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Hub proteins are central nodes in protein-protein interaction networks with critical importance to all living organisms. Recently, a new group of folded hub domains, the αα-hubs, was defined based on a shared αα-hairpin supersecondary structural foundation. The members PAH, RST, TAFH, NCBD, and HHD are found in large proteins such as Sin3, RCD1, TAF4, CBP, and harmonin, which organize disordered transcriptional regulators and membrane scaffolds in interactomes of importance to human diseases and plant quality. In this review, studies of structures, functions, and complexes across the αα-hubs are described and compared to provide a unified description of the group. This analysis expands the associated molecular concepts of "one domain-one binding site", motif-based ligand binding, and coupled folding and binding of intrinsically disordered ligands to additional concepts of importance to signal fidelity. These include context, motif reversibility, multivalency, complex heterogeneity, synergistic αα-hub:ligand folding, accessory binding sites, and supramodules. We propose that these multifaceted protein-protein interaction properties are made possible by the characteristics of the αα-hub fold, including supersite properties, dynamics, variable topologies, accessory helices, and malleability and abetted by adaptability of the disordered ligands. Critically, these features provide additional filters for specificity. With the presentations of new concepts, this review opens for new research questions addressing properties across the group, which are driven from concepts discovered in studies of the individual members. Combined, the members of the αα-hubs are ideal models for deconvoluting signal fidelity maintained by folded hubs and their interactions with intrinsically disordered ligands., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

General control nonderepressible 5 (GCN5, also known as Kat2a) and p300/CBP-associated factor (PCAF, also known as Kat2b) are two homologous acetyltransferases. Both proteins share similar domain architecture consisting of a PCAF N-terminal (PCAF_N) domain, acetyltransferase domain, and a bromodomain. PCAF also acts as a ubiquitin E3 ligase whose activity is attributable to the PCAF_N domain, but its structural aspects are largely unknown. Here, we demonstrated that GCN5 exhibited ubiquitination activity in a similar manner to PCAF and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which was not predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides new insight into not only the functional significance of GCN5 but also into ubiquitin biology., Competing Interests: Conflict of interest—All authors declare no conflict of interest in this work., (© 2020 Toma-Fukai et al.)

Protein acetylation is a prevalent posttranslational modification that is regulated by diverse acetyltransferase enzymes. Although histone acetyltransferases (HATs) have been well characterized both structurally and mechanistically, far less is known about non-histone acetyltransferase enzymes. The human ESCO1 and ESCO2 paralogs acetylate the cohesin complex subunit SMC3 to regulate the separation of sister chromatids during mitosis and meiosis. Missense mutations within the acetyltransferase domain of these proteins correlate with diseases, including endometrial cancers and Roberts syndrome. Despite their biological importance, the mechanisms underlying acetylation by the ESCO proteins are not understood. Here, we report the X-ray crystal structure of the highly conserved zinc finger-acetyltransferase moiety of ESCO1 with accompanying structure-based mutagenesis and biochemical characterization. We find that the ESCO1 acetyltransferase core is structurally homologous to the Gcn5 HAT, but contains unique additional features including a zinc finger and an ∼40-residue loop region that appear to play roles in protein stability and SMC3 substrate binding. We identify key residues that play roles in substrate binding and catalysis, and rationalize the functional consequences of disease-associated mutations. Together, these studies reveal the molecular basis for SMC3 acetylation by ESCO1 and have broader implications for understanding the structure/function of non-histone acetyltransferases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion within the N-terminal region of the ataxin-7 protein, a known subunit of the SAGA complex. Although the mechanisms of SCA7 pathogenesis remain poorly understood, previous studies have shown perturbations in SAGA histone acetyltransferase function and transcriptional alterations. We sought to determine whether and how polyQ-expanded ataxin-7 affects SAGA catalytic activity. Here, we determined that polyQ-expanded ataxin-7 directly bound the Gcn5 catalytic core of SAGA while in association with its regulatory proteins, Ada2 and Ada3. This caused a significant decrease in Gcn5 histone acetyltransferase activity in vitro and in vivo at two SAGA-regulated galactose genes, GAL1 and GAL7. However, Gcn5 occupancy at the GAL1 and GAL7 promoters was increased in these cells, revealing a dominant-negative phenotype of the polyQ-expanded ataxin-7-incorporated, catalytically inactive SAGA. These findings suggest a dominant mechanism of polyQ-mediated SAGA inhibition that potentially contributes to SCA7 disease pathogenesis.

The co-translational modification of N-terminal acetylation is ubiquitous among eukaryotes and has been reported to have a wide range of biological effects. The human N-terminal acetyltransferase (NAT) Naa50p (NAT5/SAN) acetylates the α-amino group of proteins containing an N-terminal methionine residue and is essential for proper sister chromatid cohesion and chromosome condensation. The elevated activity of NATs has also been correlated with cancer, making these enzymes attractive therapeutic targets. We report the x-ray crystal structure of Naa50p bound to a native substrate peptide fragment and CoA. We found that the peptide backbone of the substrate is anchored to the protein through a series of backbone hydrogen bonds with the first methionine residue specified through multiple van der Waals contacts, together creating an α-amino methionine-specific pocket. We also employed structure-based mutagenesis; the results support the importance of the α-amino methionine-specific pocket of Naa50p and are consistent with the proposal that conserved histidine and tyrosine residues play important catalytic roles. Superposition of the ternary Naa50p complex with the peptide-bound Gcn5 histone acetyltransferase revealed that the two enzymes share a Gcn5-related N-acetyltransferase fold but differ in their respective substrate-binding grooves such that Naa50p can accommodate only an α-amino substrate and not a side chain lysine substrate that is acetylated by lysine acetyltransferase enzymes such as Gcn5. The structure of the ternary Naa50p complex also provides the first molecular scaffold for the design of NAT-specific small molecule inhibitors with possible therapeutic applications.

Activation of transcription in response to low oxygen tension is mediated by the hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimer of two proteins: aryl hydrocarbon receptor nuclear translocator and the oxygen-regulated HIF-1 alpha. The C-terminal activation domain of HIF-1 alpha has been shown to interact with cysteine/histidine-rich region 1 (CH1) of the coactivator CBP/p300 in a hypoxia-dependent manner. However, HIF forms lacking C-terminal activation domain (naturally occurring or genetically engineered) are still able to activate transcription of target genes in hypoxia. Here, we demonstrate that the N-terminal activation domain (N-TAD) of HIF-1 alpha interacts with endogenous CBP and that this interaction facilitates its transactivation function. Our results show that interaction of HIF-1 alpha N-TAD with CBP/p300 is mediated by the CH3 region of CBP known to interact with, among other factors, p53. Using fluorescence resonance energy transfer experiments, we demonstrate that N-TAD interacts with CH3 in vivo. Coimmunoprecipitation assays using endogenous proteins showed that immunoprecipitation of CBP in hypoxia results in the recovery of a larger fraction of HIF-1 alpha than of p53. Chromatin immunoprecipitation demonstrated that at 1% O(2) CBP is recruited to a HIF-1 alpha but not to a p53 target gene. Upon activation of both pathways, lower levels of chromatin-associated CBP were detected at either target gene promoter. These results identify CBP as the coactivator directly interacting with HIF-1 alpha N-TAD and mediating the transactivation function of this domain. Thus, we suggest that in hypoxia HIF-1 alpha is a major CBP-interacting transcription factor that may compete with other CBP-dependent factors, including p53, for limiting amounts of this coactivator, underscoring the complexity in the regulation of gene expression by HIF-1 alpha.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a dual functional coregulator that facilitates transcription initiation by methylation of Arg(17) and Arg(26) of histone H3 and also dictates the subsequent coactivator complex disassembly by methylation of the steroid receptor coactivator family coactivators and p300/cAMP-response element-binding protein-binding protein. However, the regulation of CARM1 enzymatic activity and substrate specificity remains largely unknown. In this study, we report that CARM1 function is regulated by phosphorylation at Ser(217), a residue completely conserved in the type I protein arginine methyltransferase (PRMT) family of enzymes. Comparative analysis of the published CARM1 crystal structures reveals that the hydroxyl group of Ser(217) forms a strong hydrogen bond with the carbonyl oxygen atom of Tyr(154) to lock the cofactor S-adenosylmethionine inside the binding cavity. Phosphorylation of Ser(217) disrupts this hydrogen bond and subsequently abolishes S-adenosylmethionine binding and its methyltransferase activity. Importantly, Tyr(154) is also conserved in the type I PRMT family of enzymes, suggesting a general role of this hydrogen bond in maintaining the holo structure of the type I PRMT catalytic domain. Moreover, we found that phosphorylation at Ser(217) also promoted CARM1 cytoplasmic localization and that this translocation occurred mainly during mitosis. We propose that phosphorylation at Ser(217) serves as a molecular switch for controlling CARM1 enzymatic activity during the cell cycle.

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.

The hypoxic response in humans is regulated by the hypoxia-inducible transcription factor system; inhibition of hypoxia-inducible factor (HIF) activity has potential for the treatment of cancer. Chetomin, a member of the epidithiodiketopiperazine (ETP) family of natural products, inhibits the interaction between HIF-alpha and the transcriptional coactivator p300. Structure-activity studies employing both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1alpha and p300. In support of both cell-based and animal work showing that the cytotoxic effect of ETPs is reduced by the addition of Zn(2+) through an unknown mechanism, our mechanistic studies reveal that ETPs react with p300, causing zinc ion ejection. Cell studies with both natural and synthetic ETPs demonstrated a decrease in vascular endothelial growth factor and antiproliferative effects that were abrogated by zinc supplementation. The results have implications for the design of selective ETPs and for the interaction of ETPs with other zinc ion-binding protein targets involved in gene expression.

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Eukaryotic GCN5 acetyltransferases influence diverse biological processes by acetylating histones and non-histone proteins and regulating chromatin and gene-specific transcription as part of multiprotein complexes. In lower eukaryotes and invertebrates, these complexes include the yeast ADA complex that is still incompletely understood; the SAGA (Spt-Ada-Gcn5 acetylase) complexes from yeast to Drosophila that are mostly coactivators; and the ATAC (Ada Two-A containing) complex, only known in Drosophila and still poorly characterized. In contrast, vertebrate organisms, express two paralogous GCN5-like acetyltransferases (GCN5 and PCAF), which have been found so far only in SAGA-type complexes referred to hereafter as the STAGA (SPT3-TAF9-GCN5/PCAF acetylase) complexes. We now report the purification and characterization of vertebrate (human) ATAC-type complexes and identify novel components of STAGA. We show that human ATAC complexes incorporate in addition to GCN5 or PCAF (GCN5/PCAF), other epigenetic coregulators (ADA2-A, ADA3, STAF36, and WDR5), cofactors of chromatin assembly/remodeling and DNA replication machineries (POLE3/CHRAC17 and POLE4), the stress- and TGFbeta-activated protein kinase (TAK1/MAP3K7) and MAP3-kinase regulator (MBIP), additional cofactors of unknown function, and a novel YEATS2-NC2beta histone fold module that interacts with the TATA-binding protein (TBP) and negatively regulates transcription when recruited to a promoter. We further identify the p38 kinase-interacting protein (p38IP/FAM48A) as a novel component of STAGA with distant similarity to yeast Spt20. These results suggest that vertebrate ATAC-type and STAGA-type complexes link specific extracellular signals to modification of chromatin structure and regulation of the basal transcription machinery.

CITED1 is the founding member of the CITED family of cofactors that are involved in regulating a wide variety of CBP/p300-dependent transcriptional responses. In the present study, we show that the phosphorylation status of CITED1 changes during the cell cycle and affects its transcriptional cofactor activity. Tryptic mapping and mutagenesis studies identified five phosphorylated serine residues in CITED1. Phosphorylation of these residues did not affect CRM1-dependent nuclear export, but did decrease CITED1 binding to p300 and inhibited CITED1-dependent transactivation of Smad4 and p300. These results suggest that CITED1 functions as a cell cycle-dependent transcriptional cofactor whose activity is regulated by phosphorylation.

MOZ-TIF2 and MOZ-CBP are leukemogenic fusion proteins associated with therapy-induced acute myeloid leukemia. These proteins are thought to subvert normal gene expression in differentiating hematopoietic progenitor cells. We have previously shown that MOZ-TIF2 inhibits transcription by CREB-binding protein (CBP)/p300-dependent activators such as nuclear receptors and p53. Here we have shown that MOZ-TIF2 associates with the RARbeta2 promoter in vivo, resulting in altered recruitment of CBP/p300, aberrant histone modification, and down-regulation of the RARbeta2 gene. In contrast, MOZ-TIF2 up-regulated transcription mediated by the MOZ/MYST3-dependent activator AML1/RUNX1. Both wild type MOZ and MOZ-TIF2 were found to colocalize with AML1, and MOZ-TIF2 was recruited to an AML1 target promoter. A MOZ-CBP fusion protein showed similar functions to MOZ-TIF2 in that it inhibited retinoic acid receptor-mediated transcription but enhanced AML1 reporter activation. Although it contains almost the entire CBP sequence, MOZ-CBP does not appear to associate with PML bodies. In summary, our results indicate that leukemogenic MOZ fusion proteins have differential effects on the activities of CBP-dependent and MOZ-dependent activators because of their ability to alter cofactor recruitment and chromatin modification at target promoters.

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF(betaTrCP) ubiquitin ligase that binds to betaTrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.