RESUMO
Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Short linear motif (SLiM)-mediated interactions offer a unique strategy for viral intervention due to their compact interfaces, ease of convergent evolution, and key functional roles. Consequently, many viruses extensively mimic host SLiMs to hijack or deregulate cellular pathways and the same motif-binding pocket is often targeted by numerous unrelated viruses. A toolkit of therapeutics targeting commonly mimicked SLiMs could provide prophylactic and therapeutic broad-spectrum antivirals and vastly improve our ability to treat ongoing and future viral outbreaks. In this opinion article, we discuss the therapeutic relevance of SLiMs, advocating their suitability as targets for broad-spectrum antiviral inhibitors.
Assuntos
Motivos de Aminoácidos , Antivirais , Antivirais/farmacologiaRESUMO
Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.
Assuntos
Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/ultraestrutura , Sequência de Aminoácidos/genética , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X/métodos , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica/genética , Especificidade por SubstratoRESUMO
Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome.
RESUMO
The modification of intracellular proteins with O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain.
Assuntos
Proteínas , Proteômica , Animais , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , PeptídeosRESUMO
Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein-protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function.
Assuntos
Biblioteca de Peptídeos , Proteômica , Humanos , Fosforilação , ClatrinaRESUMO
The ERM (ezrin, radixin, and moesin) family of proteins and the related protein merlin participate in scaffolding and signaling events at the cell cortex. The proteins share an N-terminal FERM [band four-point-one (4.1) ERM] domain composed of three subdomains (F1, F2, and F3) with binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that displays peptides representing the intrinsically disordered regions of the human proteome, we identified a large number of novel ligands. We determined the affinities for the ERM and merlin FERM domains interacting with 18 peptides and validated interactions with full-length proteins through pull-down experiments. The majority of the peptides contained an apparent Yx[FILV] motif; others show alternative motifs. We defined distinct binding sites for two types of similar but distinct binding motifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computational peptide docking protocols and mutational analysis. We provide a detailed molecular understanding of how the two types of peptides with distinct motifs bind to different sites on the moesin FERM phosphotyrosine binding-like subdomain and uncover interdependencies between the different types of ligands. The study expands the motif-based interactomes of the ERMs and merlin and suggests that the FERM domain acts as a switchable interaction hub.
Assuntos
Domínios FERM , Neurofibromina 2 , Humanos , Neurofibromina 2/genética , Neurofibromina 2/química , Neurofibromina 2/metabolismo , Ligantes , Estrutura Terciária de Proteína , PeptídeosRESUMO
Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.
Assuntos
Proteoma , Proteômica , Sítios de Ligação , Humanos , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Proteoma/genética , Proteoma/metabolismoRESUMO
Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to mass-spectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development.
Assuntos
Fator 2 Relacionado a NF-E2 , Peptídeos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos/química , Espectrometria de Massas/métodos , Cromatografia de AfinidadeRESUMO
Cellular function is based on protein-protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismo , Sítios de Ligação , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade por SubstratoRESUMO
A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.
Assuntos
Domínios PDZ/genética , Peptídeos/genética , Mapas de Interação de Proteínas/genética , Proteoma/genética , Sequência de Aminoácidos/genética , Sítios de Ligação , Proteína 4 Homóloga a Disks-Large/genética , Humanos , Ligantes , Oligonucleotídeos/genética , Biblioteca de Peptídeos , Fosforilação , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Proteína da Zônula de Oclusão-1/genéticaRESUMO
The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.
Assuntos
Proteínas de Membrana/metabolismo , Modelos Moleculares , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Algoritmos , Sítios de Ligação , Ciclofilina A/química , Ciclofilina A/genética , Ciclofilina A/metabolismo , Humanos , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Oligopeptídeos/química , Oligopeptídeos/genética , Domínios PDZ , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosforilação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Origem de Replicação , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide-binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreatic cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action, including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage-dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads, and it provides a rich data set covering phenotypes for the inhibition of thousands of interactions.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas/métodos , Biblioteca de Peptídeos , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Lentivirus/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Mapas de Interação de Proteínas/genéticaRESUMO
The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins ß-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-µM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.
Assuntos
Bacteriófago M13/genética , Domínios PDZ/genética , Domínios PDZ/fisiologia , Biblioteca de Peptídeos , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Biologia Computacional , Primers do DNA/genética , Humanos , Análise em MicrossériesRESUMO
Interactions between modular domains and short linear motifs (3-10 amino acids peptide stretches) are crucial for cell signaling. The motifs typically reside in the disordered regions of the proteome and the interactions are often transient, allowing for rapid changes in response to changing stimuli. The properties that make domain-motif interactions suitable for cell signaling also make them difficult to capture experimentally and they are therefore largely underrepresented in the known protein-protein interaction networks. Most of the knowledge on domain-motif interactions is derived from low-throughput studies, although there exist dedicated high-throughput methods for the identification of domain-motif interactions. The methods include arrays of peptides or proteins, display of peptides on phage or yeast, and yeast-two-hybrid experiments. We here provide a survey of scalable methods for domain-motif interaction profiling. These methods have frequently been applied to a limited number of ubiquitous domain families. It is now time to apply them to a broader set of peptide binding proteins, to provide a comprehensive picture of the linear motifs in the human proteome and to link them to their potential binding partners. Despite the plethora of methods, it is still a challenge for most approaches to identify interactions that rely on post-translational modification or context dependent or conditional interactions, suggesting directions for further method development.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Motivos de Aminoácidos , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Análise Serial de Proteínas/economia , Análise Serial de Proteínas/métodos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/economia , Proteínas/química , Proteômica/economia , Proteômica/métodos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin-syndecan-PIP2-Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development.
Assuntos
Gastrulação/fisiologia , Sindecanas/metabolismo , Sinteninas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Animais , Movimento Celular/fisiologia , Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Camundongos , Dados de Sequência Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinteninas/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4PHD6) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4PHD6, and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3PHD7). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Histona-Lisina N-Metiltransferase , Ligação Proteica , Humanos , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genética , Animais , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Sítios de Ligação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genéticaRESUMO
The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes.