RESUMEN
The human transmembrane protein Transferrin Receptor-1 is regarded as a promising target for the systemic delivery of therapeutic agents, particularly of nucleic acid therapeutics, such as short double stranded RNAs. This ubiquitous receptor is involved in cellular iron uptake, keeping intracellular homeostasis. It is overexpressed in multiple cancer cell types and is internalized via clathrin-mediated endocytosis. In previous studies, a human transferrin receptor-1 RNA aptamer, identified as TR14 ST1-3, was shown to be capable of effectively internalizing into cells in culture and to deliver small, double stranded RNAs in vitro and in vivo, via systemic administration. To understand, at the molecular level, the aptamer binding to the receptor and the impact of conjugation with the therapeutic RNA, a multi-level in silico protocol was employed, including protein-aptamer docking, molecular dynamics simulations and free energy calculations. The competition for the binding pocket, between the aptamer and the natural ligand human Transferrin, was also evaluated. The results show that the aptamer binds to the same region as Transferrin, with residues from the helical domain showing a critical role. Moreover, the conjugation to the therapeutic RNA, was shown not to affect aptamer binding. Overall, this study provides an atomic-level understanding of aptamer association to human Transferrin Receptor-1 and of its conjugation with a short model-therapeutic RNA, providing also important clues for futures studies aiming to deliver other oligonucleotide-based therapeutics via Transferrin Receptor.
RESUMEN
BACKGROUND: Non-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5'-peripheral domain. Structure A is embedded in a hot spot for the 3' end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome. METHODS: To better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated. RESULTS: Our results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages. CONCLUSIONS: We showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations. GENERAL SIGNIFICANCE: The fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.
Asunto(s)
G-Cuádruplex , Simulación de Dinámica Molecular , Animales , Humanos , Conformación de Ácido Nucleico , ADN Mitocondrial/genética , ADN Mitocondrial/química , Emparejamiento Base , MamíferosRESUMEN
The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.
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Sorghum , Sorghum/genética , Sorghum/metabolismo , Lignina/metabolismo , Regulación de la Expresión Génica de las Plantas , Grano Comestible/metabolismo , Flavonoides/metabolismo , Glucósidos/metabolismo , Ésteres/metabolismoRESUMEN
The catalytic mechanism of Pdx2 was studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys-His-Glu catalytic triad to deaminate glutamine to glutamate and ammonia - the source of the nitrogen of pyridoxal 5'-phosphate (PLP). This enzyme is, therefore, a rate-limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i)â activation of Cys87 , (ii)â deamination of glutamine with the formation of the glutamyl-thioester intermediate, (iii)â hydrolysis of the formed intermediate, and (iv)â enzymatic turnover. The kinetic data available in the literature (19.1-19.5â kcal mol-1 ) agree very well with the calculated free energy barrier of the hydrolytic step (18.2â kcal.mol-11 ), which is the rate-limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i)â it requires the activation of the nucleophile Cys87 to a thiolate; ii)â the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu198 does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.
Asunto(s)
Glutaminasa , Malaria , Catálisis , Ácido Glutámico , Glutaminasa/química , Glutaminasa/metabolismo , Glutamina/metabolismo , Humanos , Fosfato de Piridoxal/químicaRESUMEN
Many fungal quinazolinone metabolites, which contain the methyl-indole pyrazino [1,2-b]quinazoline-3,6-dione core, have been found to possess promising antitumor activity. The purpose of this work was to synthesize the enantiomeric pairs of two members of this quinazolinone family, to explore their potential as antitumor and their ability to revert multidrug resistance. The marine natural product fiscalin B (4c), and antienantiomers (4b, 5b, and 5c) were synthesized via a one-pot approach, while the syn enantiomers (4a, 4d, 5a, and 5d) were synthetized by a multi-step procedure. These strategies used anthranilic acid (i), chiral N-protected α-amino acids (ii), and tryptophan methyl esters (iii) to form the core ring of pyrazino[2,1-b]quinazoline-3,6-dione scaffold. Four enantiomeric pairs, with different enantiomeric purities, were obtained with overall yields ranging from 7 to 40%. Compounds 4aâ»d and 5aâ»d were evaluated for their growth inhibitory effect against two tumor cell lines. Differences between enantiomeric pairs were noted and 5aâ»d displayed GI50 values ranging from 31 to 52 µM, which are lower than those of 4aâ»d. Nevertheless, no effect on P-glycoprotein (P-gp) modulation was observed for all compounds. This study disclosed new data for fiscalin B (4c), as well as for its analogues for a future development of novel anticancer drug leads.
Asunto(s)
Alcaloides/farmacología , Antineoplásicos/farmacología , Productos Biológicos/química , Quinazolinonas/farmacología , Alcaloides/síntesis química , Alcaloides/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Organismos Acuáticos , Línea Celular Tumoral , Humanos , Estructura Molecular , Quinazolinonas/síntesis química , Quinazolinonas/químicaRESUMEN
We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product.