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1.
Immunity ; 54(8): 1745-1757.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34348118

RESUMO

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas contra Escherichia coli/imunologia , Gastroenteropatias/imunologia , Intestino Delgado/imunologia , Linfócitos T Reguladores/imunologia , Administração Oral , Animais , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Escherichia coli/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Gastroenteropatias/microbiologia , Gastroenteropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Vacinação
2.
Mol Cell ; 82(24): 4611-4626.e7, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36476474

RESUMO

PALI1 is a newly identified accessory protein of the Polycomb repressive complex 2 (PRC2) that catalyzes H3K27 methylation. However, the roles of PALI1 in cancer are yet to be defined. Here, we report that PALI1 is upregulated in advanced prostate cancer (PCa) and competes with JARID2 for binding to the PRC2 core subunit SUZ12. PALI1 further interacts with the H3K9 methyltransferase G9A, bridging the formation of a unique G9A-PALI1-PRC2 super-complex that occupies a subset of G9A-target genes to mediate dual H3K9/K27 methylation and gene repression. Many of these genes are developmental regulators required for cell differentiation, and their loss in PCa predicts poor prognosis. Accordingly, PALI1 and G9A drive PCa cell proliferation and invasion in vitro and xenograft tumor growth in vivo. Collectively, our study shows that PALI1 harnesses two central epigenetic mechanisms to suppress cellular differentiation and promote tumorigenesis, which can be targeted by dual EZH2 and G9A inhibition.


Assuntos
Neoplasias , Complexo Repressor Polycomb 2 , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Neoplasias/genética , Epigênese Genética
3.
Mol Cell ; 80(4): 726-735.e7, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33049227

RESUMO

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.


Assuntos
Cromatina/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Mutação , Proteínas Oncogênicas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regulação Alostérica , Animais , Ilhas de CpG , Drosophila melanogaster , Humanos , Camundongos , Proteínas Oncogênicas/genética , Complexo Repressor Polycomb 2/genética
4.
Cereb Cortex ; 34(7)2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38960704

RESUMO

The Polycomb Repressive Complex 2 (PRC2) regulates corticogenesis, yet the consequences of mutations to this epigenetic modifier in the mature brain are poorly defined. Importantly, PRC2 core genes are haploinsufficient and causative of several human neurodevelopmental disorders. To address the role of PRC2 in mature cortical structure and function, we conditionally deleted the PRC2 gene Eed from the developing mouse dorsal telencephalon. Adult homozygotes displayed smaller forebrain structures. Single-nucleus transcriptomics revealed that glutamatergic neurons were particularly affected, exhibiting dysregulated gene expression profiles, accompanied by aberrations in neuronal morphology and connectivity. Remarkably, homozygous mice performed well on challenging cognitive tasks. In contrast, while heterozygous mice did not exhibit clear anatomical or behavioral differences, they displayed dysregulation of neuronal genes and altered neuronal morphology that was strikingly different from homozygous phenotypes. Collectively, these data reveal how alterations to PRC2 function shape the mature brain and reveal a dose-specific role for PRC2 in determining glutamatergic neuron identity.


Assuntos
Ácido Glutâmico , Neurogênese , Neurônios , Complexo Repressor Polycomb 2 , Animais , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Camundongos , Neurogênese/fisiologia , Ácido Glutâmico/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Masculino , Camundongos Endogâmicos C57BL , Feminino , Camundongos Transgênicos
5.
Am J Med Genet A ; 194(2): 374-382, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37840385

RESUMO

EED is a core component of polycomb repressive complex 2 (PRC2) with EZH2 and SUZ12. PRC2 has H3K27 methyltransferase activity (HMTase) that catalyzes the addition of up to three methyl groups on histone 3 at lysine residue 27 (H3K27). Germline heterozygous variants in EED, SUZ12, and EZH2 have been identified in patients with overgrowth and multiple dysmorphic features. The clinical manifestations of these syndromes significantly overlap: generalized overgrowth, intellectual disability, and scoliosis. To date, 11 unrelated patients have been published with missense variants in EED at highly conserved amino acids. We report three affected members in a family with a previously reported missense variant. All three affected members manifested very similarly, and this represents a homogenous clinical phenotype associated with EED related intellectual disability and overgrowth. This disorder is appropriately called Cohen-Gibson syndrome.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Deficiência Intelectual , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Complexo Repressor Polycomb 2/genética , Histonas/genética , Mutação de Sentido Incorreto/genética
6.
Genes Chromosomes Cancer ; 62(3): 152-160, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36445224

RESUMO

Endometrial stromal sarcomas (ESS) are morphologically and molecularly heterogeneous. We report novel gene fusions (EPC1::EED, EPC1::EZH2, ING3::PHF1) identified by targeted RNA sequencing in five cases. The ING3::PHF1-fusion positive ESS presented in a 58-year-old female as extrauterine mesocolonic, ovarian masses, and displayed large, monomorphic ovoid-to-epithelioid cells arranged in solid sheets. The patient remained alive with disease 13 months after surgery. The three ESS with EPC1::EED occurred in the uterine corpus in patients with a median age of 58 years (range 27-62 years). One tumor showed a uniform epithelioid nested morphology, while the other two were composed of monomorphic spindle cells in fascicles with elevated mitotic figures, focal tumor cell necrosis, and lymphovascular invasion. At a median follow-up of 20 months, two patients developed local recurrence, including one with concomitant distant metastasis, while one patient remained free of disease. All three patients were alive at the last follow-up. The EPC1::EZH2-fusion positive ESS presented in a 52-year-old female in the uterus, and displayed uniform spindled cells arranged in short fascicles, with focally elevated mitotic activity but without necrosis. The patient remained free of disease 3 months after surgery. All cases were diffusely positive for CD10; four diffusely express estrogen and progesterone receptors. Our study expands the molecular spectrum of EPC1 and PHF1-related gene fusions in ESS to include additional novel subunits of the PRC2 and/or NuA4/TIP60 complexes. These cases displayed a monomorphic epithelioid or spindled phenotype, spanning low-grade and high-grade cytomorphology, all expressing CD10 and commonly ER and PR, and are prone to local and/or distant spread.


Assuntos
Neoplasias do Endométrio , Sarcoma do Estroma Endometrial , Feminino , Humanos , Sarcoma do Estroma Endometrial/genética , Sarcoma do Estroma Endometrial/cirurgia , Sarcoma do Estroma Endometrial/patologia , Montagem e Desmontagem da Cromatina , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/cirurgia , Neoplasias do Endométrio/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fusão Gênica , Proteínas de Homeodomínio/genética , Proteínas Supressoras de Tumor/genética
7.
Am J Hum Genet ; 106(5): 596-610, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32243864

RESUMO

Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.


Assuntos
Anormalidades Múltiplas/genética , Hipotireoidismo Congênito/genética , Anormalidades Craniofaciais/genética , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Mutação , Complexo Repressor Polycomb 2/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Masculino , Mosaicismo , Mutação de Sentido Incorreto/genética , Proteínas de Neoplasias , Reprodutibilidade dos Testes , Fatores de Transcrição , Adulto Jovem
8.
Development ; 147(21)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32376680

RESUMO

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule neuron progenitors (CGNPs) and its mis-regulation is linked to various disorders, including the cerebellar cancer medulloblastoma (MB). We recently identified RNF220, a ubiquitin E3 ligase promoting K63-linked polyubiquitylation and nuclear exportation of Gli transcription factors, as an Shh/Gli regulator involved in ventral neural patterning. Here, we report that RNF220 is required for the proliferation of CGNPs and Daoy cells (an Shh-grouped MB cell line), working as a positive regulator of Shh signaling. Mechanistic investigation demonstrated that RNF220 promotes Shh target gene expression by targeting the PRC2 component EED, and alters levels of epigenetic modification marks on Shh target promoters. We provided evidence that RNF220+/-; Ptch1+/- mice showed lower spontaneous MB occurrence compared with Ptch1+/- mice. Furthermore, in human clinical MB samples, RNF220 expression correlated well with that of GAB1, an Shh-group MB marker. Our findings provide new insights into the epigenetic regulation of Shh signaling and identify RNF220 as a potential new diagnostic marker and therapeutic target for Shh-group MB.


Assuntos
Cerebelo/embriologia , Progressão da Doença , Epigênese Genética , Proteínas Hedgehog/genética , Meduloblastoma/genética , Meduloblastoma/patologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cerebelo/patologia , Grânulos Citoplasmáticos/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Neurocrit Care ; 2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38030878

RESUMO

BACKGROUND: Our objective was to test the association between hematoma volume and long-term (> 72 h) edema extension distance (EED) evolution and the association between peak EED and early EED increase with functional outcome at 3 months in patients with intracerebral hemorrhage (ICH). METHODS: This retrospective cohort study included patients with spontaneous supratentorial ICH between January 2006 and January 2014. EED, an edema measure defined as the distance between the hematoma border and the outer edema border, was calculated by using absolute hematoma and edema volumes. We used multivariable logistic regression accounting for age, ICH volume, and location and receiver operating characteristic analysis for assessing measures associated with functional outcome and EED evolution. Functional outcome after 3 months was assessed by using the modified Rankin Scale (0-3 = favorable, 4-6 = unfavorable). To identify properties associated with peak EED multivariable linear and logistic regression analyses were conducted. RESULTS: A total of 292 patients were included. Median age was 70 years (interquartile range [IQR] 62-78), median ICH volume on admission 17.7 mL (IQR 7.9-40.2), median peak perihemorrhagic edema (PHE) volume was 37.5 mL (IQR 19.1-60.6), median peak EED was 0.67 cm (IQR 0.51-0.84) with an early EED increase up to 72 h (EED72-0) of 0.06 cm (- 0.02 to 0.15). Peak EED was found to be independent of ICH volume (R2 = 0.001, p = 0.6). In multivariable analyses, peak EED (odds ratio 0.224, 95% confidence interval [CI] [0.071-0.705]) and peak PHE volume (odds ratio 0.984 [95% CI 0.973-0.994]) were inversely associated with favorable functional outcome at 3 months. Receiver operating characteristic analysis identified a peak PHE volume of 26.8 mL (area under the curve 0.695 [95% CI 0.632-0.759]; p ≤ 0.001) and a peak EED of 0.58 cm (area under the curve 0.608 [95% CI 0.540-0.676]; p = 0.002) as best predictive values for outcome discrimination. CONCLUSIONS: Compared with absolute peak PHE volume, peak EED represents a promising edema measure in patients with ICH that is largely hematoma volume-independent and nevertheless associated with functional outcome.

10.
Molecules ; 28(24)2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-38138487

RESUMO

Inhibition of the Embryonic Ectoderm Development (EED) subunit in Polycomb Repressive Complex 2 (PRC2) can inhibit tumor growth. In this paper, we selected six experimentally designed EED competitive Inhibitors of the triazolopyrimidine derivatives class. We investigated the difference in the binding mode of the natural substrate to the Inhibitors and the effects of differences in the parent nuclei, heads, and tails of the Inhibitors on the inhibitory capacity. The results showed that the binding free energy of this class of Inhibitors was close to or lower compared to the natural substrate, providing an energetic basis for competitive inhibition. For the Inhibitors, the presence of a strong negatively charged group at the 6-position of the parent nucleus or the 8'-position of the head would make the hydrogen atom on the head imino group prone to flip, resulting in the vertical movement of the parent nucleus, which significantly decreased the inhibitory ability. When the 6-position of the parent nucleus was a nonpolar group, the parent nucleus would move horizontally, slightly decreasing the inhibitory ability. When the 8'-position of the head was methylene, it formed an intramolecular hydrophobic interaction with the benzene ring on the tail, resulting in a significant increase in inhibition ability.


Assuntos
Ectoderma , Simulação de Dinâmica Molecular , Ectoderma/metabolismo , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo
11.
J Cell Mol Med ; 26(14): 4061-4075, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35734954

RESUMO

Polycomb repressive complex 2 (PRC2) is a multicomponent complex with methyltransferase activity that catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3). Interaction of the epigenetic reader protein EED with EZH2, a catalytic unit of PRC, allosterically stimulates PRC2 activity. In this study, we investigated the role and underlying mechanism of the PRC2 in acute kidney injury (AKI) by using EED226, a highly selective PRC2 inhibitor, to target EED. Administration of EED226 improved renal function, attenuated renal pathological changes, and reduced renal tubular cell apoptosis in a murine model of cisplatin-induced AKI. In cultured renal epithelial cells, treatment with either EED226 or EED siRNA also ameliorated cisplatin-induced apoptosis. Mechanistically, EED226 treatment inhibited cisplatin-induced phosphorylation of p53 and FOXO3a, two transcriptional factors contributing to apoptosis, and preserved expression of Sirtuin 3 and PGC1α, two proteins associated with mitochondrial protection in vivo and in vitro. EED226 was also effective in enhancing renal tubular cell proliferation, suppressing expression of multiple inflammatory cytokines, and reducing infiltration of macrophages to the injured kidney. These data suggest that inhibition of the PRC2 activity by targeting EED can protect against cisplatin-induced AKI by promoting the survival and proliferation of renal tubular cells and inhibiting inflammatory response.


Assuntos
Injúria Renal Aguda , Complexo Repressor Polycomb 2 , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Animais , Cisplatino , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Complexo Repressor Polycomb 2/metabolismo
12.
Am J Med Genet A ; 188(1): 292-297, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34533271

RESUMO

Cohen-Gibson syndrome is a rare genetic disorder, characterized by fetal or early childhood overgrowth and mild to severe intellectual disability. It is caused by heterozygous aberrations in EED, which encodes an evolutionary conserved polycomb group (PcG) protein that forms the polycomb repressive complex-2 (PRC2) together with EZH2, SUZ12, and RBBP7/4. In total, 11 affected individuals with heterozygous pathogenic variants in EED were reported, so far. All variants affect a few key residues within the EED WD40 repeat domain. By trio exome sequencing, we identified the heterozygous missense variant c.581A > G, p.(Asn194Ser) in exon 6 of the EED-gene in an individual with moderate intellectual disability, overgrowth, and epilepsy. The same pathogenic variant was detected in 2 of the 11 previously reported cases. Epilepsy, however, was only diagnosed in one other individual with Cohen-Gibson syndrome before. Our findings further confirm that the WD40 repeat domain represents a mutational hotspot; they also expand the clinical spectrum of Cohen-Gibson syndrome and highlight the clinical variability even in individuals with the same pathogenic variant. Furthermore, they indicate a possible association between Cohen-Gibson syndrome and epilepsy.


Assuntos
Epilepsia , Deficiência Intelectual , Pré-Escolar , Epilepsia/genética , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação , Complexo Repressor Polycomb 2/genética , Sequenciamento do Exoma
13.
Xenobiotica ; 52(1): 65-78, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34761729

RESUMO

MAK683 (N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridin-3-yl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine) is a potent and orally bioavailable EED inhibitor for the potential treatment in oncology. Pharmacokinetics (PK) in preclinical species are characterised by low to moderate plasma clearances, high oral exposure, and moderate to high oral bioavailability at the dose of 1-2 mg/kg.A species comparison of the metabolic pathways of MAK683 has been made using [14C]MAK683 incubations with liver microsomes and hepatocytes from rat, dog, cynomolgus monkey, and human. Overall, the in vitro hepatic metabolism pathway of MAK683 in all five species was very complex. A total of 60 metabolites with 19 metabolites >1.5% of the total integrated area in the radiochromatogram of at least one species were identified in five species (rat, mouse, dog, monkey, and human).The primary in vitro hepatic oxidative metabolism pathway identified in humans involved 2-hydroxylation of the dihydrofuran ring to form alcohol (M28), which was in a chemical equilibrium favouring the formation of its aldehyde form. The aldehyde was then oxidised to the carboxylic acid metabolite (M26) or reduced to the O-hydroxyethylphenol (M29). N-dealkylation (M1), 3-hydroxylation of the dihydrofuran ring (M27), N-oxidation of the pyridine moiety (M53), and sulphate conjugation of M28 to form M19 were also important biotransformation pathways in human hepatocytes. The above major human hepatic metabolic pathways were also observed across the animal species (rat, mouse, dog, and monkey) mostly providing precursors for the formation of other metabolites via further oxygenation, glucuronidation, and sulphation pathways.No human-specific metabolites were observed. In addition, in vivo biotransformation was also conducted in bile-duct cannulated (BDC) rat. The metabolism in BDC rat was similar to those observed the in vitro hepatocytes.


Assuntos
Ectoderma , Neoplasias , Animais , Cães , Hepatócitos/metabolismo , Macaca fascicularis , Camundongos , Microssomos Hepáticos/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Ratos
14.
J Nutr ; 151(12): 3689-3700, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34718665

RESUMO

BACKGROUND: Intestinal inflammation and malabsorption in environmental enteric dysfunction (EED) are associated with early childhood growth faltering in impoverished settings worldwide. OBJECTIVES: The goal of this study was to identify candidate biomarkers associated with inflammation, EED histology, and as predictors of later growth outcomes by focusing on the liver-gut axis by investigating the bile acid metabolome. METHODS: Undernourished rural Pakistani infants (n = 365) with weight-for-height Z score (WHZ) < -2 were followed up to the age of 24 mo and monitored for growth, infections, and EED. Well-nourished local children (n = 51) were controls, based on consistent WHZ > 0 and height-for-age Z score (HAZ) > -1 on 2 consecutive visits at 3 and 6 mo. Serum bile acid (sBA) profiles were measured by tandem MS at the ages of 3-6 and 9 mo and before nutritional intervention. Biopsies and duodenal aspirates were obtained following upper gastrointestinal endoscopy from a subset of children (n = 63) that responded poorly to nutritional intervention. BA composition in paired plasma and duodenal aspirates was compared based on the severity of EED histopathological scores and correlated to clinical and growth outcomes. RESULTS: Remarkably, >70% of undernourished Pakistani infants displayed elevated sBA concentrations consistent with subclinical cholestasis. Serum glycocholic acid (GCA) correlated with linear growth faltering (HAZ, r = -0.252 and -0.295 at the age of 3-6 and 9 mo, respectively, P <0.001) and biomarkers of inflammation. The proportion of GCA positively correlated with EED severity for both plasma (rs = 0.324 P = 0.02) and duodenal aspirates (rs = 0.307 P = 0.06) in children with refractory wasting that underwent endoscopy, and the proportion of secondary BA was low in both undernourished and EED children. CONCLUSIONS: Dysregulated bile acid metabolism is associated with growth faltering and EED severity in undernourished children. Restoration of intestinal BA homeostasis may offer a novel therapeutic target for undernutrition in children with EED. This trial was registered at clinicaltrials.gov as NCT03588013.


Assuntos
Transtornos da Nutrição Infantil , Transtornos da Nutrição do Lactente , Ácidos e Sais Biliares , Criança , Pré-Escolar , Transtornos do Crescimento/etiologia , Humanos , Lactente , Intestino Delgado
15.
Bioorg Med Chem Lett ; 39: 127904, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33684441

RESUMO

Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Purinas/farmacologia , Termodinâmica , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Ligantes , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Complexo Repressor Polycomb 2/metabolismo , Purinas/síntese química , Purinas/química , Teoria Quântica , Relação Estrutura-Atividade
16.
J Comput Aided Mol Des ; 35(5): 601-611, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33635506

RESUMO

Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is overexpressed in many cancers. Numerous EZH2 inhibitors have been developed as anticancer agents, but recent studies have also focused on protein-protein interaction (PPI) between embryonic ectoderm development (EED) and EZH2 as a novel drug discovery target. Because EED indirectly enhances EZH2 enzymatic activity, EED-EZH2 PPI inhibitors suppress the methyltransferase activity and inhibit cancer growth. By contrast to the numerous promising EZH2 inhibitors, there are a paucity of EED-EZH2 PPI inhibitors reported in the literature. Here, we aimed to discover novel EED-EZH2 PPI inhibitors by first identifying possible binders of EED using an in-house knowledge-based in silico fragment mapping method. Next, 3D pharmacophore models were constructed from the arrangement pattern of the potential binders mapped onto the EED surface. In all, 16 compounds were selected by 3D pharmacophore-based virtual screening followed by docking-based virtual screening. In vitro evaluation revealed that five of these compounds exhibited inhibitory activities. This study has provided structural insights into the discovery and the molecular design of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Complexo Repressor Polycomb 2/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Simulação por Computador , Desenho de Fármacos , Descoberta de Drogas , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo
17.
Development ; 144(3): 400-408, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28049658

RESUMO

Urothelium is the protective lining of the urinary tract. The mechanisms underlying urothelial formation and maintenance are largely unknown. Here, we report the stage-specific roles of PRC2 epigenetic regulators in embryonic and adult urothelial progenitors. Without Eed, the obligatory subunit of PRC2, embryonic urothelial progenitors demonstrate reduced proliferation with concomitant dysregulation of genes including Cdkn2a (p16), Cdkn2b (p15) and Shh. These mutants display premature differentiation of keratin 5-positive (Krt5+) basal cells and ectopic expression of squamous-like differentiation markers. Deletion of Ezh2, the major enzymatic component of PRC2, causes upregulation of Upk3a+ superficial cells. Unexpectedly, Eed and Eed/Ezh2 double mutants exhibit delayed superficial cell differentiation. Furthermore, Eed regulates the proliferative and regenerative capacity of adult urothelial progenitors and prevents precocious differentiation. Collectively, these findings uncover the epigenetic mechanism by which PRC2 controls urothelial progenitor cell fate and the timing of differentiation, and further suggest an epigenetic basis of urothelial maintenance and regeneration.


Assuntos
Complexo Repressor Polycomb 2/fisiologia , Regeneração/fisiologia , Bexiga Urinária/crescimento & desenvolvimento , Bexiga Urinária/fisiologia , Urotélio/crescimento & desenvolvimento , Urotélio/fisiologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/genética , Subunidades Proteicas , Regeneração/genética , Bexiga Urinária/embriologia , Urotélio/embriologia
18.
Chemphyschem ; 21(3): 263-271, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31816138

RESUMO

Polycomb Polycomb repressive complex 2 (PRC2) plays a key role in silencing epigenetic gene through trimethylation of lysine 27 on histone 3 (H3K27). Dysregulations of PRC2 caused by overexpression and mutations of the core subunits of PRC2 have been implicated in many cancers. The core subunits EZH1/2 are histone-lysine N-methyltransferases that function as the enzymatic component of PRC2. While the core subunit EED is a scaffolding protein to support EZH1/2 and binds JARID2K116me3/H3K27me3 to enhance the enzymatic activity of PRC2 through allosteric activation. Recently, several small molecules that compete with JARI2K116me3 and H3K27me3 have been reported. These molecules selectively bind to the JARID2K116me3/H3K27me3-binding pocket of EED, thereby preventing the allosteric regulation of PRC2. These first-in-class PRC2 inhibitors show robust suppression in DLBCL cell lines, demonstrating anticancer drugs that target the EED subunit of PRC2 are viable. In this study, we used the recently developed MM/GBSA_IE and the alanine scanning method to analyze the hot spots in EED/inhibitor interactions. The analysis of these hot and warm spots helps us to understand the fundamental differences between inhibitors. Our results give a quantitative explanation on why the binding affinities of EED/A-395 interactions are stronger than that of EED/EED226 while their binding modes are similar and provide valuable insights for rational design of novel EED inhibitors.


Assuntos
Indanos/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Sulfonamidas/metabolismo , Sulfonas/metabolismo , Triazóis/metabolismo , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Complexo Repressor Polycomb 2/química , Ligação Proteica , Termodinâmica
19.
Chem Pharm Bull (Tokyo) ; 68(1): 58-63, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31685780

RESUMO

Polycomb repressive complex 2 (PRC2) is an attractive drug target for anti-cancer treatment. Among the three core subunits (EZH2, EED and SUZ12) of PRC2, EZH2 is the catalytic subunit that methylates histone H3 lysine 27 (H3K27), while EED is the regulatory subunit. Besides the small-molecule inhibitors of EZH2, those targeting the protein-protein interaction (PPI) between EZH2 and EED have also been reported. Here, for the first time, we have identified the key residues that contributed most to the EED-EZH2 binding affinity by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations based on the 200 ns molecular dynamics simulation. Moreover, we report the identification of two novel and potent small-molecule inhibitors (35 and 49) of EZH2-EED interaction (bottom interaction surface) by virtual screening and biological evaluations. Binding modes of the two identified molecules with EED were probed by molecular docking. Additionally, 35 and 49 displayed cellular antiproliferative activity against diffuse large B-cell lymphoma (DLBCL) cancer cell line Toledo whose cell growth was driven by aberrant PRC2 activity. Our findings have provided structural insights for the design of novel EZH2-EED interaction inhibitors to regulate the activity of PRC2 complex.


Assuntos
Simulação de Acoplamento Molecular , Complexo Repressor Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Ligação Competitiva , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Humanos , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
20.
Dev Biol ; 433(1): 47-60, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29137925

RESUMO

Polycomb repressive complexes maintain transcriptional repression of genes encoding crucial developmental regulators through chromatin modification. Here we investigated the role of Polycomb repressive complex 2 (PRC2) in retinal development by inactivating its key components Eed and Ezh2. Conditional deletion of Ezh2 resulted in a partial loss of PRC2 function and accelerated differentiation of Müller glial cells. In contrast, inactivation of Eed led to the ablation of PRC2 function at early postnatal stage. Cell proliferation was reduced and retinal progenitor cells were significantly decreased in this mutant, which subsequently caused depletion of Müller glia, bipolar, and rod photoreceptor cells, primarily generated from postnatal retinal progenitor cells. Interestingly, the proportion of amacrine cells was dramatically increased at postnatal stages in the Eed-deficient retina. In accordance, multiple transcription factors controlling amacrine cell differentiation were upregulated. Furthermore, ChIP-seq analysis showed that these deregulated genes contained bivalent chromatin (H3K27me3+ H3K4me3+). Our results suggest that PRC2 is required for proliferation in order to maintain the retinal progenitor cells at postnatal stages and for retinal differentiation by controlling amacrine cell generation.


Assuntos
Complexo Repressor Polycomb 2/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Neurogênese , Neuroglia/metabolismo , Retina/metabolismo , Retina/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo
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