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Coffee cherry is a rich source of chlorogenic acids (CGAs) and caffeine. In this study we examined the potential antioxidant activity and enzyme inhibitory effects of whole coffee cherries (WCC) and their two extracts on α-amylase, α-glucosidase and acetylcholinesterase (AChE) activities, which are targets for the control of diabetes and Alzheimer's diseases. Whole coffee cherry extract 40% (WCCE1) is rich in chlorogenic acid compounds, consisting of a minimum of 40% major isomers, namely 3-caffeoylquinic acids, 4-caffeoylquinic acids, 5-caffeoylquinic acids, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 4-feruloylquinc acid, and 5-feruloylquinc acid. Whole coffee cherry extract 70% (WCCE2) is rich in caffeine, with a minimum of 70%. WCCE1 inhibited the activities of digestive enzymes α-amylase and α-glucosidase, and WCCE2 inhibited acetylcholinesterase activities with their IC 50 values of 1.74, 2.42, and 0.09 mg/mL, respectively. Multiple antioxidant assays-including DPPH, ABTS, FRAP, ORAC, HORAC, NORAC, and SORAC-demonstrated that WCCE1 has strong antioxidant activity.

Leafy green vegetables contain essential nutrients and are frequently consumed for their perceived health benefits. The purpose of this study was to profile the nutritional and natural bioactive phytochemical compounds extracted from freeze-dried spinach and kale and compare them with our previously published freeze-dried purslane results. Novel research suggests that these leafy greens contain an abundance of fatty acids, amino acids, organic acids, vitamins, and minerals, which can reduce the risk of many chronic diseases. LC-MS/MS screening identified 69 and 103 compounds in spinach and kale, respectively, including flavonoids, glucosinolates, and phenolic and organic acids. Out of a total of 26 flavonoids identified in the current study, only three were found in spinach. All three leafy greens showed nutritional and health benefits and the unique phytochemical compounds found only in purslane provide a strong basis to incorporate its distinct dietary benefits.

The current work assessed the phytochemical contents of Arabica whole coffee cherry (WCC) and its two commercially available extracts: a minimum 40% chlorogenic acid (CGA; WCCE-1) and 70% caffeine (WCCE-2). Mass spectrometry analyses tentatively identified 219 phytochemicals in the three coffee samples, which is, so far, the largest number of identifications in a single study. A new group of CGA derivative namely caffeoylvaleroylquinic acid (CVQA) was identified in the three samples. Moreover, three 5-hydroxytryptamide derivatives (C 20 -5HT, C 22 -5HT, and C 24 -5HT) were identified in WCC but not in the extracts. Two groups of atractyligenin derivatives (carboxyatractyligenin and noncarboxyatractyligenin) were identified in the three samples. Furthermore, our results show that both extracts retained a large number of phenolic and other potentially bioactive compounds that were naturally present in whole coffee cherry (WCC).

Purslane (Portulaca oleracea) is a weed naturally found in driveways, lawns, and fields and edible in many regions of Europe, Asia, the Middle East, Africa, and Australia. The purpose of this study was to compare the nutritional and phytochemical components of cultivated and wild purslane. Omega-3 contents of both purslane genotypes were comparable with 189.16 ± 25.52 mg/100 g dry weight and 188.48 ± 6.35 mg/100 g dry weight in cultivated and wild purslane leaves, respectively. Omega-6/omega-3 ratio (1:1-1:3) were low in both genotypes. However, high levels of oxalic acid were observed. Cultivated contained greater amounts of amino acids and vitamins than wild purslane. Of the 184 compounds identified in both genotypes by LC-MS/MS, including phenolic acids, organic acids, flavonoids, alkaloids, and betanin, more than 80 showed greater than two-fold abundance in the wild compared to cultivated purslane. Purslane has the potential to be cultivated as a food ingredient for nutraceutical applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Both gain-of-function enhancer of zeste homolog 2 (EZH2) mutations and inactivating histone acetyltransferases mutations, such as CREBBP and EP300, have been implicated in the pathogenesis of germinal center (GC)-derived lymphomas. We hypothesized that direct inhibition of EZH2 and histone deacetyltransferase (HDAC) would be synergistic in GC-derived lymphomas., Experimental Design: Lymphoma cell lines ( n = 21) were exposed to GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor. Synergy was assessed by excess over bliss. Western blot, mass spectrometry, and coimmunoprecipitation were performed. A SU-DHL-10 xenograft model was utilized to validate in vitro findings. Pretreatment RNA-sequencing of cell lines was performed. MetaVIPER analysis was used to infer protein activity., Results: Exposure to GSK126 and romidepsin demonstrated potent synergy in lymphoma cell lines with EZH2 dysregulation. Combination of romidepsin with other EZH2 inhibitors also demonstrated synergy suggesting a class effect of EZH2 inhibition with romidepsin. Dual inhibition of EZH2 and HDAC led to modulation of acetylation and methylation of H3K27. The synergistic effects of the combination were due to disruption of the PRC2 complex secondary to acetylation of RbAP 46/48. A common basal gene signature was shared among synergistic lymphoma cell lines and was characterized by upregulation in chromatin remodeling genes and transcriptional regulators. This finding was supported by metaVIPER analysis which also revealed that HDAC 1/2 and DNA methyltransferase were associated with EZH2 activation., Conclusions: Inhibition of EZH2 and HDAC is synergistic and leads to the dissociation of PRC2 complex. Our findings support the clinical translation of the combination of EZH2 and HDAC inhibition in EZH2 dysregulated lymphomas., (©2019 American Association for Cancer Research.)

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair., Competing Interests: The authors declare no conflict of interest.

Histone deacetylases (HDACs) catalyze the removal of acetylation marks from lysine residues on histone and nonhistone substrates. Their activity is generally associated with essential cellular processes such as transcriptional repression and heterochromatin formation. Interestingly, abnormal activity of HDACs has been reported in various types of cancers, which makes them a promising therapeutic target for cancer treatment. In the current study, we aim to understand the mechanisms underlying the function of HDACs using an in-depth quantitative analysis of changes in histone acetylation levels in Schizosaccharomyces pombe (S. pombe) lacking major HDAC activities. We employed a targeted quantitative mass spectrometry approach to profile changes of acetylation and methylation at multiple lysine residues on the N-terminal tail of histones H3 and H4. Our analyses identified a number of histone acetylation sites that are significantly affected by S. pombe HDAC mutations. We discovered that mutation of the Class I HDAC known as Clr6 causes a major increase in the abundance of triacetylated H4 molecules at K5, K8, and K12. A clr6-1 hypomorphic mutation also increased the abundance of multiple acetyl-lysines in histone H3. In addition, our study uncovered a few crosstalks between histone acetylation and methylation upon deletion of HDACs Hos2 and Clr3. We anticipate that the results from this study will greatly improve our current understanding of the mechanisms involved in HDAC-mediated gene regulation and heterochromatin assembly.

Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein-protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors.

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage., (Copyright © 2015 by the Genetics Society of America.)

Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme-to-substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. This article is part of a Special Issue entitled: From protein structures to clinical applications., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

Background: Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined., Methodology/principal Findings: Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line., Conclusions/significance: Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.