies. The porosity of the tetrahedral cage assembly was determined by gas adsorption studies, which show a higher uptake capacity for CO2 over N2 and H2. The tetrahedral cage was shown to encapsulate a wide range of neutral guest solvents from polar to nonpolar such as dimethyl sulfoxide, benzene, dichloromethane, chloroform, carbon tetrachloride, and cyclopentane as observed by mass spectral and single-crystal X-ray diffraction studies. The (1)H two-dimensional diffusion ordered spectroscopy NMR analysis shows that the host and guest molecules exhibit similar diffusion coefficients in all the studied host-guest systems. Further, the tetrahedral cage shows selective binding of benzene, CCl4, and cyclopentane among other solvents from their categories as evidenced from mass spectral analysis. A preliminary density functional theory analysis gave a highest binding energy for benzene among the other solvents that were structurally shown to be encapsulated at the intrinsic cavity of the tetrahedral cage.