In this report, we demonstrate glucose-derived carbon nanospheres to be an emerging class of intracellular carriers. The surfaces of these spheres are highly functionalized and do not need any further modification. Besides, the intrinsic fluorescence property of carbon nanospheres helps in tracking their cellular localization without any additional fluorescent tags. The spheres are found to target the nucleus of the mammalian cells, causing no toxicity. Interestingly, the in vivo experiments show that these nanospheres have an important ability to cross the blood-brain barrier and localize in the brain besides getting localized in the liver and the spleen. There is also evidence to show that they are continuously being removed from these tissues over time. Furthermore, these nanospheres were used as a carrier for the membrane-impermeable molecule CTPB (N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxybenzamide), the only known small-molecule activator of histone acetyltransferase (HAT) p300. Biochemical analyses such as Western blotting, immunohistochemistry, and gene expression analysis show the induction of the hyperacetylation of histone acetyltransferase (HAT) p300 (autoacetylation) as well as histones both in vitro and in vivo and the activation of HAT-dependent transcription upon CTPB delivery. These results establish an alternative path for the activation of gene expression mediated by the induction of HAT activity instead of histone deacetylase (HDAC) inhibition.
Although the brain functions of specific acetyltransferases such as the CREB-binding protein (CBP) and p300 have been well documented using mutant transgenic mice models, studies based on their direct pharmacological activation are still missing due to the lack of cell-permeable activators. Here we present a small-molecule (TTK21) activator of the histone acetyltransferases CBP/p300, which, when conjugated to glucosebased carbon nanosphere (CSP), passed the blood-brain barrier, induced no toxicity, and reached different parts of the brain. After intraperitoneal administration in mice, CSP-TTK21 significantly acetylated histones in the hippocampus and frontal cortex. Remarkably, CSP-TTK21 treatment promoted the formation of long and highly branched doublecortin-positive neurons in the subgranular zone of the dentate gyrus and reduced BrdU incorporation, suggesting that CBP/p300 activation favors maturation and differentiation of adult neuronal progenitors. In addition, mRNA levels of the neuroD1 differentiation marker and BDNF, a neurotrophin required for the terminal differentiation of newly generated neurons, were both increased in the hippocampus concomitantly with an enrichment of acetylated-histone on their proximal promoter. Finally, we found that CBP/p300 activation during a spatial training, while not improving retention of a recent memory, resulted in a significantextensionofmemoryduration.ThisreportisthefirstevidenceforCBP/p300-mediatedhistoneacetylationinthebrainbyanactivator molecule, which has beneficial implications for the brain functions of adult neurogenesis and long-term memory. We propose that direct stimulation of acetyltransferase function could be useful in terms of therapeutic options for brain diseases.
Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.
Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.
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