Negative regulation of the acetyltransferase TIP60-p53 interplay by UHRF1 (ubiquitin-like with PHD and RING finger domains 1).

Numerous studies indicate the importance of acetylation in p53-mediated stress responses upon DNA damage. We and others previously showed that TIP60 (Tat-interacting protein of 60 kDa)-mediated acetylation of p53 at K120 is crucial for p53-dependent apoptotic responses. Nevertheless, it remains unclear how TIP60-mediated effects on p53 are dynamically regulated in vivo. Here, we report that UHRF1 (ubiquitin-like with PHD and RING finger domains 1) interacts with TIP60 both in vitro and in vivo and induces degradation-independent ubiquitination of TIP60. Moreover, UHRF1 expression markedly suppresses the ability of TIP60 to acetylate p53. In contrast, RNAi-mediated knockdown of UHRF1 increases the endogenous levels of p53 acetylation at K120 and p53-mediated apoptosis is significantly enhanced in UHRF1-depleted cells. To elucidate the mechanisms of this regulation, we found that the interaction between TIP60 and p53 is severely inhibited in the presence of UHRF1, suggesting that UHRF1 modulates TIP60-mediated functions in both K120 acetylation-dependent and -independent manners. Consistent with this notion, UHRF1 knockdown promotes activation of p21 and PUMA but not MDM2. These findings demonstrate that UHRF1 is a critical negative regulator of TIP60 and suggest that UHRF1-mediated effects on p53 may contribute, at least in part, to its role in tumorigenesis.

From Epimedium to Neuroprotection: Exploring the Potential of Wushanicaritin.

Epimedium has been used for functional foods with many beneficial functions to human health. Wushanicaritin is one of the most important chemicals int Epimedium. This study investigated the neuroprotective effects of wushanicaritin and potential underlying mechanisms. The results demonstrated that wushanicaritin possessed superior intercellular antioxidant activity compared to icaritin. Wushanicaritin, with an EC50 value of 3.87 µM, showed better neuroprotective effect than quercetin, a promising neuroprotection agent. Wushanicaritin significantly reversed lactate dehydrogenase release, reactive oxygen species generation, cell apoptosis, and mRNA expression related to cell apoptosis and oxidative defense, in glutamate-induced PC-12 cells. Wushanicaritin could also maintain the enzymatic antioxidant defense system and mitochondrial function. The suppression of caspase-3 activation and amelioration of mitochondrial membrane potential loss and nucleus morphology changes were involved in the antiapoptotic effect of wushanicaritin. These findings suggested that wushanicaritin possesses excellent intercellular antioxidant and neuroprotective activities, showing potential promise in functional foods.

The high-quality genome of Grona styracifolia uncovers the genomic mechanism of high levels of schaftoside, a promising drug candidate for treatment of COVID-19.

Recent study has evidenced that traditional Chinese medicinal (TCM) plant-derived schaftoside shows promise as a potential drug candidate for COVID-19 treatment. However, the biosynthetic pathway of schaftoside in TCM plants remains unknown. In this study, the genome of the TCM herb Grona styracifolia (Osbeck) H.Ohashi & K.Ohashi (GSO), which is rich in schaftoside, was sequenced, and a high-quality assembly of GSO genome was obtained. Our findings revealed that GSO did not undergo recent whole genome duplication (WGD) but shared an ancestral papilionoid polyploidy event, leading to the gene expansion of chalcone synthase (CHS) and isoflavone 2'-hydroxylase (HIDH). Furthermore, GSO-specific tandem gene duplication resulted in the gene expansion of C-glucosyltransferase (CGT). Integrative analysis of the metabolome and transcriptome identified 13 CGTs and eight HIDHs involved in the biosynthetic pathway of schaftoside. Functional studies indicated that CGTs and HIDHs identified here are bona fide responsible for the biosynthesis of schaftoside in GSO, as confirmed through hairy root transgenic system and in vitro enzyme activity assay. Taken together, the ancestral papilionoid polyploidy event expanding CHSs and HIDHs, along with the GSO-specific tandem duplication of CGT, contributes, partially if not completely, to the robust biosynthesis of schaftoside in GSO. These findings provide insights into the genomic mechanisms underlying the abundant biosynthesis of schaftoside in GSO, highlighting the potential of GSO as a source of bioactive compounds for pharmaceutical development.

FvbHLH1 Regulates the Accumulation of Phenolic Compounds in the Yellow Cap of Flammulina velutipes.

Flammulina velutipes is a renowned edible and medicinal fungus. Commercially cultivated F. velutipes occurs in two distinct phenotypes: white and yellow. However, the underlying mechanism contributing to the yellow phenotype and high nutritional value remain uncertain. We reconfirmed that the browning process in F. velutipes is attributable to melanin accumulation, although the initial yellow cap seemed unrelated to melanin. A transcriptomic and metabolomic joint analysis revealed that 477 chemical compounds categorized into 11 classes, among which 191 exhibited significantly different levels of accumulation between different phenotypes. Specifically, 12 compounds were unique to the yellow F. velutipes, including ferulic acid, and 3-Aminosalicylic acid. Free fatty acids and xanthine were identified as the primary compounds correlating with the yellow and oily cap. A total of 44,087 genes were identified, which were more homologous to Pleurotus ostreatus PC15. Structural genes such as PAL (phenylalanine ammonialyase), C4H (cinnamate 4-hydroxylase), C3H (Coumarin-3-hydroxylase), AoMT (caffeoyl coenzyme A-O-methyltransferase), and 4CL (4-coumarate: CoA ligase) were up-regulated, thereby activating the lignin biosynthesis and metabolism pathway. Additionally, FvbHLH1 can lead to the consumption of a huge amount of phenylalanine while generating flavonoids and organic acid compounds. Meanwhile, ferulic acid biosynthesis was activated. Therefore, this study clarifies the chemical and molecular bases for the yellow phenotype and nutritional value of F. velutipes.

CBP and p300 are cytoplasmic E4 polyubiquitin ligases for p53.

p300 and CREB-binding protein (CBP) act as multifunctional regulators of p53 via acetylase and polyubiquitin ligase (E4) activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. Consistent with the cytoplasmic localization of its E3/E4 activity, CBP deficiency specifically stabilized cytoplasmic, but not nuclear p53. The N-terminal 616 aa of CBP, which includes the conserved Zn(2+)-binding C/H1-TAZ1 domain, was the minimal domain sufficient to destabilize p53 in vivo, and it included within an intrinsic E3 autoubiquitination activity and, in a two-step E4 assay, exhibited robust E4 activity for p53. Cytoplasmic compartmentalization of p300/CBP's ubiquitination function reconciles seemingly opposed functions and explains how a futile cycle is avoided-cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while physically separate nuclear p300/CBP activities, such as p53 acetylation, activate p53.

Selection and validation of reference genes for RT-qPCR analysis in Desmodium styracifolium Merr.

Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function. To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Desmodium styracifolium Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for D. styracifolium. In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression. The expression stability of currently used Leguminosae ACT6 was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of PP + UFM1, CCRP4 + BRM and NFD6 + NCLN1 were the most stable reference genes in leaf, stem and root tissues, respectively. The most stable reference gene combination for all tissues was CCRP4 + CUL1. In addition, the most stable reference genes for different experimental conditions were distinct, for instance SMUP1 for MeJA treatment, ERDJ2A + SMUP1 for SA treatment, NCLN1 + ERDJ2A for ABA treatment and SF3B + VAMP721d for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in D. styracifolium. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02954-x.

The antioxidant activity and neuroprotective mechanism of isoliquiritigenin.

Glycyrrhizae radix has been widely accepted as a functional food in Asia. Isoliquiritigenin is a characteristic bioactive chemical in this medicinal plant. In this work, the neuroprotective effect of isoliquiritigenin and the possible mechanisms were investigated. The results revealed that isoliquiritigenin exhibited better neuroprotective and antioxidant activities than quercetin, a commercial natural antioxidant. Isoliquiritigenin significantly inhibited the release of lactate dehydrogenase, and the generation of reactive oxygen species in H2O2-treated cells. The activities of superoxide dismutase, glutathione peroxidase and catalase were improved. The mRNA expression levels related to oxidative defense and cell apoptosis were reversed by isoliquiritigenin. Moreover, isoliquiritigenin might inhibit the cell apoptosis via ameliorating the loss of mitochondrial membrane potential and the change of nucleus morphology.

Identification of two novel prenylated flavonoids in mulberry leaf and their bioactivities.

Mulberry leaf is a vegetable used in daily diet. It can bring delicious taste and multiple health benefits. However, the chemicals responsible for these health benefits remain unveiled. In this work, two novel prenylated flavonoids were isolated from mulberry leaf. Their structures were identified and named as morachalcone D and morachalcone E. The protective effects of these two compounds were investigated, against endogenous oxidative damage (oxytosis/ferroptosis) induced by glutamate and erastin in HT22 cells. The results revealed that morachalcone D was much more potent in preventing from glutamate- and erastin-induced cell death than morachalcone E. The neuroprotective effect of morachalcone D was related to the prevention of ROS production, glutathione depletion, and iron accumulation. Morachalcone D upregulated the expression of genes involved in antioxidant defense, including GPx4, CAT, SOD2, Nrf2, HMOX1 and SLC7A11. These findings indicated that morachalcone D was responsible for the health benefits of mulberry leaf, and could be a potent neuroprotective agent for use in dietary supplements and functional foods.

Connecting the navigational clock to sun compass input in monarch butterfly brain.

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.

Immunomodulatory mechanism of α-d-(1→6)-glucan isolated from banana.

Banana is a delicious fruit with potent immunomodulatory function. In this study, α-d-(1→6)-glucan was purified from banana pulp. It could significantly promote pinocytic activity and production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The mRNA expression of nitric oxide synthase (iNOS), IL-6 and TNF-α was increased in RAW264.7 macrophages. α-d-(1→6)-glucan could not only increase the expression levels of p-p65 and p-IκBα, but also induce the translocation of nuclear factor-kappa B (NF-κB) p65 into the nucleus. Moreover, mitogen-activated protein kinases (MAPKs), including p-ERK, p-JNK and p-p38, were upregulated. These results suggested that NF-κB and MAPK signaling pathways were involved in the immunomodulatory mechanisms of α-d-(1→6)-glucan. The results revealed that α-d-(1→6)-glucan might be the critical component responsible for the health benefits of banana.

Sodium para-aminosalicylate delays pericarp browning of litchi fruit by inhibiting ROS-mediated senescence during postharvest storage.

The effect of sodium para-aminosalicylate (PAS-Na) on litchi pericarp browning and the potential regulating mechanism was investigated in this study. Results showed that 0.3 g L-1 PAS-Na significantly inhibited the development of pericarp browning and reduced respiration rate of litchi fruit. PAS-Na inhibited the production of reactive oxygen species (ROS) and decreased the expression level of senescence-related genes. Additionally, PAS-Na treatment enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), which might contribute to the scavenging of ROS. Meanwhile, PAS-Na treatment maintained membrane integrity as indicated by reduced relative membrane leakage rate and malondialdehyde (MDA) content, as well as lower activities of membrane lipids-degrading enzymes: lipase and lipoxygenase (LOX). Amino acids, especially GABA, Glu, Met contents were also significantly affected by PAS-Na treatment. Taken together, we postulated that PAS-Na treatment might be a promising method for controlling postharvest browning and prolonging shelf-life of harvested litchi fruit.

Mechanism of Cell Wall Polysaccharides Modification in Harvested 'Shatangju' Mandarin (Citrus reticulate Blanco) Fruit Caused by Penicillium italicum.

Modification of cell wall polysaccharide in the plant plays an important role in response to fungi infection. However, the mechanism of fungi infection on cell wall modification need further clarification. In this study, the effects of Penicillium italicum inoculation on 'shatangju' mandarin disease development and the potential mechanism of cell wall polysaccharides modification caused by P. italicum were investigated. Compared to the control fruit, P. italicum infection modified the cell wall polysaccharides, indicated by water-soluble pectin (WSP), acid-soluble pectin (ASP), hemicellulose and lignin contents change. P. italicum infection enhanced the activities of polygalacturonase (PG), pectin methylesterase (PME), and the expression levels of xyloglucanendotransglucosylase/hydrolase (XTH) and expansin, which might contribute to cell wall disassembly and cellular integrity damage. Additionally, higher accumulation of reactive oxygen species (ROS) via decreasing antioxidant metabolites and the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) also contributed to the cell wall polysaccharides modification. Meanwhile, the gene expression levels of hydroxyproline-rich glycoprotein (HRGP) and germin-like protein (GLP) were inhibited by pathogen infection. Altogether, these findings suggested that cell wall degradation/modification caused by non-enzymatic and enzymatic factors was an important strategy for P. italicum to infect 'shatangju' mandarin.

Identification of moracin N in mulberry leaf and evaluation of antioxidant activity.

Mulberry leaf is a newly accepted vegetable for daily diet. It tastes good and has multiple health benefits, including antioxidant and anti-inflammatory activities. However, the chemicals responsible for these health benefits remain unveiled. Prenylated phenolics are characteristic bioactive compounds in mulberry leaf, which are recognized as good antioxidants. In this work, moracin N was purified from mulberry leaf. It showed better antioxidant activities than resveratrol. The EC50 value of cellular antioxidant activity was 24.92 µM, and the IC50 value against DPPH radical was 40.00 µM. The prenyl group rendered the molecule more membrane affinity which improved the bioavailability. The furan ring was critical for the antioxidant behaviour. The cell viability test revealed that moracin N had a good safety. These results pointed out that moracin N contributed to the antioxidant activity of mulberry leaf.

PCDH10, a novel p53 transcriptional target in regulating cell migration.

Cell cycle arrest, senescence and apoptosis are commonly regarded as the major tumor suppression mechanisms of p53. However, accumulating evidence indicates that loss of these canonical functions is not sufficient for tumor formation, highlighting the complexity of p53-mediated tumor suppression. PCDH10 belongs to a proto cadherin protein family and is a potential tumor suppressor protein as the dysregulation of PCDH10 gene frequently existed in multiple human tumors. Here, we found that PCDH10 is a transcriptional target of p53 and that the levels of PCDH10 expression can be induced by wild type p53 but not mutant p53 in a number of human cancer cell lines. Moreover, we identified a p53 consensus binding site located in the PCDH10 promoter region that is responsive to p53 regulation. Although upregulation of PCDH10 has no obvious effect on growth arrest or apoptosis in human cells, PCDH10 exhibits inhibitory roles in cancer cell motility and cell migration. These results suggest an important role of p53 in regulating tumor cell migration through activating PCDH10 expression and support the notion that non-canonical activities of p53 may contribute to its tumor suppressor function in vivo.

p53 Ubiquitination and proteasomal degradation.

p53 levels and activity are controlled in large part through regulated ubiquitination and subsequent destruction by the 26S proteasome. Monoubiquitination of p53 is mediated primarily by the RING-finger E3 ubiquitin ligase MDM2 and impacts p53 activity through modulation of p53 localization and transcription activities. Recently, several E4 ubiquitin ligases (E4s) have been identified which serve to extend these monoubiquitin chains. The ubiquitin ligase activity of these factors toward p53, and their contribution to p53 degradation, can be studied using a variety of in vitro and in vivo methods and reagents which will be described in this chapter. These methods include in vivo ubiquitination of p53 using HA-ubiquitin or his-ubiquitin; the in vitro E3 ubiquitin ligase assay, in which ubiquitin reaction components (URC) are incubated with a purified E3 or E4 ligase; a one-step E4 assay, in which URC are incubated with a substrate, E3, and E4; and a two-step E4 assay in which p53 is monoubiquitinated in an E3 reaction, and subsequently purified and incubated with an E4. Finally, we will describe an in vitro degradation assay in which ubiquitinated p53 is incubated with purified 26S proteasomes. Together, these assays can be used to provide insight into the biochemical nature of p53 ubiquitination and degradation.

Dual Roles of MDM2 in the Regulation of p53: Ubiquitination Dependent and Ubiquitination Independent Mechanisms of MDM2 Repression of p53 Activity.

MDM2 oncogenic protein is the principal cellular antagonist of the p53 tumor suppresser gene. p53 activity needs exquisite control to elicit appropriate responses to differential cellular stress conditions. p53 becomes stabilized and active upon various types of stresses. However, too much p53 is not beneficial to cells and causes lethality. At the steady state, p53 activity needs to be leashed for cell survival. Early studies suggested that the MDM2 oncoprotein negatively regulates p53 activity through the induction of p53 protein degradation. MDM2 serves as an E3 ubiquitin ligase of p53; it catalyzes polyubiquitination and subsequently induces proteasome degradation to downregulate p53 protein level. However, the mechanism by which MDM2 represses p53 is not a single mode. Emerging evidence reveals another cellular location of MDM2-p53 interaction. MDM2 is recruited to chromatin, specifically the p53 responsive promoter regions, in a p53 dependent manner. MDM2 is proposed to directly inhibit p53 transactivity at chromatin. This article provides an overview of the mechanism by which p53 is repressed by MDM2 in both ubiquitination dependent and ubiquitination independent pathways.

The histone acetyltransferase PCAF regulates p21 transcription through stress-induced acetylation of histone H3.

The activity of p53 as a tumor suppressor primarily depends on its ability to transactivate specific target genes in response to genotoxic and other potentially mutagenic stresses. Several histone acetyl transferases (HATs), including p300, CBP, PCAF and GCN5 have been implicated in the activation of p53-dependent transcription of the cyclin-dependent kinase (cdk) inhibitor p21 as well as other target genes. Here we show that PCAF, but not CBP or p300, is a critical regulator of p53-dependent p21 expression in response to multiple p53-activating stresses. PCAF was required for the transcriptional activation of p21 in response to exogenous p53 in p53-null cells, nutlin-3, DNA damaging agents and p14(ARF) expression, suggesting a broad requirement for PCAF in p53 signaling to p21 after stress. Importantly, cells lacking PCAF failed to undergo cell cycle arrest in response to nutlin-3 treatment or p14(ARF) expression, consistent with a physiologically important role for PCAF in this p53 function. Surprisingly, the role for PCAF in induction of p21 was independent of p53 lysine 320 acetylation, a previously suggested target of PCAF-mediated acetylation. Though p21 promoter occupancy by p53 was not altered by PCAF knockdown, activation of p21 transcription required an intact PCAF HAT domain, and induction of chromatin marks acetyl-H3K9 and acetyl-H3K14 at the p21 promoter by p53 was dependent upon physiologic levels of PCAF. Together, our experiments indicate that PCAF is required for stress-responsive histone 3 acetylation at the p21 promoter, p53-directed transcription of p21 and the resultant growth arrest.

Ubiquitin becomes ubiquitous in cancer: emerging roles of ubiquitin ligases and deubiquitinases in tumorigenesis and as therapeutic targets.

By virtue of its ability to regulate both protein turnover and non-proteolytic signalling functions, ubiquitin protein conjugation has been implicated in the control of multiple cellular processes, including protein localization, cell cycle control, transcription regulation, DNA damage repair, and endocytosis. Ubiquitin metabolism enzymes have been identified as either oncogenes or tumor suppressors in a variety of cancers. Given that ubiquitin metabolism is governed by enzymes--E1, E2, E3, E4, deubiquitinases (DUBs), and the proteasome- the system as a whole is ripe for target and drug discovery in cancer. Of the ubiquitin/proteasome system components, the E3's and DUBs can recognize substrates with the most specificity, and are thus of key interest as drug targets in cancer. This review examines the molecular role in cancer, relevant substrates, and potential for pharmacologic development, of E3's and DUBs that have been associated thus far with human malignancies as oncogenes or tumor suppressors.

Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves a post-ubiquitination mechanism.

Inhibition of the MDM2-p53 feedback loop is critical for p53 activation in response to cellular stresses. The ribosomal proteins L5, L11, and L23 can block this loop by inhibiting MDM2-mediated p53 ubiquitination and degradation in response to ribosomal stress. Here, we show that L11, but not L5 and L23, leads to a drastic accumulation of ubiquitinated and native MDM2. This effect is dependent on the ubiquitin ligase activity of MDM2, but not p53, and requires the central MDM2 binding domain (residues 51-108) of L11. We further show that L11 inhibited 26 S proteasome-mediated degradation of ubiquitinated MDM2 in vitro and consistently prolonged the half-life of MDM2 in cells. These results suggest that L11, unlike L5 and L23, differentially regulates the levels of ubiquitinated p53 and MDM2 and inhibits the turnover and activity of MDM2 through a post-ubiquitination mechanism.