scholarly journals PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Siddhant U. Jain ◽  
Truman J. Do ◽  
Peder J. Lund ◽  
Andrew Q. Rashoff ◽  
Katharine L. Diehl ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Cpg Islands ◽  
Inhibitory Protein ◽  
Allosteric Activation ◽  
Conserved Sequence ◽  
H3k27 Methylation ◽  
Necessary And Sufficient

Abstract Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP ‘oncohistone-mimic’, that dysregulate gene silencing to promote tumorigenesis.

scholarly journals PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

2019 ◽  
Author(s):  
Siddhant U. Jain ◽  
Truman J. Do ◽  
Peder J. Lund ◽  
Andrew Q. Rashoff ◽  
Marcin Cieslik ◽  
...  
Keyword(s):  
Cpg Islands ◽  
Polycomb Group ◽  
Inhibitory Protein ◽  
Conserved Sequence ◽  
H3k27 Methylation ◽  
C Terminus ◽  
Diffuse Intrinsic Pontine Gliomas ◽  
Necessary And Sufficient

ABSTRACTPolycomb group (PcG) proteins are essential for development and are frequently misregulated in human cancers. Polycomb Repressive Complexes (PRC1, PRC2) function in a collaborative epigenetic cross-talk with H3K27me3 to initiate and maintain transcriptional silencing. Diffuse intrinsic pontine gliomas (DIPGs) have extremely low H3K27me3 levels mediated by H3 K27M oncohistone. Posterior fossa type A (PFA) ependymomas also exhibit very low H3K27 methylation but lack the K27M oncohistone. Instead, PFA tumors express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). We find that a highly conserved sequence within the C-terminus of EZHIP is necessary and sufficient to inhibit the catalytic activity of PRC2 in vitro and in vivo. Our biochemical experiments indicate that EZHIP directly interacts with the active site of the EZH2 subunit in a mechanism that is remarkably similar to the K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promote similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at the sites of PRC2 recruitment. Importantly, we find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a potential mechanism for loss of H3K27me3 spreading from CpG islands in vivo. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors– the K27M oncohistone and the EZHIP ‘oncohistone-mimic’– that dysregulate gene silencing to promote tumorigenesis.

scholarly journals Set2-Dependent K36 Methylation Is Regulated by Novel Intratail Interactions within H3

2009 ◽  
Vol 29 (24) ◽  
pp. 6413-6426 ◽  
Author(s):  
James N. Psathas ◽  
Suting Zheng ◽  
Song Tan ◽  
Joseph C. Reese
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Transcription Initiation ◽  
Gene Activation ◽  
N Terminus

ABSTRACT Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

scholarly journals A Conserved Motif in Goosecoid Mediates Groucho-Dependent Repression in Drosophila Embryos

1999 ◽  
Vol 19 (3) ◽  
pp. 2080-2087 ◽  
Author(s):  
Gerardo Jiménez ◽  
C. Peter Verrijzer ◽  
David Ish-Horowicz
Keyword(s):  
Transcriptional Repressors ◽  
Multiprotein Complex ◽  
Biological Functions ◽  
Conserved Sequence ◽  
Peptide Motifs ◽  
Necessary And Sufficient ◽  
Target Promoters ◽  
Drosophila Embryos

ABSTRACT Surprisingly small peptide motifs can confer critical biological functions. One example is the WRPW tetrapeptide present in the Hairy family of transcriptional repressors, which mediates recruitment of the Groucho (Gro) corepressor to target promoters. We recently showed that Engrailed (En) is another repressor that requires association with Gro for its function. En lacks a WRPW motif; instead, it contains another short conserved sequence, the En homology region 1 (eh1)/GEH motif, that is likely to play a role in tethering Gro to the promoter. Here, we characterize a repressor domain from the Goosecoid (Gsc) developmental regulator that includes an eh1/GEH-like motif. We demonstrate that this domain (GscR) mediates efficient repression in Drosophila blastoderm embryos and that repression by GscR requires Gro function. GscRand Gro interact in vitro, and the eh1/GEH motif is necessary and sufficient for the interaction and for in vivo repression. Because WRPW- and eh1/GEH-like motifs are present in different proteins and in many organisms, the results suggest that interactions between short peptides and Gro represent a widespread mechanism of repression. Finally, we investigate whether Gro is part of a stable multiprotein complex in the nucleus. Our results indicate that Gro does not form stable associations with other proteins but that it may be able to assemble into homomultimeric complexes.

scholarly journals H3 K27M and EZHIP impede H3K27-methylation spreading by inhibiting allosterically stimulated PRC2

2020 ◽  
Author(s):  
Siddhant U. Jain ◽  
Andrew Q. Rashoff ◽  
Samuel D. Krabbenhoft ◽  
Dominik Hoelper ◽  
Truman J. Do ◽  
...  
Keyword(s):  
Cpg Islands ◽  
Methyltransferase Activity ◽  
Polycomb Repressive Complex 2 ◽  
H3k27 Methylation ◽  
K27m Mutation ◽  
Mechanistic Insight ◽  
Competitive Inhibitors ◽  
Lysine Methyltransferase

AbstractDiffuse midline gliomas and posterior fossa type-A ependymomas contain the highly recurrent histone H3 K27M mutation and the H3 K27M-mimic EZHIP, respectively. In vitro, H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3K27me3 levels, however residual peaks of H3K27me3 remain at CpG islands through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with an allosterically activated form of PRC2 in vivo. The formation of H3 K27M- and EZHIP-PRC2 complexes occurs at CpG islands containing H3K27me3 and impedes PRC2 and H3K27me3 spreading. While EZHIP is not found outside of placental mammals, we find that expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved molecular mechanism. Our results highlight the mechanistic similarities between EZHIP and H3 K27M in vivo and provide mechanistic insight for the retention of residual H3K27me3 in tumors driven by these oncogenes.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals YAP inhibits autophagy and promotes progression of colorectal cancer via upregulating Bcl-2 expression

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Lan Jin ◽  
Yunhe Chen ◽  
Dan Cheng ◽  
Zhikai He ◽  
Xinyi Shi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Death ◽  
Transcriptional Coactivator ◽  
Inhibitory Protein ◽  
Potential Therapeutic Target ◽  
Related Cell ◽  
Autophagy Inhibition

AbstractColorectal cancer (CRC) is one of the most aggressive and lethal cancers. The role of autophagy in the pathobiology of CRC is intricate, with opposing functions manifested in different cellular contexts. The Yes-associated protein (YAP), a transcriptional coactivator inactivated by the Hippo tumor-suppressor pathway, functions as an oncoprotein in a variety of cancers. In this study, we found that YAP could negatively regulate autophagy in CRC cells, and consequently, promote tumor progression of CRC in vitro and in vivo. Mechanistically, YAP interacts with TEAD forming a complex to upregulate the transcription of the apoptosis-inhibitory protein Bcl-2, which may subsequently facilitate cell survival by suppressing autophagy-related cell death; silencing Bcl-2 expression could alleviate YAP-induced autophagy inhibition without affecting YAP expression. Collectively, our data provide evidence for YAP/Bcl-2 as a potential therapeutic target for drug exploration against CRC.

scholarly journals ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Ove Alexander Høgmoen Åstrand ◽  
Christopher Fröhlich ◽  
Adam Heikal ◽  
Susann Skagseth ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Functional Space ◽  
Bactericidal Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

scholarly journals ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

2005 ◽  
Vol 79 (20) ◽  
pp. 12721-12731 ◽  
Author(s):  
Ákos Putics ◽  
Witold Filipowicz ◽  
Jonathan Hall ◽  
Alexander E. Gorbalenya ◽  
John Ziebuhr
Keyword(s):  
Active Site ◽  
Biological Significance ◽  
Virus Titer ◽  
Site Directed Mutagenesis ◽  
Replicase Gene ◽  
Active Site Residues ◽  
Nonstructural Proteins ◽  
Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

scholarly journals PGC7 suppresses TET3 for protecting DNA methylation

2013 ◽  
Vol 42 (5) ◽  
pp. 2893-2905 ◽  
Author(s):  
Chunjing Bian ◽  
Xiaochun Yu
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanism ◽  
Biological Process ◽  
Cpg Islands ◽  
Binding Motifs ◽  
Genome Wide Analysis ◽  
Dna Motif ◽  
Genome Wide

Abstract Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation.

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